Physiol Rev
83: 117–161, 2003; 10.1152/physrev.00018.2002.
Molecular Physiology of Low-Voltage-Activated
T-type Calcium Channels
EDWARD PEREZ-REYES
Department of Pharmacology, University of Virginia, Charlottesville, Virginia
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I. Introduction
II. Electrophysiology of Native T-type Currents
A. Low-theshold calcium spikes
B. Neuronal
C. Heart
D. Kidney
E. Smooth muscle
F. Skeletal muscle
G. Sperm
H. Endocrine tissues
I. Cell lines
J. Single-channel recordings
III. Regulation
A. Hormonal inhibition
B. Hormonal stimulation
C. Guanine nucleotides
D. Protein kinases
E. Voltage
IV. Molecular Cloning of T-type Channels
A. Cloning
B. Structure
C. Distribution
D. Electrophysiology of recombinant channels
E. Auxiliary subunits?
V. Pharmacology
A. Antihypertensives
B. Antiepileptics
C. Anesthetics
D. Antipsychotics
VI. Conclusions
A. Physiological roles
B. Future directions
Perez-Reyes, Edward. Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels. Physiol Rev 83:
117–161, 2003; 10.1152/physrev.00018.2002.—T-type Ca2⫹ channels were originally called low-voltage-activated
(LVA) channels because they can be activated by small depolarizations of the plasma membrane. In many neurons
Ca2⫹ influx through LVA channels triggers low-threshold spikes, which in turn triggers a burst of action potentials
mediated by Na⫹ channels. Burst firing is thought to play an important role in the synchronized activity of the
thalamus observed in absence epilepsy, but may also underlie a wider range of thalamocortical dysrhythmias. In
addition to a pacemaker role, Ca2⫹ entry via T-type channels can directly regulate intracellular Ca2⫹ concentrations,
which is an important second messenger for a variety of cellular processes. Molecular cloning revealed the existence
of three T-type channel genes. The deduced amino acid sequence shows a similar four-repeat structure to that found
in high-voltage-activated (HVA) Ca2⫹ channels, and Na⫹ channels, indicating that they are evolutionarily related.
Hence, the ␣1-subunits of T-type channels are now designated Cav3. Although mRNAs for all three Cav3 subtypes are
expressed in brain, they vary in terms of their peripheral expression, with Cav3.2 showing the widest expression. The
electrophysiological activities of recombinant Cav3 channels are very similar to native T-type currents and can be
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EDWARD PEREZ-REYES
differentiated from HVA channels by their activation at lower voltages, faster inactivation, slower deactivation, and
smaller conductance of Ba2⫹. The Cav3 subtypes can be differentiated by their kinetics and sensitivity to block by
Ni2⫹. The goal of this review is to provide a comprehensive description of T-type currents, their distribution,
regulation, pharmacology, and cloning.
I. INTRODUCTION
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Rises in intracellular calcium trigger a variety of processes including muscle contraction, chemotaxis, gene
expression, synaptic plasticity, and secretion of hormones and neurotransmitters. Although there are many
channels and pumps involved in controlling intracellular
Ca2⫹ levels, voltage-gated Ca2⫹ channels play a key role
in this process (50). Calcium is not only an important
second messenger, but its entry can also depolarize the
plasma membrane, and thereby activate other voltagegated ion channels. This property is especially important
for neuronal T-type channels, which can generate lowthreshold spikes that lead to burst firing and oscillatory
behavior (175, 377). This activity is especially prominent
in the thalamus, where it plays an important role in sensory gating, sleep, and arousal (277). The term thalamocortical dysrhythmias has been coined to describe pathological changes in these oscillations, and they have been
implicated in a wide range of neurological disorders including absence epilepsy, the tremor associated with Parkinson’s disease, tinnitus, neuropsychiatric disorders, and
neurogenic pain (186, 242).
The ability of neurons to fire low-threshold Ca2⫹
spikes suggested the existence of low-voltage-activated
(LVA) Ca2⫹ channels. Since 1975 it has been recognized
that there were at least two distinct types of Ca2⫹ channel
(reviewed in Ref. 153). Hagiwara, Ozawa, and Sand, using
two-microelectrode voltage-clamp recordings from starfish eggs, found channels that were activated after small
depolarizations of the membrane, LVA, and other channels that required larger depolarizations of the membrane,
high voltage activated (HVA) (154). LVA currents inactivated faster, more completely, and at more negative membrane potentials than HVA currents. Similarly, LVA and
HVA currents were described in the marine pileworm
Neanthes (123). Of historical note, the first recordings of
mammalian LVA channels were made by Moolenar and
Spector in 1978 (290), who used the two-microelectrode
voltage-clamp technique on the mouse neuroblastoma
cell line N1E-115. However, these authors only observed
one type of Ca2⫹ channel, which in 10 mM Ca2⫹ activated
at ⫺55 mV and peaked at ⫺20 mV. Subsequent studies
have shown that N1E-115 cells provide a convenient system to study the properties of native T-type currents (see
sect. III). Using quinidine to block K⫹ currents, Fishman
and Spector reported in 1981 (120) on the existence of
two types of mammalian Ca2⫹ channel. One type activated at ⫺50 mV, peaked at ⫺20 mV, and inactivated
rapidly (␶ ⬃15–30 ms). The second type peaked at 0 mV
and inactivated slowly (␶ ⬃2,000 ms). The existence of
LVA currents was firmly established by the work of many
groups, including those headed by Kostyuk (115, 420),
Carbone and Lux (62), Armstrong (12), Bean (32), Feltz
(54), and Tsien (303, 306). Whole cell patch-clamp recordings showed that depolarizations in the ⫺60 to ⫺20 mV
range elicited rapidly inactivating currents, while higher
depolarizations elicited noninactivating currents. Plots of
the current versus test potential (I-V) showed two components, with the LVA component appearing as a hump
on the back of a larger HVA component. LVA currents
also turned off, or deactivated, slower than HVA currents,
producing slow tail currents (62). This property was studied in great detail in GH3 cells, where slowly deactivating
(SD) tail currents were found to activate at lower thresholds than fast deactivating (FD) currents (12). Tsien and
colleagues (306) extended the classification of dorsal root
ganglion (DRG) channels and proposed that these channels be called T type for transient, L type for long lasting,
and N type for neither T nor L type. LVA currents were
also found to be resistant to rundown, unlike HVA currents that required cAMP, ATP, and Mg2⫹ in the pipette
for stability (115). T- and L-type channels were also described in cardiac myocytes from guinea pig ventricle and
dog atrium and could be distinguished by their voltage
dependence, kinetics, single-channel currents, pharmacology, and conductance of Ca2⫹ and Ba2⫹ (32, 303). The
observation that T-type currents inactivate at lower membrane potentials than L-type currents led to the development of an assay to separate these two components (32).
The activity of both channels was recorded from a wellhyperpolarized potential such as ⫺90 mV, then the activity of L-type channels was recorded at a potential where
T-type channels are inactivated and L type are not (typically ⫺50 mV), then the L-type currents are subtracted
from the T- plus L-type currents to isolate the T-type
current. Additional methods, such as tail current analysis,
are required to isolate T-type currents in most other tissues due to the presence of HVA channels that also inactivate at ⫺50 mV. Another possible contamination is voltage-gated Na⫹ currents that appear to conduct Ca2⫹
(ICa,TTX); however, these can be differentiated by their
sensitivity to tetrodotoxin (TTX) (162). Hallmark features
of T-type channel electrophysiology are as follows: 1)
they begin to open after small depolarizations of the
plasma membrane (LVA); 2) their currents during a sustained pulse are transient; 3) they close slowly upon
repolarization of the membrane, generating a SD tail cur-
T-TYPE CALCIUM CHANNELS
2⫹
FIG. 1. Evolutionary tree of voltage-gated Ca
channels. Tree is
based on an alignment of the putative membrane-spanning regions and
pore loops of the human channels. Alignment of the full-length sequences yields a similar pattern, although all the branch points are
shifted to the left due to inclusion of nonconserved sequences. Lowvoltage-activated (LVA) channels appear to have diverged from an ancestral Ca2⫹ channel before the bifurcation of the high-voltage-activated
(HVA) channel family into Cav1 and Cav2 subfamilies. (Adapted from
Perez-Reyes E, Cribbs LL, Daud A, Yang J, Lacerda AE, Barclay J,
Williamson MP, Fox M, Rees M, and Lee J-H. Molecular characterization
of T-type calcium channels. In: Low Voltage-activated T-type Calcium
Channels, edited by Tsien RW, Clozel J-P, and Nargeot J. Chester, UK:
Adis International, 1998, p. 290 –305.)
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gests that gene duplication and divergence of an ancestral Ca2⫹ channel gene gave rise to LVA and HVA
subfamilies. Further duplication of the HVA gene gave
rise to Cav1 and Cav2 subfamilies. This duplication
must have occurred well over 500 million years ago,
since the nematode Caenorhabditis elegans contains
one member of each subclass. Eventually the Cav1
subfamily evolved into four genes, while both the Cav2
and Cav3 subfamilies evolved into three. Cloning and
expression studies have established that the Cav3 family encodes LVA T-type channels (sect. IV), while the
Cav1 subfamily encodes L-type channels (although expression of Cav1.4 has not been reported yet). These
channels play a critical role in excitation-contraction
coupling in cardiac, skeletal, and smooth muscle. In
fact, the Cav1.1 gene has evolved beyond a Ca2⫹ channel, and its physiological role in skeletal muscle is as a
voltage sensor, coupling membrane depolarization directly to calcium release channels of the sarcoplasmic
reticulum (SR) (340). In contrast, cardiac and smooth
muscle contraction requires influx of extracellular Ca2⫹
through Cav1.2 channels. In heart, this influx activates a
larger release of Ca2⫹ from intracellular pools via a
mechanism called calcium-induced calcium release
(CICR) (37). Cav1.2 channels are an important therapeutic target in the control of blood pressure and cardiac rhythm. The physiological roles of Cav1.3 channels
have been difficult to discern since pharmacological
tools have not been described that can separate its
activity from Cav1.2, and because these channels appear to be coexpressed in a number of tissues. In
contrast, the activity of Cav2 family members can be
selectively blocked by peptide toxins. Splice variation
of the Cav2.1 gene results in channels that differ in their
sensitivity to the spider toxin ␻-Aga-IVA and therefore
encode both P- and Q-type channels (55). The Cav2.2
gene encodes N-type channels, which are characterized
by their irreversible and potent block by the snail toxin
␻-conotoxin-GVIA. Studies with these toxins indicate
that Cav2.1 and Cav2.2 channels mediate the Ca2⫹ influx
into presynaptic terminals that trigger neurotransmitter
release. In contrast to these channels, it has been difficult to determine which native Ca2⫹ current is carried
by Cav2.3 channels. Recombinant Cav2.3 channels resemble T-type channels in their sensitivity to nickel and
their voltage dependence of inactivation, leading to the
early suggestion that they might encode T-type channels (376). However, Cav2.3 channels require stronger
depolarizations for channel opening, i.e., are HVA channels, and they deactivate 10-fold faster than T-type
channels (228). Studies with SNX-482, a tarantula toxin
that selectively blocks recombinant Cav2.3 channels,
suggests that these channels mediate R-type currents in
some tissues (299).
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rent; 4) they have a tiny, and equivalent, single-channel
conductance of Ba2⫹ and Ca2⫹; 5) they are relatively
insensitive to dihydropyridines; and 6) their steady-state
inactivation occurs over a similar voltage range as activation. In fact, T-type channels display a window current,
i.e., there is a small range of voltages where T-type channels can open, but do not inactivate completely. This
property may be particularly important in controlling intracellular Ca2⫹ levels (45). In conclusion, studies on
native channels have provided a toolkit for separating
Ca2⫹ channel subtypes, and these tools have proven useful in the characterization of both native and recombinant
channels.
T-type channels are expressed throughout the body,
including nervous tissue, heart, kidney, smooth muscle,
sperm, and many endocrine organs. These channels have
been implicated in variety of physiological processes including neuronal firing, hormone secretion, smooth muscle contraction, myoblast fusion, and fertilization. In general, the electrophysiological properties of T-type
currents recorded from various cell types are similar, but
differences have been noted in how they inactivate and in
their pharmacology. This heterogeneity can be explained
in part by the existence of three T-type channels that are
encoded on separate genes (90, 228, 324).
Voltage-gated Ca2⫹ channels have been classified
by their electrophysiological and pharmacological
properties, and more recently by their amino acid sequence identity. There are at least 10 genes encoding
␣1-subunits of voltage-gated Ca2⫹ channels (Fig. 1).
Alignment of their deduced amino acid sequences sug-
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EDWARD PEREZ-REYES
II. ELECTROPHYSIOLOGY OF NATIVE
T-TYPE CURRENTS
A. Low-Threshold Calcium Spikes
Low-threshold calcium spikes (LTS) have been described in slices and in isolated neurons from a variety of
brain nuclei such as inferior olive (243, 244), thalamic
relay (96, 183), medial pontine reticular formation (146),
lateral habenula (437), septum (10), deep cerebellar nuclei (241), CA1-CA3 of the hippocampus (163, 307), association cortex (122), paraventricular (399) and preoptic
nuclei of the hypothalamus (385), dorsal raphe (60), globus pallidus (296), and the subthalamic nucleus (40).
Typically the spike is crowned by a burst of action potentials (Fig. 2). Addition of TTX to block voltage-gated Na⫹
channels abolishes the fast action potentials, effectively
isolating the slowly activating and inactivating LTS. Numerous studies have shown that the LTS is mediated by a
2⫹
FIG. 2. Low-threshold Ca
spikes generate burst firing. A: many neurons can generate two distinct patterns of action
potential firing in response to a depolarizing stimulus. Regular, or tonic, firing is elicited when the neuron is depolarized
from a resting membrane potential near ⫺55 mV. In contrast, when the membrane potential is below ⫺70 mV, the same
depolarizing stimulus triggers a high-frequency burst of action potentials. [From Huguenard JR. Low-voltage-activated
(T-type) calcium-channel genes identified. Trends Neurosci 21: 451– 452, 1998, with permission from Elsevier Science.]
B: a representative example of currents that generate burst firing. Depolarization of the plasma membrane by hyperpolarization-activated current (Ih) leads to activation of T-type currents (IT), and a second phase of depolarization called
the low-threshold Ca2⫹ spike. Riding on top of the low-threshold Ca2⫹ spike are a burst of Na⫹ spikes mediated by fast
voltage-gated Na⫹ channels. High-threshold Ca2⫹ and K⫹ currents can also be activated by the low-threshold calcium
spikes. Ca2⫹ entry during the burst leads to activation of Ca2⫹-activated K⫹ currents, which in combination with
voltage-gated K⫹ channels repolarize the membrane. (From Bal T and McCormick DA. Synchronized oscillations in the
inferior olive are controlled by the hyperpolarization-activated cation current Ih. J Neurophysiol 77: 3145–3156, 1997.)
C: thalamic neurons of transgenic mice lacking expression of Cav3.1 do not fire bursts. Current-clamp recordings are
from neurons held at ⫺60, ⫺70, or ⫺80 mV, then depolarized or hyperpolarized by current injections of varying
magnitudes as indicated below the set of traces. When the resting membrane potential is ⫺60 mV, a depolarizing stimulus
triggers tonic firing in both wild-type (⫹/⫹) and transgenic (⫺/⫺) animals. When the membrane potential (Vm) is lowered
to ⫺70 mV, a hyperpolarizing stimulus triggers burst firing in neurons from wild-type, but not transgenic animals. When
the resting membrane potential is ⫺80 mV, a depolarizing stimulus only triggers burst firing in neurons from wild-type
animals. (From Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, and Shin HS. Lack of the burst firing
of thalamocortical relay neurons and resistance to absence seizures in mice lacking ␣1G T-type Ca2⫹ channels. Neuron
31: 35– 45, 2001, with permission from Elsevier Science.)
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Electrophysiological studies of recombinant channels show that Cav3.1 (formerly ␣1G) and Cav3.2 (␣1H)
have similar activation and inactivation kinetics, but can
be differentiated by their recovery from inactivation and
sensitivity to block by nickel (209, 230, 351). The kinetic
properties of these ␣1-subunits closely resemble native
T-type channels. In contrast, recombinant Cav1 and Cav2
channels require auxiliary subunits for normal gating behavior. This suggests that native T-type channels may be
formed by a single ␣1-subunit. Cav3.3 (␣1I) channels also
generate LVA currents; however, these currents activate
and inactivate much more slowly than T-type channels.
Native currents with these properties have only been
described in a limited number of studies (19, 99, 177, 316,
398). Although there is no drug that is highly selective
(⬎10-fold) for T-type over other ion channels, block of
T-type channels appears to contribute to the therapeutic
usefulness of antihypertensives, antiepileptics, anesthetics, and possibly antipsychotics. Therefore, T-type channels provide a novel target for drug development.
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T-TYPE CALCIUM CHANNELS
TABLE
These Na⫹-dependent action potentials are brief (1–2 ms)
and of high amplitude (⌬80 mV), and in some thalamic
neurons of very high frequency (⬎400 Hz). High-threshold
Ca2⫹ channels will also begin to open at potentials more
positive than ⫺40 mV (385). This will lead to more influx
of Ca2⫹ and, if present, activation of Ca2⫹-dependent K⫹
channels, which can lead to an afterhyperpolarization
(AHP) (40, 244). Low-threshold channels can recover
from inactivation during the AHP and could begin to fire
again as the membrane potential returns to its resting
level. Hyperpolarizations can also activate Ih channels,
which allow the influx of both Na⫹ and K⫹, thereby depolarizing the cell and directly triggering another LTS (Fig. 2).
In this scenario Ih are the primary pacemaker current (254).
Clearly there are a variety of ionic conductances that mediate pacemaker activity, and it is an oversimplification to
implicate a single channel as the mediator of a pacemaker
current. This is especially true in the heart (61).
Another way that low-threshold channels are involved in generating oscillations includes reciprocal connections with an inhibitory interneuron. Due to its important role in controlling brain activity, the best studied of
these circuits involves the GABAergic neurons of the
thalamic reticular nucleus that regulate the activity of
thalamic relay neurons (377). Interestingly, the firing patterns of thalamic relay neurons vary with the state of
consciousness (277, 378). In the awake state and during
rapid-eye-movement sleep, they fire in tonic mode: the
resting membrane potential is relatively depolarized, the
LTS is inactivated, and excitatory inputs faithfully trigger
action potentials. In deep sleep, or during thalamocortical
dysrhythmias, these neurons fire in an oscillatory mode
called burst firing; the resting membrane potential is hy-
1. Properties of neuronal low-threshold Ca2⫹ spikes
Brain Region
Subthalamic nucleus
Septal nucleus
Hippocampus, CA1–CA3
Thalamic nuclei
Lateral thalamic nuclei
Lateral geniculate nuclei
Lateral geniculate nuclei
Lateral habenular nucleus
Paraventricular nucleus
Medial preoptic nucleus
Dorsal raphe nucleus
Cerebellar nuclei
Cerebellar nuclei
Inferior olivary nucleus
Hypoglossal motoneurons
Resting
Membrane
Potential, mV
⫺60
⫺60
⫺64
⫺60
⫺60
⫺62
⫺66
⫺67
⫺57
⫺58
⫺64
⬍⫺60
Threshold,
mV
Amplitude,
mV
⫺50
20
14
11
23
20
30
35
30
40
34
⫺63
⫺54
⫺65
⫺68
⫺65
⫺60
⫺60
⫺60
⫺65
⫺65
⫺65
24
20
35
10
Deinact V
Threshold,
mV
Deinact V
Midpt,
mV
Deinact Time
Midpt,
ms
Deinact Time
Max,
ms
⫺78
⫺63
⫺77
80
70
120
175
⫺55
⫺69
⫺75
⫺64
⫺65
⫺65
60
170
50
⫺73
⫺70
⫺72
⫺72
⫺70
⫺83
⫺77
200
120
400
300
400
800
200
40
200
400
75
400
⫺66
⫺72
⫺68
⫺78
⫺77
⫺75
Reference
Nos.
40
10
307
183
96
457
456
437
399
385
60
241
2
243, 244
421
The properties of low-threshold Ca2⫹ spikes (LTS) were measured using current-clamp recordings. Threshold was defined by the voltage
where a depolarizing pulse triggers a LTS. The amplitude of the LTS is shown and does not include the fast Na⫹-dependent action potential. The
voltage and time dependence for deinactivation (recovery) of the LTS spike is shown. Deinact., deinactivation; midpt, midpoint; CA1–CA3,
hippocampal pyramidal neurons.
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Ca2⫹ conductance; it is not observed in Ca2⫹-free external
solutions, and it can be blocked by divalent cations such
as Co2⫹ and Ni2⫹. Definitive proof that thalamic LTS are
mediated by T-type channels was provided by studies on
transgenic mice, where knockout of the Cav3.1 gene abolished these spikes and burst firing (Fig. 2C) (206).
With a few notable exceptions (7, 191), LTS cannot
be triggered by depolarization of the neuron from the
resting membrane potential, which is typically between
⫺60 and ⫺65 mV. However, LTS can be observed after a
hyperpolarizing pulse is delivered. This process has been
called “deinactivation” and is due to recovery of channels
from inactivation. Table 1 lists a number of studies where
the voltage and time dependence for recovery of these
spikes has been examined in detail. LTS began to appear
after the neuronal membrane was hyperpolarized below
⫺69 mV, and full-amplitude spikes were observed when
the membrane was below ⫺73 mV. The rate of recovery
was measured by varying the duration of a hyperpolarizing pulse, then testing for the presence of an LTS upon
return to the resting membrane potential. These studies
found that the LTS deinactivated relatively quickly, with
half of it recovering in ⬃100 ms and full recovery after 200
ms. Therefore, one physiological role of the LTS is as a
pacemaker. Although increases in intracellular concentrations of Ca2⫹ act as a second messenger for a variety of
processes, in this case the important property is that
influx of the divalent cation leads to direct depolarization
of the membrane. The amplitude of this depolarization
was typically 25 mV (Table 1), which raised the membrane potential to approximately ⫺40 mV. Since the
threshold of many voltage-gated Na⫹ channels is around
⫺55 mV, the LTS can trigger a burst of action potentials.
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perpolarized, allowing full-amplitude low-threshold spiking (Fig. 2A). Such firing is thought to underlie spike-wave
discharges observed during absence seizures (176). Similar patterns of brain activity have been detected in a
variety of neurological disorders by magnetoencephalography and have been termed thalamocortical dysrhythmias (242).
B. Neuronal
1. Isolated neurons
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LVA T-type currents have been characterized using
whole cell voltage-clamp recording of neurons isolated
from a variety of brain regions. Very large T-type currents
(⬎1 nA) have been recorded from cholinergic neurons of
the basal forebrain (6), from relay neurons of the ventrobasal thalamus (222), and from sensory neurons of dorsal
root ganglia (436). Prominent T-type currents have been
found in neurons from many brain regions (Table 2). I-V
relationships are typically measured by holding the cell at
⫺90 mV, and then currents are elicited by depolarizing
pulses that increase in 10-mV increments. T-type currents
begin to activate when the membrane is depolarized
above ⫺60 mV. At threshold potentials, the currents activate relatively slowly, requiring many milliseconds to
reach peak, and also inactivate relatively slowly. At higher
potentials both activation and inactivation are faster. This
produces a stereotypical pattern in the family of current
traces where successive records cross each other, reminiscent of voltage-gated Na⫹ channels. In general, HVA
channels do not produce this criss-crossing pattern because inactivation rates are much slower than activation
rates (334). I-V curves commonly show two components,
with the LVA currents peaking at ⫺30 mV while the HVA
currents peak around 0 mV. In many neurons, T-type
currents can be measured in isolation with test pulses to
⫺30 mV and below. However, at higher potentials both
LVA and HVA currents are activated, complicating estimates of the LVA activation curve. Both I-V curves show
a peak then decrease at more positive potentials, which is
due to a reduction of the electrochemical gradient for
calcium ions as the test potential approaches the theoretical reversal potential (⬃100 mV; see Fig. 7 for example).
Therefore, the fraction of channels activated by a test
pulse continues to increase beyond the peak of the I-V
curve. This explains why estimates of the midpoint of
channel activation (V0.5) derived by normalizing the current at each test potential to the maximum current observed (I/Imax) produces values that are more negative
(⫺50 mV) than estimates that take into account changes
in driving force (⫺41 mV). To circumvent this problem,
activation curves have also been estimated by measuring
the amplitude of slowly deactivating tail currents at a
constant voltage. Most of these studies used a test pulse
of constant duration (isochronal), which assumes that the
rates of channel activation and inactivation are voltage
independent. However, this is not the case, so this method
can underestimate channel activation at negative potentials where channels open slowly. It can also underestimate activation if channels inactivate during the test
pulse. This problem can be overcome by varying the
duration of the activating pulse, so that the tail current is
elicited at the peak of the current (288). So far this
method has only been applied to recombinant channels.
Another experimental variable that affects the position of
the activation curve is the concentration and choice of
charge carrier. Millimolar concentrations of positively
charged divalent cations can bind to surface charges on
both the channel and the membrane, thereby reducing the
effective transmembrane voltage gradient (164). An effect
called surface charge screening. Increasing external
[Ca2⫹] from 2 to 10 mM shifts the apparent gating of
T-type channels by ⬃10 mV (128).
A hallmark of T-type currents is that they are transient; currents reach a peak then decay. In solutions
where K⫹ channels are blocked, this decay is due to
channel inactivation. The inactivation rate is measured by
fitting the current trace with an exponential function and
is typically reported with a ␶ in milliseconds. Plots of
inactivation rate versus test potential reveal two phases:
inactivation is slow at ⫺60, gets faster between ⫺50 and
⫺30, then is constant above ⫺30 mV. Activation rates
have a similar voltage dependence, leading to the notion
that channels must activate before inactivating, and that
the inactivation process is essentially voltage-independent. Although most T-type currents inactivate relatively
rapidly (⬍30 ms), there are also reports of slowly inactivating currents (Table 2). This variability was interpreted
as evidence for multiple T-type channel genes (179). This
hypothesis was supported by the cloning of T-type channels that differed in this property; Cav3.1 and 3.2 display
fast inactivation, while Cav3.3 displays slowly inactivating
currents. Notably, Cav3.3 mRNA is expressed in the same
brain regions as the slow currents (177, 228, 393). Excluding these cases, the average inactivation ␶ from 22 studies
is 20 ms.
The voltage dependence of inactivation is also measured at “steady-state” by holding the membrane at varying potentials (prepulse), then assaying for available Ttype channels with a test pulse to ⫺30 mV (h⬁). To
approximate steady-state, many studies have used a prepulse duration of 1 s, which is considerably longer than
the time required to inactivate open channels. However,
increasing the prepulse duration to 10 s results in a
⫺10-mV shift in the apparent h⬁ curve (53, 389). Despite
the use of different prepulses and concentration of divalent cations, there is good agreement between the 34
studies shown in Table 2, and the average midpoint of the
h⬁ curve is ⫺77 mV (Table 2). Somewhat surprisingly this
123
T-TYPE CALCIUM CHANNELS
TABLE
2. Properties of T-type currents in isolated neurons
Tissue
CA1, 3 pyramidal
Thalamus
Ventrobasal
Ventrobasal
Ventrobasal
LGN
LGN
LGN-interneuron
LGN-relay
Reticular
Reticular
Lateral habenula
Hypothalamus
Hypothalamus
Hypothalamus
Hypothalamus
Supraoptic
Supraoptic
Paraventricular
Midbrain and pons
Area postrema
Cochlear nucleus
Cerebellum
Purkinje
Dorsal root
ganglion
DRG
DRG
Nodose ganglion
Nodose
Pelvic ganglion
Olfactory
Olfactory
Retina
Rod bipolar
Cone bipolar
Oligodendrocytes
Threshold,
mV
I–V Peak,
mV
Imax,
pA
V0.5,
mV
h ⬁,
mV
5 Ca
5 Ca
2.5 Ca
2 Ba
10 Ca
⫺60
⫺64
⫺54
⫺70
⫺60
⫺40
⫺33
⫺40
⫺20
⫺90
⫺51
⫺1,320
⫺500
⫺20
⫺53M
⫺43C
⫺40M
⫺59M
⫺38C
⫺88
⫺80
⫺49
⫺84
⫺70
2 Ca
5 Ca
⫺60
⫺60
⫺30
⫺30
⫺47
⫺250
⫺44T
⫺86
⫺86
22
2 Ca
2 Ca
⫺50
⫺60
⫺30
⫺30
⫺224
⫺100
⫺48M
⫺65
⫺84
25
12
10 Ca
⫺60
⫺30
⫺150
⫺46C
⫺79
15
2 Ba
⫺57
⫺76
25
3 Ca
3 Ca
5 Ca
10 Ca
5 Ca
2 Ca
2 Ca
3 Ca
2 Ca
1 Ca
⫺60
⫺60
⫺70
⫺48
⫺60
⫺70
⫺70
⫺50
⫺70
⫺65
⫺25
⫺30
20
30
15
36
20
20
20
80
25
58W
251
350
353
299
615
218
206
570
275
507
10 Ca
10 Ca
5 Ca
2 Ca
2 Ca
10 Ca
40
15
20
940
215
⫺100
⫺52T
⫺59T
⫺10
⫺50
⫺35
⫺350
⫺280
⫺1,400
⫺186
⫺200
⫺110
⫺289
⫺131
⫺128
⫺150
⫺45M
⫺55M
⫺64M
⫺49T
⫺64T
⫺62T
⫺84
⫺81
⫺96
⫺67
⫺64
⫺81
⫺86
⫺78
⫺85
⫺81
⫺65
⫺60
⫺50
⫺64
⫺60
⫺59
⫺30
⫺30
⫺20
⫺60
⫺30
⫺33
⫺150
⫺400
⫺250
⫺400
⫺402
⫺46C
⫺93
⫺79
5 Ca
2 Ca
⫺65
⫺75
⫺10
⫺40
⫺36
⫺400
⫺59M
⫺72
⫺89
10 Ca
⫺60
⫺20
⫺400
⫺40C
⫺79
2.5 Ca
10 Ba
5 Ca
10 Ca
10 Ca
2.6 Ca
3 Ca
⫺65
⫺60
⫺60
⫺55
⫺50
⫺57
⫺70
⫺35
⫺25
⫺20
⫺15
⫺25
⫺36
⫺40
⫺2,000
10 Ca
10 Ca
20 Ba
⫺60
⫺60
⫺50
⫺30
⫺20
⫺25
⫺35
⫺66
⫺150
⫺15
⫺23
⫺400
⫺400
⫺500
⫺50
⫺60
⫺65
⫺67
⫺41C
⫺29C
⫺38T
⫺45C
⫺36T
⫺35T
⫺82
⫺60
⫺48
⫺44
⫺57
⫺82
⫺66
⫺59
⫺60
⫺63
␶recov, ms
Nickel, ␮M
288
397
16
5
300
400
100
50 (28%)
400
430 (63%)
7,000
820
42
12
23
3,300
25
30
30
330
400
170
80
6
147
188
354
257
458
126
389
400
200 (66%)
500 (83%)
50 (100%)
200 (54%)
50 (38%)
600
85
179
222
387
159
318
318
179
409
177
50 (50%)
200
50 (84%)
100 (67%)
4
389
293
110
119
253
10 (100%)
500 (50%)
157
101
110
189
100 (95%)
436
295
54
226
231
429
200, 201
10
100 (71%)
30
55
10
Reference
Nos.
1,000 (80%)
1,000 (85%)
316
316
47
The divalent cation concentration used to measure the currents is shown. Properties of the current-voltage relationship are described in terms
of the voltage threshold where currents become detectable, the voltage where they peak (I–V peak), and the maximum amplitude observed (Imax).
Methods used to calculate midpoints of the activation curves (V0.5) are denoted with the following superscripts: C, chord conductance; T, tail
currents; M, I/Imax. The midpoint of the steady-state inactivation curve is reported (h⬁). Kinetics of inactivation (␶inact) can vary with test potentials;
therefore, the values shown represent the voltage-independent rate, which was typically measured at ⫺10 mV. WCurrents decayed in a biexponential
manner; to simplify comparisons, a weighted tau was calculated according to the following equation ␶W ⫽ A1␶1 ⫹ A2␶2, where A represents the
fraction of current decaying with the respective time constant. The values shown for recovery from inactivation represent the voltage-independent
rate, which was typically measured at ⫺90 mV. Tail current kinetics continuously vary with repolarization potential, so when possible the values
shown represent those obtained at ⫺90 mV. In some cases recovery from inactivation was found to be biphasic, so the values represent the tau of
the fast component, the fraction of channels that recover fast (in parentheses), and the tau of the slow component. Nickel responses represent either
the IC50 value obtained or the concentration tested and the percent block observed (in parentheses). LGN, lateral geniculate nucleus; MDN,
magnocellular dorsal nucleus; L–M, lacunosum-moleculare of hippocampus; DRG, dorsal root ganglion; inact., inactivation; recov, recovery.
Physiol Rev • VOL
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Basal forebrain
Striatum
Accumbens
Septum
Septum
Amygdala
Cerebral cortex
Temporal
Piriform
Hippocampus
Dentate granule
L–M
interneurons
CA1 pyramidal
␶inact,
ms
Divalent,
mM
124
EDWARD PEREZ-REYES
Physiol Rev • VOL
more selective than it is. In summary, block by 10 –50 ␮M
Ni2⫹ can be used to implicate Cav3.2 channels, but additional evidence, such as a slowly deactivating tail current,
is required to rule out Cav2.3 channels. Both these criteria, plus PCR, were recently applied to show the expression of Cav3.2 in sympathetic ganglion neurons (231).
The temperature sensitivity of T-type currents has
been measured in thalamic neurons (85), DRG neurons
(305), N1E-115 neuroblastoma cells (298), and GH3 cells
(343). Heating from 22 to 37°C caused over twofold increases in current amplitudes and accelerated channel
activation and inactivation. In contrast, heating did not
affect the position of the I-V curve (305). The effects of
temperature have been found to be nonlinear, making it
difficult to calculate the effect of a 10°C increment (Q10)
in temperature (298, 343). In the linear range, Q10 values
with an average of 2.5 have been found for effects on
amplitude, activation kinetics, and inactivation kinetics.
The pH sensitivity of T-type currents has been studied in CA1 hippocampal neurons (405), ventrobasal thalamic neurons (365), and cardiac myocytes (83, 414).
Acidification of the external solution decreased current
amplitudes, whereas alkalinization had the opposite effect. In contrast, T-type currents are relatively insensitive
to changes in intracellular pH (405). The effect of external
pH is in part mediated by changes in the single-channel
conductance, which when measured with 110 mM Ca2⫹
increased from 3.5 pS at pH 6 to 10.8 pS at pH 9 (414).
Small changes in pH have also been shown to shift the
voltage dependence of activation and inactivation; changing pH from 7.3 to 6.9 shifted both of these parameters by
⫹3 mV, while increasing the pH to 7.7 had the opposite
effect (365). Larger shifts in the voltage dependence of
activation (⫺15 mV) have been observed using larger
shifts in pH (from 7.5 to 9.8) (83). The observed pKa
values were ⬃7, indicating that T-type currents will be
affected by modest changes in extracellular pH (365, 414).
2. Slices
LVA currents have been characterized in slices prepared from numerous brain regions (Table 3). The most
striking difference between these data and that obtained
with isolated neurons is that currents are much larger in
slices. This difference has been attributed to loss of channels that were localized on dendrites (97). In fact, T-type
channels appear to be preferentially localized to dendrites
(77, 135, 195, 199, 328).
T-type currents recorded from slices also appear to
activate and inactivate at more negative potentials than
observed in isolated neurons. The average threshold in
slices was ⫺70 mV, and peak currents were observed at
⫺45 mV (Table 3), while in isolated neurons threshold
was ⫺60 and the peak occurred at ⫺30 mV (Table 2). This
difference has been attributed to poor voltage clamp of
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value is considerably more negative than the apparent
threshold of activation, indicating that channels can inactivate at potentials where they do not appear to open.
Similar h⬁ values have been obtained with the recombinant Cav3 channels (see Table 8), where this closed-state
inactivation has been studied in greater detail (127). Although most HVA channels inactivate at more positive
potentials, R-type and recombinant Cav2.3 channels inactivate over a similar voltage range as T-type channels (334).
The time course of recovery from inactivation is an
important property of T-type channels. LTS are often
triggered after an inhibitory postsynaptic potential
(IPSP), and this is due to fast recovery of T-type channels
during the IPSP (deinactivation), followed by their opening as the membrane returns to its resting potential. Recovery is often measured by varying the time between
(interpulse) an inactivating pulse and a test pulse. In most
studies the interpulse voltage was ⫺90 mV, and similar
results are obtained at more negative voltages. In contrast, recovery is slower at potentials where channels can
also inactivate. There is considerable variability in the
reported values for recovery, ranging from 100 to 3,300 ms
(Table 2). Although most studies found that recovery
followed a monoexponential time course, others have
found evidence for a biexponential process. This discrepancy might be due to differences in the duration of the
inactivating pulse, where long pulses allow channels to
accumulate in a second inactivated state from which they
recover slowly. As observed in sensory neurons (53),
recombinant channels recover with a monoexponential
time course from short pulses and with a slower, biexponential time course from long pulses, although this property is less pronounced for Cav3.3. Differences in recovery
kinetics of T-type currents between different neurons can
also be due to which Cav3 isoform they express (209). Of
the three isoforms, Cav3.1 channels recover the fastest
(⬃120 ms from short pulses), and this time course is
similar to that observed for recovery of both native T-type
currents (⬃300 ms; Table 2) and LTS (⬃150 ms; Table 1).
Early studies found that LVA channels were more
sensitive to block by 50 –100 ␮M nickel than HVA channels (124, 152, 298). Subsequent studies found that many
neuronal T-type channels required ⬎10-fold higher concentrations for block, with many studies reporting IC50
values of ⬃300 ␮M (Table 2). Studies on recombinant
channels provided an explanation for this diversity; only
Cav3.2 channels are highly nickel sensitive (IC50 ⬃10 ␮M),
while Cav3.1 and Cav3.2 are 20-fold less sensitive (230).
Recombinant HVA channels also differ in their Ni2⫹ sensitivity, with Cav2.3 being relatively sensitive (455). Although the mechanisms of block of recombinant HVA and
LVA channels are complex and subject to multiple interpretations, a consistent finding is that block is greatest at
potentials near threshold. This voltage dependence would
exaggerate block of LVA channels, making nickel appear
125
T-TYPE CALCIUM CHANNELS
TABLE
3. Properties of T-type currents in brain slices
Brain Region
Cerebellum
Thalamus
LGN
LGN
Laterodorsal, IT,f
Laterodorsal, IT,s
Hypothalamus MDN
Brain stem respiratory
Dorsal horn
Threshold,
mV
I–V
Peak,
mV
Imax,
pA
0.5
⫺60
⫺33
1
2
2 Ca
2 Ca
2
1.5
2 Ba
⫺65
⫺70
⫺80
⫺80
⫺83
⫺65
⫺54
⫺53
⫺45
⫺55
⫺65
⫺50
⫺40
⫺20
Divalent,
mM
V0.5,
mV
h⬁,
mV
␶inact,
ms
⫺690
⫺51M
⫺86
14
⫺1,500
⫺1,812
⫺670
⫺630
⫺1,051
⫺550
⫺1,000
⫺61M
⫺61M
⫺77
⫺84
⫺86
⫺98
⫺82
⫺81
⫺86
26
17
32
55, 69
12
⫺70M
⫺59M
⫺45M
14
␶recov,
ms
100
100
Nickel, ␮M
Reference
Nos.
100 (19%)
292
200 (53%)
93
151
398
398
302
107
347
25 (50%)
500 (100%)
200 (50%)
200 (91%)
neurons in slices (97). Consistent with this suggestion, the
voltage dependence of single T-type channels recorded
from dendrites is similar to that observed in isolated
neurons (256).
In summary, T-type currents are found in neurons
throughout the brain, with particularly large currents
found in thalamic, septal, and sensory neurons. Their
activation near the resting membrane potential and their
fast recovery from inactivation allow them to generate
LTS. Their preferential localization in dendrites suggests
T-type channels play an important role in synaptic integration.
C. Heart
Cardiac T-type currents have been the focus of many
studies due to their possible role as a pacemaker current.
These studies have established that T-type current densities are highest in conduction and pacemaker cells, and
much reduced or nonexistent in ventricular myocytes.
T-type currents are highest near birth and gradually decline but may reappear in pathological conditions. Currents are larger in lower species and have a wider distribution. For example T-type currents are prominent
throughout the guinea pig and hamster heart but virtually
absent in adult ventricular myocytes of rat, cat, and dog.
Although Cav3.2 was cloned from a human heart cDNA
library, T-type currents have not been detected in human
myocytes (39, 235, 313).
Cardiac T-type currents were initially described in
dog atrium (32) and guinea pig ventricle (283, 303). These
studies showed that cardiac T-type currents resembled
those recorded from neurons, but differed from L-type
channels in terms of their kinetics (transient), pharmacology (dihydropyridine insensitive), conductance of divalent cations (Ca2⫹ ⫽ Ba2⫹), lack of regulation by ␤-adrenergic agonists, and smaller single-channel conductance of
Ba2⫹. Subsequent work has established their presence in
Physiol Rev • VOL
myocytes isolated from dog Purkinje (367), rabbit sinoatrial node (152), cat atrium and latent pacemaker cells
(462), hamster ventricle (362), and rat atrium (446). In
general, the currents were small, displaying a peak current density below ⫺3 pA/pF. In contrast, prominent Ttype currents (⬎⫺10 pA/pF) have been recorded from
myocytes isolated from shark (271), chick (202), finch
(48), and mollusks (452).
Calcium channels were originally classified by their
inactivation kinetics: rapidly inactivating or transient
channels were called T type, while slowly inactivating, or
long-lasting channels were called L type (303, 306). It
should be noted that this only holds for currents carried
by Ba2⫹, as cardiac L-type currents carried by Ca2⫹ can
inactivate at a similar rate as T-type currents. This is due
to calcium-induced inactivation of the L-type channel,
which appears to be mediated by calmodulin tethered to
the carboxy terminus of the channel (111). This inhibition
is triggered with every heartbeat as Ca2⫹ entry triggers a
larger release of Ca2⫹ from internal stores (37). In contrast, Ca2⫹ currents through T-type channels inactivate a
bit slower than Ba2⫹ currents (116, 410), which excludes
a similar regulation by calmodulin. Another major difference between these channels is that L-type channels conduct Ba2⫹ threefold better than Ca2⫹, whereas T-type
channels conduct both equally well (discussed further in
sect. IIJ).
A proposed physiological role for cardiac T-type
channels is as a pacemaker current during diastolic depolarization. These studies have been hampered by the
lack of a selective blocker, relying heavily on the ability of
40 ␮M nickel to block T- but not L-type currents (152,
298). Current-clamp studies of spontaneous action potentials showed that nickel slowed the late phase of depolarization, and hence slowed the firing of rabbit sinoatrial
nodal cells (SAN). Similar results have been obtained by a
number of groups using rabbit SAN (98, 353) or cat latent
pacemaker cells (462). Tetramethrin (0.1 ␮M) was re-
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The voltage dependence of activation was estimated by normalizing the current observed at each test potential to the maximum ( M ) (I/Imax),
fit with a Boltzmann function, then reported as the midpoint voltage (V0.5). A single concentration of nickel was tested, and the percent block is
shown in parentheses. LGN, lateral geniculate nucleus; MDN, magnocellular dorsal nucleus.
126
EDWARD PEREZ-REYES
TABLE
nantly express Cav3.2 channels. In contrast, in situ hybridization of mouse heart suggests that Cav3.1 channels predominate, although both isoforms were readily detected
and both were enriched in SAN relative to atrium (49). It
should be noted that distinct in situ probes might differ in
their ability to detect their cognate sequence, making
such comparisons difficult. PCR amplification of mouse
heart detected the expression of a splice variant of Cav3.1
that differs in the III-IV loop (91).
Expression of T-type currents in developing heart
has also been studied. T-type currents are readily detectable in neonatal hearts, increase slightly to a peak between postnatal days 4 and 8, then decline slowly to a
steady-state (236, 246). In other studies T-type currents
were only detected in neonatal rats, disappearing by postnatal day 21 (236). In contrast to L type, the biophysical
properties of T-type currents are unchanged during neonatal development. Changes in nickel sensitivity have not
been investigated but may be informative. No difference
in T-type current density was detected in SAN myocytes
from newborn (3–10 days old) and adult (41– 48 days old)
rabbits (329). Expression of Cav3.2 mRNA is higher in
fetal human heart than adult, whereas Cav1.2 shows the
opposite pattern (331).
4. Properties of T-type currents in peripheral tissues
Tissue
Divalent,
mM
Threshold,
mV
I–V Peak,
mV
Heart
Atrium
Atrium
Atrium
Atrium
Atrium
SAN
Latent pacemaker
Purkinje
Purkinje
5 Ca
5.4 Ca
2 Ca
1.8 Ca
2.5 Ca
2.5 Ca
2.7 Ca
2 Ca
5 Ca
⫺50
⫺50
⫺50
⫺50
⫺60
⫺47
⫺50
⫺60
⫺40
⫺30
⫺30
⫺30
⫺30
⫺40
⫺10
⫺20
⫺30
⫺25
5.4 Ca
20 Ba
1.8 Ca
10 Ca
⫺50
⫺50
⫺55
⫺45
10 Ca
Ventricle
Ventricle
Skeletal muscle
Spermatocytes
Adrenal
Glomerulosa
Fasciculata
Pituitary
Pars intermedia
Lactotropes
Melanotropes
Gonadotropes
Pancreas
␤-Cell
␤-Cell
pA/pF
⫺0.3
⫺1.0
⫺2.6
Imax,
pA
⫺24
⫺75
V0.5,
mV
h⬁ ,
mV
⫺40M
⫺42C
⫺24T
⫺38C
⫺23C
⫺31C
⫺48C
⫺47M
⫺57
⫺68
⫺59
⫺59
⫺75
⫺57
⫺68
⫺37
⫺77
⫺63
⫺41
␶inact,
ms
20
15
13
␶deact,
ms
⫺2.1
⫺3.3
⫺1.7
⫺2.9
⫺10
⫺30
⫺5.8
⫺100
⫺858
⫺15
⫺3.6
⫺400
⫺43C
⫺42C
⫺24C
⫺60
⫺20
⫺6.5
⫺218
⫺47C
⫺64
7
1.6
20 Ba
10 Ca
⫺45
⫺50
⫺15
⫺8.2
⫺30
⫺272
⫺7T
⫺17G
⫺56
⫺50
18
7
1.7
10 Ca
5 Ca
11 Ba
20 Ca
⫺50
⫺48
⫺50
⫺40
⫺15
⫺12
⫺20
⫺20
⫺150
⫺90
⫺30
⫺75
5T
⫺28M
⫺66
⫺61
⫺61
15
19
15
3
10 Ca
15 Ba
⫺40
⫺60
⫺30
⫺35
21
2.5
⫺16T
⫺14M
10
3.2
10
4
Nickel, ␮M
197
50 (66%)
160
23
144
40 (100%)
40 (54%)
100 (90%)
50 (47%)
1.3
⫺350
⫺115
⫺88
⫺90
⫺391
⫺700
⫺27C
␶recov, ms
100
83 (67%)
444
11
13
100 (100%)
16
224 (65%)
5,160
118
400 (50%)
4,800
13
5.4
200 (75%)
20
1.8
100 (80%)
100 (100%)
Reference
Nos.
32
444
236
323
116
152
462
165
410
129
362
45
38
350
82
287
84
240
204
262
320
25
Definitions are as in Table 2. Methods used to calculate activation curves are denoted with the following superscripts: C, chord conductance;
T, tail currents; G, constant-field equation of Goldman-Hodgkin-Katz; M, I/Imax. In some cases recovery from inactivation was found to be biphasic,
so the values represent the tau of the fast component, the fraction of channels that recover fast (in parenthesis), and the tau of the slow component.
SAN, sinoatrial node.
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ported to produce selective block as well, and to produce
similar effects on pacemaker cycle (152); however, subsequent studies failed to confirm this selectivity (165).
Similarly, a number of studies have found lower sensitivity and selectivity for nickel (Table 4). Two possible
explanations for these disparate results are 1) that heart
expresses two isoforms of the T-type channel, Cav3.1 and
Cav3.2, and these isoforms differ in their sensitivity to
nickel, and 2) that isoform expression is age and species
dependent. Cloned Cav3.2 channels are blocked at 20-fold
lower concentrations than Cav3.1 (or Cav3.3), displaying
an IC50 of ⬃10 ␮M (230). There is a strong correlation
between nickel sensitivity and Cav3.2 expression, suggesting that native Cav3.2 channels are as sensitive as the
cloned channel (323). This suggests that nickel (⬍100
␮M) can be used to implicate Cav3.2 channel expression.
Expression in atrium appears to be species specific:
Cav3.1 appears to predominate in rat and mice (91, 236),
while in guinea pigs it is Cav3.2 (323). In fact, careful
measurement of the nickel dose response in guinea pig
atrium revealed a biphasic response, with 80% of the
channels being blocked with an apparent IC50 of 23 ␮M,
while 20% of the channels were inhibited with an IC50 of
1,350 ␮M. Rabbit and cat SAN cells appear to predomi-
127
T-TYPE CALCIUM CHANNELS
TABLE
that lower blood pressure (356). In contrast, T-type channels may be localized near SR in atrial pacemaker cells of
the cat (180). These authors suggested a novel pacemaking mechanism where Ca2⫹ influx through the T-type
channel triggers subsarcolemmal Ca2⫹ sparks, which in
turn activate Na⫹/Ca2⫹ exchange currents that depolarize
the membrane to threshold.
A role for T-type channels in triggering atrial natriuretic peptide (ANP) secretion has been inferred from the
effects of mibefradil (236, 434). Evidence that mibefradil
acted on T-type channels was provided by the observation
that L-type blockers were much less potent blockers, or
stimulated ANP secretion.
D. Kidney
Relatively little is known about the physiological
roles of T-type channels in kidney. T-type currents have
only been recorded from smooth muscle cells (SMC)
isolated from rat interlobular and arcuate arteries (143).
Heterogeneity in Ca2⫹ currents was observed, with approximately one-third of the freshly isolated myocytes
displaying only T-type currents (“T-rich”), one-third only L
type, and the rest a mixture. Currents in T-rich cells were
some of the largest ever recorded for cardiovascular
smooth muscle (⫺156 pA in 2.5 mM Ca2⫹; Table 5), which
allowed their recording in physiological Ca2⫹ solutions.
Consistent with their assignment as T-type channels, currents activated and inactivated at voltages 20 mV more
negative than observed for L-type currents. In contrast to
most T-type currents, these SMC currents activated (I-V
peak ⫺10 mV) and inactivated (h⬁ ⫽ ⫺50 mV) at voltages
that were slightly more positive, which might be characterized as mid-voltage activated. They also inactivated
about twofold slower during a pulse (␶inact ⫽ 46 ms at ⫺10
5. Properties of LVA currents in smooth muscle
Tissue
Arteries
Coronary
Coronary
Aortic
Aortic
Aortic
Mesenteric
Rabbit ear
Rat tail
Veins
Portal
Organs
Lung
Uterus
Bladder
Colon
Imax, pA
h⬁ ,
mV
␶inact,
ms
⫺125
⫺250
⫺140
⬍⫺100
⫺65
⫺55
⫺80
⫺65
14
⫺30
⫺20
⫺38
⫺63
⫺55
⫹10
⫺263
⫺50
⫺10
⫺30
⫺10
⫺10
⫺200
⫺300
⫺200
⫺385
⫺70
⫺60
⫺58
Divalent,
mM
Threshold,
mV
I–V Peak,
mV
10 Ba
20 Ba
20 Ca
1.5 Ca
20 Ba
10 Ba
110 Ba
20 Ba
⫺60
⫺40
⫺60
⫺60
⫺40
⫺40
⫺30
⫺50
⫺10
⫺30
⫺30
⫺30
⫺12
5 Ca
⫺45
10 Ca
1.8 Ca
1.8 Ca
2 Ca
⫺50
⫺60
⫺56
⫺60
⫹10
Definitions are as in Table 2.
Physiol Rev • VOL
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Nickel, ␮M
Reference
Nos.
46
130, 131
332
3
339
339
372
34
428
13
246
20
448
453
382
445
20
600
24
100 (100%)
30 (50%)
Downloaded from http://physrev.physiology.org/ by 10.220.33.4 on July 4, 2017
Cardiac hypertrophy appears to trigger a return to
the neonatal pattern of gene expression (388), leading to
reexpression of T-type channels in rat and cat ventricular
myocytes (173, 268, 308). RNase protection assays indicate that Cav3.1 transcripts are upregulated in a rat model
where hypertrophy is induced by infarction (173). Although levels of Cav3.2 transcripts were not examined,
the sensitivity of reexpressed currents to nickel block
suggests a contribution of Cav3.2 channels as well (173,
268). T-type currents are also reexpressed in dedifferentiated rat ventricular myocytes (114). T-type currents
have been found to be increased twofold in cardiomyopathic Syrian hamsters relative to other hamster strains
(46, 362). Studies using 8-mo-old animals found that the
voltage dependence of activation and inactivation was
shifted to more negative potentials in ventricular myocytes from cardiomyopathic animals (362). In contrast,
studies using 1-day-old hamsters found that only inactivation was shifted (46). The relevance of these observations
to human pathologies is unclear as T-type currents have
not been detected in myocytes isolated from normal
atrium (235) or diseased ventricle (39, 337).
Calcium influx through voltage-gated channels activates intracellular Ca2⫹ release channels, leading to larger
elevations in intracellular Ca2⫹ and hence muscle contraction. The role of L-type channels in this process is well
established. T-type channels are also capable of triggering
this CICR, albeit much more weakly in both cardiac (370,
461) and skeletal muscle (133). T-type current-induced
contraction developed more slowly, suggesting that these
channels are not localized near SR stores as are L-type
channels. This observation coupled with the lower expression of T-type channels in working myocytes indicates that they do not play a major role in excitationcontraction coupling. Consistent with this notion,
mibefradil has little effect on cardiac inotropy at doses
128
EDWARD PEREZ-REYES
Physiol Rev • VOL
nifedipine affected glomerular filtration rate. Mibefradil
has also been reported to decrease renin secretion in rats
with renal artery clips, but had no effect on renin secretion from isolated juxtaglomerular cells (423). In conclusion, these studies suggest that T-type channels on afferent and efferent glomerular arterioles may be an
important therapeutic target (89). A possible advantage to
a T-selective antihypertensive drug is that it may also
prevent glomerular damage (192).
E. Smooth Muscle
In addition to L-type currents, SMCs isolated from
arteries, veins, and organs have also been reported to
have LVA currents. With the exception of the kidney
results noted above, cardiovascular SMCs have tiny LVA
currents. Therefore, most studies have had to use very
high concentrations of charge carrier, which shifts gating
to positive potentials, and currents have only been characterized in terms of their I-V relations and sensitivity to
holding potential. These characteristics are not sufficient
to establish an LVA current as T type, since HVA currents
actually represent a spectrum of mid- to high-voltageactivated currents. HVA currents also inactivate over a
wide range of potentials, with some R-type currents inactivating over the same voltage as T-type channels. These
concerns take on additional weight with the finding that
some vascular SMCs express non-L-type HVA currents
(291) such as P-type currents (155). Native P-type currents can be mid-voltage activated (33). In addition, a
nickel- and mibefradil-sensitive LVA current has been
described in colonic myocytes that gates similar to T-type
channels but has different permeability properties (214).
LVA channels can be classified as T type by their slow tail
currents, which surprisingly have not been reported, and
by their single-channel properties, which have been reported (34, 130, 450). Because T-type channels inactivate
at the resting membrane potentials of most SMCs (⫺75 to
⫺60 mV, reviewed in Ref. 168), it has been hard to discern
their physiological role. Perhaps they contribute to basal
intracellular Ca2⫹ concentrations via their window current, or after transient hyperpolarizations induced by
spontaneous transient outward currents (STOCs).
LVA currents have been found in the following arterial SMCs: coronary (130, 281, 332), aortic (3), mesenteric
(150, 311, 372), rabbit ear (34), rat tail (327, 428), and
middle cerebral arterioles (167). RT-PCR detected the
expression of both Cav3.1 and Cav3.2 in rat mesenteric
arterioles (150). Single-channel studies have established
the presence of 7.5-pS T-type channels in freshly isolated
guinea pig coronary SMCs (130). This study also reported
that whole cell LVA currents could only be observed in
half of the cells. In contrast, LVA currents were never
detected in freshly isolated coronary SMCs from rabbits
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mV). Atypical currents with similar properties have been
reported for SMC isolated from rat portal vein (315) and
from the terminal branches of guinea pig mesenteric arteries (291).
Quite surprisingly, the human tissue that expresses
the most mRNA for Cav3.2 is the kidney (90, 438). In
contrast, both Cav3.1 and Cav3.3 are predominantly expressed in brain (228, 288, 289, 324). Skøtt and co-workers
(11, 156) have studied the distribution Cav3.1 and 3.2 in
kidney and concluded that Cav3.1 was in the tubules,
while Cav3.2 was in renal smooth muscle (11, 156). RTPCR indicated that mRNAs for Cav3.2, and to a lesser
extent Cav3.1, were expressed in rat (but not rabbit)
glomerular afferent and efferent vessels, and vasa recta.
The same study reported that K⫹-induced contraction of
perfused rabbit afferent arterioles could be blocked by
low concentrations of mibefradil (IC50 ⬃10 nM) that
should be selective for T-type channels. Nickel could also
block this response; however, the concentrations required (1 mM) would be expected to block almost all Ca2⫹
channels. Similar results were obtained with rat isolated
perfused hydronephrotic kidneys: either 1 ␮M mibefradil
or 100 ␮M nickel (a relatively selective dose) dilated
afferent and efferent arterioles constricted with ANG II
(314). Selectivity of mibefradil for T-type channels was
demonstrated by coadministration with the L-type
blocker nifedipine. In these experiments 1 ␮M nifedipine
caused a modest dilation of the efferent arteriole but did
not prevent the more pronounced dilation induced by
mibefradil.
Cav3.1 was detected in dot blots of human fetal, but
not adult, kidney (288). RNase protection assays of rat
kidney regions indicated that Cav3.1 was predominantly
expressed in the inner medulla (11). RT-PCR of microdissected nephron segments indicated that Cav3.1 was expressed in distal convoluted tubules, connecting tubules,
and collecting ducts. This study also examined the distribution of Cav3.1 protein using a polyclonal antibody
raised against the first 22 amino acids of the deduced
Cav3.1 sequence (11). Immunoreactivity was detected in
the apical domains of the distal convoluted tubules and in
principal cells of connecting tubules and inner medullary
collecting ducts. Preincubation of the antibody with peptide blocked the signal; however, Western blots were not
presented to demonstrate the selectivity of the antibody.
These results suggested that T-type channels, along with
endothelial calcium channels (ECaC), might be involved
in Ca2⫹ reabsorption.
Calcium channel blockers are widely used in the
treatment of hypertension. It is generally accepted that
they work by blocking L-type channels in vascular smooth
muscle, leading to vasodilation. Recent studies suggest
that part of their effect may be mediated by changes in
renal hemodynamics (172). In intact dogs, both nifedipine
and mibefradil increased renal blood flow, whereas only
T-TYPE CALCIUM CHANNELS
F. Skeletal Muscle
Newborn rodent skeletal muscle was also used in
early studies to establish the existence of T-type channels
Physiol Rev • VOL
as separate from L-type channels (30, 81). Developmental
studies of mice and rats have shown that the T-type
current is only found in embryonic and newborn muscle
and disappears by 3 wk of age (29, 38, 142). Concomitantly, the slow L-type current increases. Studies on the
muscular dysgenesis (mdg) mouse clearly established
that the T- and L-type channels were encoded on separate
genes (30, 67, 212), and it is the L-type channel (Cav1.1)
that plays a critical role in excitation-contraction coupling
(395). Recent studies have shown that the T-type current
is important for myoblast fusion (45). In particular, it was
shown that T-type channels could generate sufficient window currents to alter intracellular Ca2⫹ concentrations,
and trigger fusion. This hypothesis was further supported
by pharmacological experiments using low concentrations of Ni2⫹ and amiloride, and by antisense oligonucleotides directed against Cav3.2 (45). The selective expression of Cav3.2 in skeletal muscle fibers has also been
demonstrated using single-cell PCR (38). The electrophysiological properties of these currents (Table 4) and Ni2⫹
sensitivity closely resemble recombinant Cav3.2 currents,
although differences have been noted in kinetics of activation, inactivation, and recovery from inactivation (38).
Early studies also noted kinetic differences as a function
of development (142), suggesting that these differences
are not due to rapid events such as phosphorylation, but
rather slower events such as gene transcription of auxiliary subunits (see sect. IVE).
G. Sperm
Calcium channels appear to play a role in mediating
the egg-induced acrosome reaction that precedes fusion
(for review, see Ref. 94). T-type channels have been implicated in this process, although other Ca2⫹ conductances may be involved (134, 435). One reason for this
uncertainty is that it is virtually impossible to patch clamp
mature sperm (335). Therefore, electrophysiological studies have used spermatogenic cells, which are primary
spermatocytes in the pachytene stage. The only voltagedependent Ca2⫹ channels in these cells are T type, and
their biophysical (350) and pharmacological properties
(15) have been well characterized. PCR results indicate
that Cav3.2 is the predominant isoform expressed in human testis (182, 375). Consistent with this result is the
high sensitivity (IC50 ⫽ 34 ␮M) with which nickel blocks
the spermatocytic T-type current (15), although it should
be noted that Sertoli cells also express nickel-sensitive
T-type currents (225). Similar concentrations of nickel
also block the acrosome reaction (121, 375). Similarly, the
following compounds have been found to block spermatocyte T-type channels and the acrosome reaction at
similar concentrations: isradipine (PN 200 –110), nifedipine, pimozide, amiloride, mibefradil, W-7, and trifluoper-
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(269) or humans (332). LVA currents were found if the
cells were cultured, and these currents appear to be T
type based on their voltage dependence and 1:1 conductance of Ca2⫹ and Ba2⫹ (332). Similar results have been
obtained with aortic SMCs; T-type currents are expressed
in proliferating cultures, but neither in confluent cultures
(3, 339) nor in freshly isolated cells (220). In fact, T-type
currents were only detected in cells that were in G0 or G1
phases (220), leading to the hypothesis that they might
play a role in proliferating SMCs (338). Consistent with
this notion is the finding that mibefradil could reduce
neointima formation following balloon injury to rat carotid arteries (355). However, it should be noted that
T-type currents were not observed in SMCs prepared from
injured rat aorta, although a transient downregulation of
L-type channels was documented (333).
LVA currents have also been detected in SMCs isolated from veins, such as saphenous (450), portal (246),
and azygous (282). T-type single-channel currents were
recorded in saphenous vein SMCs (450). Isradipine (PN
200 –110) was found to block T-type currents of rat portal
vein with an IC50 of 0.5 ␮M, while L-type currents were
blocked at 1 nM (246). The sensitivity of these T-type
currents was greater than observed in other preparations
(see sect. VA), suggesting that Cav3 isoforms may differ in
their pharmacology. Current-clamp recordings revealed
that portal vein SMCs could fire action potentials that
resembled those observed with cardiac myocytes (246).
Isradipine (10 nM) inhibited the plateau phase with no
effect on the rising phase, suggesting that T-type channels
might play a pacemaker role in these cells.
LVA currents have been described in SMCs from
bronchi (185, 448), ileum (373), colon (445), bladder
(382), and uterus (453). The results with pregnant human
uterine SMCs are notable because the T-type currents (⫺3
pA/pF in 1.8 mM Ca2⫹) were larger than the L-type currents (453). These currents activated and inactivated
(h⬁ ⫽ ⫺70 mV) at potentials typical of most T-type currents (Table 5). Current-clamp recordings indicated that a
depolarizing pulse could trigger an action potential; however, the threshold (⫺20 mV) was higher than observed
with neuronal LTS. Sizable (⫺200 pA) T-type currents
were also described for porcine bronchial smooth muscle,
being detected in 30% of the cells, but absent from tracheal SMCs (448). LVA currents (⫺1.4 pA/pF) were found
to disappear when bladder SMCs were cultured (382).
Concomitant with this loss was a shift in the threshold for
action potential generation from ⫺25 to ⫺15 mV, suggestive of a role for T-type channels.
129
130
EDWARD PEREZ-REYES
azine (13, 15, 121, 247, 350, 375). Spermatocytic T-type
channels appear to be regulated by tyrosine and calmodulin-dependent protein kinases (14, 247).
H. Endocrine Tissues
1. Adrenal
2. Pituitary
LVA Ca2⫹ currents have been recorded from cells of the
anterior and intermediate lobes of the pituitary gland including (84, 392, 439) lactotropes (240), corticotropes (261),
gonadotropes (379), and thyrotropes (204). These cells genPhysiol Rev • VOL
3. Pancreas
T-type channels appear to play a similar pacemaker role
in insulin secretion from pancreatic ␤-cells of the islets of
Langerhans. Increases in plasma glucose and its metabolism
in ␤-cells leads to increases in cellular ATP and inhibition of
KATP channels (300). Closure of KATP channels initiates the
pacemaker cycle by depolarizing the ␤-cell membrane to
about ⫺55 mV. Riding on top of this slow plateau depolarization are rapid calcium-dependent spikes (reviewed in Ref.
352). Insulin secretion can be blocked by a wide variety of
agents, indicating that these spikes activate L-, P-, and R-type
currents (238, 258, 418).
T-type currents in pancreatic ␤-cells have been characterized at the whole cell (Table 4; Refs. 25, 320) and
single-channel level (17, 348). Expression is species dependent, with little or no expression in rodents, but
readily detectable in humans (25, 426). Therefore, it is
notable that LVA currents were found in ␤-cells from
diabetes-prone NOD mice (426). The expression of LVA
currents in mouse cells has been reported to be stimulated by a 6-h treatment with cytokines (25 U/ml of interleukin-1␤ plus 300 U/ml of interferon-␥; Ref. 427). However, the I-V curve for the cytokine-induced current
peaked between ⫹10 and ⫹20, while under similar recording conditions this group found that bona fide, slowly
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T-type channels have been implicated in the secretion of aldosterone (76) and cortisol (109). Calcium currents, and their regulation by secretagogues, have been
extensively studied in cells isolated from bovine adrenal
zona glomerulosa and fasciculata. Glomerulosa cells express a mixture of T- and L-type channels (82), whereas
fasciculata cells predominantly express T-type channels
(22, 287). Although these cells can be considered nonexcitable due to their lack of Na⫹ channels, they are still
capable of generating Ca2⫹-dependent action potentials
(22). In contrast, adrenal chromaffin cells express HVA
Ca2⫹ and Na⫹ currents but no LVA currents (16).
Serum K⫹ is regulated by secretion of aldosterone
from adrenal glomerulosa cells. Glomerulosa cells are
excellent K⫹ sensors and depolarize after small increases
in serum K⫹, which in turn leads to the activation of
T-type channels, Ca2⫹ influx, and stimulation of aldosterone synthesis and release. For example, increasing K⫹
from 2 to 5 mM causes the resting membrane potential of
isolated cells to depolarize from ⫺97 to ⫺78 mV (76).
Aldosterone secretion is also stimulated by ANG II, an
effect mediated by stimulation of T-type channel activity
(discussed in sect. IIIB).
Cortisol secretion from adrenal zona fasciculata cells
is regulated by ACTH, which depolarizes these cells by
inhibiting a K⫹ conductance (286). Depolarization would
activate T-type channels, leading to a rise in intracellular
Ca2⫹, and ultimately cortisol synthesis and secretion. Cortisol secretion can be inhibited by a variety of blockers
including mibefradil, and this block occurs over the same
concentration range as their block of the T-type channels
(109, 141, 345). ACTH may also regulate the expression of
T-type channels (22).
Adrenal T-type channels are encoded by Cav3.2. In
situ hybridization of rat and bovine glands detected high
levels of Cav3.2 expression in the cortex, with only trace
amounts of Cav3.1 and Cav3.3 (358). The biophysical properties and sensitivity to Ni2⫹ block of both glomerulosa
and fasciculata cells are also consistent with their being
encoded by Cav3.2 (287, 358).
erate spontaneous action potentials and Ca2⫹ spikes, leading to secretion of hormone (see Ref. 406 and references
therein). Hormonal regulation of these LVA currents supports the idea that these currents are involved in secretion
(see sect. III). The voltage- and time-dependent properties of
these currents are similar, but not identical to other native
T-type currents (Tables 2 and 4). Notably the peak of the I-V
curve is slightly depolarized. In addition, one study reported
that the LVA current was fivefold larger in Ba2⫹ than Ca2⫹
(379), a property typically ascribed to HVA currents. As
suggested previously, not all LVA currents are carried by
T-type channels, and other criteria must be applied (19, 33,
376). One such discriminating property is their tail deactivation kinetics, and slowly deactivating T-type currents are
clearly present in pituitary cells (84). Similarly, these channels can be distinguished at the single-channel level by their
conductance for Ba2⫹, and again, pituitary T-type currents
have been clearly identified (203). In situ hybridization studies suggest all three Cav3 isoforms are expressed, although
Cav3.2 was the predominant isoform detected (393). Consistent with this result, T-type currents were reported to be
nickel sensitive, with 80% of the current blocked by 100 ␮M
NiCl2 (240). Currents recorded from GH3 cells are not nickel
sensitive (IC50 ⫽ 777 ␮M), suggesting that this tumor-derived cell line most likely expresses Cav3.1 channels (160). In
conclusion, T-type channels appear to play a pacemaker role
in anterior pituitary, although they may also be involved in
stimulus-secretion coupling (258).
131
T-TYPE CALCIUM CHANNELS
sively T-type currents, allowing characterization of these
channels in the absence of contaminating HVA currents
(Table 6). The study of T-type currents in cell lines has
advanced our understanding of gating, permeation, and
pharmacology (73, 160, 368). For example, Chen and Hess
(73) developed models of gating using recordings from
3T3 fibroblasts. They also showed that the T-type currents
of NG108 –15 cells had different kinetics, leading them to
predict the existence of multiple channel isoforms. A
similar conclusion was reached from studies on TT cells,
which also recovered from inactivation much more
slowly than observed previously (44). Differences in the
pharmacology of T-type channels also suggested the existence of distinct channel isoforms (160). The cloning of
multiple Cav3 isoforms that differ in their kinetics, pharmacology, and recovery from inactivation supports this
hypothesis (209, 228, 404).
I. Cell Lines
J. Single-Channel Recordings
T-type currents have been reported in a number of
cell lines such as 3T3 fibroblasts (73) and many lines of
tumor origin, such as neoplastic B lymphocytes (128), the
related mouse neuroblastoma-derived lines N1E-115 (298,
368), NG108 –15 (196, 334), 140 –3 (144), N18 (397), and
ND7–23 (213); human neuroblastoma lines IMR-32 (65)
and SK-N-MC (18); rat pituitary lines GH3 (160, 270);
human TT cells (also called h-MTC) and rat 6 –23 (clone 6)
cells from medullary thyroid tumors (43, 44); human Y79
retinoblastoma cells (24); AT-1 from mouse atrium (351);
rat smooth muscle lines A7r5 and A10 (272); and the
pancreatic insulinoma lines INS-1 (rat; Ref. 42), HIT-T15
(hamster; Ref. 259), and NIT-1 (mouse; Ref. 426). Many of
these cell lines were reported to express almost exclu-
Single-channel studies provided conclusive evidence
that T-type channels were distinct from HVA Ca2⫹ channels (63, 303, 306). T-type channels were distinguished by
their smaller currents, insensitivity to dihydropyridines,
and voltage dependence of activation and inactivation. In
addition to providing criteria for resolving T-type currents, single-channel studies have provided insights into
both hormonal regulation of activity and gating.
With isotonic Ba2⫹ as the charge carrier (110 mM),
the single-channel current at a test potential of 0 mV is
⫺0.3 pA for T-type channels (Table 7) and ⫺1.5 pA for
L-type channels. Single-channel conductance is defined as
the slope of the line relating single-channel amplitudes
versus test potential and is commonly reported in pico-
TABLE
6. Properties of T-type currents in cell lines
Cell Line
Divalent,
mM
Threshold,
mV
I–V Peak,
mV
Imax,
pA
V0.5,
mV
h⬁,
mV
␶inact,
ms
2
1,900
2
5
1,260
200 (80%)
1,000
Human
TT
IMR-32
Y79
Rat
C-cell 6-23
GH3
10 Ca
10 Ca
20 Ba
⫺40
⫺50
⫺50
⫺15
⫺20
⫺20
⫺211
⫺80
⫺87
⫺27T
⫺51
⫺32C
⫺40
16
15
20
10 Ca
10 Ca
⫺50
⫺50
⫺15
⫺20
⫺500
⫺600
⫺31M
⫺33G
⫺57
⫺71
20
20
Mouse
MAb-7B
N1E-115
NG108-15
ND7-23
3T3
AT-1
2.5 Ca
50 Ba
10 Ba
10 Ba
20 Ba
2.5
⫺65
⫺50
⫺60
⫺50
⫺50
⫺60
⫺35
⫺20
⫺15
⫺30
⫺20
⫺35
⫺80
⫺300
⫺140
⫺119
⫺500
⫺500
⫺82
16
⫺27C
⫺48
C
⫺53
⫺67
⫺65
⫺64
␶deact,
ms
␶recov, ms
Nickel, ␮M
5 (57%)
200 (52%)
44
65
24
5 (22%)
777
43
160
47
20
18, 32
10
14
2
2
2.5
845
100
150 (73%)
1,200
Reference
Nos.
100 (60%)
128
298
334
213
73
351
Definitions are as in Table 2. Methods used to calculate activation curves are denoted with the following superscripts: C, chord conductance;
T, tail currents; G, constant-field equation of Goldman-Hodgkin-Katz; M, I/Imax.
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deactivating, T-type currents peaked between ⫺20 and
⫺10 mV (426). Human ␤-cells express both LVA and HVA
currents, and in 20% of the cells tested, the LVA current
was dominant (25). Voltage-clamp recordings indicated
that 100 ␮M Ni2⫹ could block the LVA current, while
current-clamp recordings indicated that it slowed action
potential frequency. Similar studies using the rat insulinoma cell line INS-1 found that low concentrations of
nickel could completely block T-type currents (IC50 ⫽ 30
␮M) and partially block insulin secretion (42). Nickelsensitive T-type currents (IC50 ⫽ 3 ␮M) have also been
recorded from the mouse insulinoma cell line NIT-1 (426).
Li and co-workers (463) also cloned a splice variant of
Cav3.1 from an INS-1 cDNA library. The high sensitivity to
nickel block suggests that Cav3.2 channels are also expressed, and this conclusion is supported by Northern
analysis (438).
132
EDWARD PEREZ-REYES
TABLE
they approach from the intracellular side of the channel
(255, 363). The selectivity sequence for monovalent
cations through T-type channels is Li⫹ⱖ Na⫹ ⬎ K⫹ ⱖ
Rb⫹ ⬎ Cs⫹ (128, 255). In fact, T-type channels begin to
pass outward K⫹ currents at physiological concentrations
of Ca2⫹ and voltage (⫹28 mV; Ref. 128). HVA channels
can also pass outward currents, but this requires depolarization of the membrane beyond ⫹70 mV (161). Our understanding of Ca2⫹ channel permeation is far from complete, and subject to multiple interpretations (275, 442).
Single-channel currents have also been studied at
Ca2⫹ concentrations closer to the physiological levels,
displaying a single-channel conductance of ⬃2 pS in 5 mM
(64) and 4.6 pS in 10 mM Ca2⫹ (21). Whole cell studies
using varying concentrations of Ca2⫹ indicate that currents increase sharply over the 1–10 mM range, then
saturate at higher concentrations. These studies have allowed estimates of the affinity of the open channel for
Ca2⫹. The average KD value from seven studies is 4.9 mM,
with considerable variability (range 0.3⫺10 mM) (4, 22,
54, 152, 160, 389, 429).
The transitions between open and closed states have
been studied in detail in DRG neurons (64), cardiac myocytes (104), fibroblasts (73), and N1E-115 neuroblastoma
cells (368). T-type channels respond to depolarizing
pulses by opening in bursts, that is, channels open and
close many times, become silent for a while, and at some
voltages may burst again. The fast opening and closings
7. Single-channel properties
Source
Divalent, mM
Current at 0
mV, pA
Conductance,
pS
Rat DRG
Chick DRG
Mouse DRG
Hippocampal CA3 pyramidal
Septum and diagonal band
Cerebellar Purkinje
Pituitary menalotropes
Hypoglossal motoneuron
Retinal ganglion
Cardiac ventricle
Cardiac Purkinje
Ear artery SMC
Coronary artery SMC
Saphenous vein
Adrenal glomerulosa
Pancreatic ␤-cells
Neuroblastoma N1E-115
Neuroblastoma ND7-23
Neuroblastoma 140-3
Low divalent
DRG
Cardiac ventricle
Coronary artery SMC
Coronary artery SMC
40 Ca
110 Ba
60 Ba
95 Ba
100 Ba
110 Ba
110 Ba
110 Ba
96 Ba
110 Ca
110 Ba
110 Ba
110 Ba
90 Ba
110 Ba
100 Ba
60 Ba
110 Ba
110 Ba
⫺0.2
⫺0.4
⫺0.25
⫺0.3
⫺0.2
⫺0.3
⫺0.4
⫺0.3
⫺0.36
⫺0.33
⫺0.4
⫺0.3
⫺0.38
⫺0.35
⫺0.25
⫺0.3
⫺0.25
⫺0.25
⫺0.3
5.2
8
7.2
7.8
7.8
9.0
8.1
7
8
6.8
9.0
6.9
7.5
8
8.7
6.4
7.2
7.9
6.84
5 Ca
10 Ca
10 Ca
10 Ba
⫺0.16
⫺0.16
⫺0.2
2
4.7
4.7
4.6
Selectivity
Ca ⫽ Ba
Ba ⬎ Sr
Ba ⫽ Ca
Ba ⫽ Ca
Ba ⫽ Ca
Ca ⫽ Ba ⫽ Sr
Reference
Nos.
64
124
217
118
147
52
203
417
194
104
367
34
130
450
27
348
368
213
144
64
21
21
130
Single-channel properties were measured from cell-attached patches using the indicated concentration of divalent cation as charge carrier. The
slope conductance of high-voltage-activated channels is similar to T-type channels when recorded in Ca2⫹ solutions but can be distinguished by their
current amplitudes at a test potential of 0 mV. DRG, dorsal root ganglion; SMC, smooth muscle cell.
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seimens (pS). When recorded in Ba2⫹, the conductance of
T-type channels is 7– 8 pS (Table 7), which is smaller than
that observed for either N-type (13–15 pS) or brain L-type
channels (25–28 pS) (118, 125, 417). Somewhat surprisingly, all three families have a similar Ca2⫹ conductance.
For example, the L-type channel conductance drops
threefold in Ca2⫹ (149, 367). Recombinant Cav2.1 and
Cav2.2 channels also preferentially conduct Ba2⫹ (⬎2fold), whereas Cav2.3 conducts both ions equally well
(56). In contrast, native T-type channels conduct Ca2⫹ and
Ba2⫹ (and Sr2⫹) equally well (64, 104, 367, 368). The
conductance plots differ in their relative position, with
T-type channels gating at more negative ranges, and extrapolating to a reversal potential of approximately ⫹40
mV, while HVA currents appear to reverse at ⫹60 mV (34,
52, 118, 124, 194, 203, 213, 367, 417).
Estimates of permeability based on reversal potentials yield the opposite rank order, with Ca2⫹ being more
permeable than Ba2⫹ (128). Similar results have been
obtained using recombinant T-type (363) and native Ltype channels (161) and suggest that Ca2⫹ binds with high
affinity in the pore and permeates when displaced by a
second Ca2⫹. A very interesting property of all voltagegated Ca2⫹ channels is that they become highly permeable to Na⫹ in the absence of Ca2⫹. Apparently Na⫹ are
not capable of displacing the bound Ca2⫹, and hence do
not permeate. This block is unidirectional, since monovalent cations can displace Ca2⫹ bound in the pore when
T-TYPE CALCIUM CHANNELS
FIG. 3. Simplified gating scheme. The model represents transitions
of T-type channels between rested (R), closed (C), inactive (I), and open
states (O). Changes in the membrane potential are thought to induce
movement of the S4 voltage sensors, which leads to rearrangements in
the channel structure. Movement of three of the four S4 regions is
sufficient to induce closed states that inactivate relatively quickly. The
proximal closed state (C4) can also show closed state inactivation or
lead to channel opening. Single T-type channels show a propensity to
open in bursts during a prolonged depolarization, and this represents
transitions between C4 and O states. The model has been simplified from
the version presented by Frazier et al. (127) by omission of C1, C2, I1, and
I2. The effects of voltage suggest that S4 sensors must move back toward
their original position before channels can recover from inactivation.
Physiol Rev • VOL
the whole cell observation that steady-state inactivation
can be observed at potentials more negative than channel
opening. This inactivation is slow and voltage dependent
(364). Inactivation from open (or the proximal closed
state C4) has similar kinetics and limits the channel to one
burst per sweep. Whole cell recordings suggest that this
rate is relatively voltage independent at potentials above
⫺20 mV. Several models of T-type channel gating have
been proposed that can simulate the observed activity.
One reason these models differ is that they are based on
disparate results. Unresolved questions include the following: Does the voltage dependence of the mean open
time explain the slowly deactivating tail currents observed at the whole cell level (64)? One study found that
open times were shorter at more negative test potentials
(64), another found no difference (104), while two found
that they increased (217, 368). To complicate matters
more, two studies have found evidence for a second type
of opening of longer duration (27, 147). Discrepancy in
mean open time measurements has been attributed to the
low signal-to-noise ratio of channel openings (73). A related question is: Does the burst duration vary with test
potential? Most studies report the burst duration at a
single voltage, although one study found that it increased
from 5 ms at ⫺40 mV to 14 ms at ⫺10 mV (104). These
models might also differ because of T-type channel heterogeneity, as evidenced by the distinct gating behavior of
the three recombinant Cav3 channels (228). Recently, a
model has been proposed that can simulate the gating
behavior of both recombinant Cav3.1 and Cav3.3 channels,
although different rate constants for the transitions are
required (127, 364). The model assumes that depolarization leads to sequential movement of each of the four S4
voltage sensors (see sect. IVB), culminating in a final
closed state that precedes the open state. The open state
can either deactivate (return to C4) or inactivate (IO).
Closed state inactivation is allosterically coupled to S4
movement and becomes prominent after three S4 sensors
have moved (Fig. 3). Movement of the fourth voltage
sensor does not affect the inactivation rate, so open channels inactivate at the same rate as closed channels in C3
and C4 states. The speed of voltage sensor movement and
subsequent transitions among closed states appears to be
slower in Cav3.3 than Cav3.1, leading to slower activation
kinetics. The channels also differ in their equilibrium
between C4 and O states, with Cav3.3 favoring C4 while
Cav3.1 favors O. Therefore, Cav3.1 channels inactivate
more from open states, whereas Cav3.3 channels inactivate more from closed states. The model accounts for
most, but not all, of the observed properties of T-type
channels; in particular, it does not attempt to explain
multiple open states or ultraslow (⬎1 s) inactivation
(127). In summary, voltage-gated Ca2⫹ channels can be
distinguished by their single-channel properties, with T-
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can be modeled as transitions between a closed state C
(C4 in Fig. 3) and an open state O. The transition rates out
of the open state determine the mean open time, which is
typically 0.5–2 ms (Table 7). Multiple bursts are seen
during depolarizing pulses just above the threshold for
channel opening. At more depolarized potentials a single
burst is observed, indicating that channels entered an
inactivated state. Analysis of the time a channel spends in
closed states reveals a bimodal distribution; closings
within a burst are short (⬃1 ms), whereas the time between bursts is longer (⬃10 ms). Channels can also open
partially, leading to a subconductance state that has a
smaller current (27, 64, 104). In contrast to other ion
channels (66), the gating behavior of this subconductance
state was found to be similar to the main conductance
state (64).
A distinguishing kinetic feature of T-type channels is
that whole cell current traces recorded during an I-V
protocol produce a criss-crossing pattern, where successive traces cross each other. This is largely due to slow
activation kinetics at threshold potentials (⫺60 to ⫺40
mV). At the single-channel level, T-type channels show a
long latency to the first opening, especially at threshold
potentials, where 40 ms may pass before the first opening.
This delay presumably reflects slow transitions between
closed states and can lead to a sigmoidal rise in the whole
cell current. This rate is voltage dependent, such that
channels open faster at more depolarized potentials (104).
In most sweeps (⬃70%) the channel does not open at
all, resulting in a blank sweep (73, 104). This suggests that
channels might inactivate without opening. Such closed
state inactivation has been observed in other channels
and is particularly prominent in Cav2 channels (321).
Closed state inactivation also provides an explanation for
133
134
EDWARD PEREZ-REYES
type channels showing a smaller conductance in Ba2⫹ and
equal conductance of Ca2⫹ and Ba2⫹.
III. REGULATION
A. Hormonal Inhibition
Many hormones and neurotransmitters have been
reported to inhibit T-type currents, including dopamine,
serotonin, somatostatin, opioids, ANP, and ANG II. Although most of these studies were performed on isolated
neurons, regulation has also been noted in secretory cells
of the pituitary and adrenal.
Dopamine inhibition of T-type currents has been described in DRG neurons (260), adrenal glomerulosa cells
(312), pituitary melanotrophs (204), lactotrophs (240),
and cells from the pars intermedia (309). Chick DRG
currents were shown to be inhibited 60% by either 10 ␮M
dopamine or norepinephrine (260). Inhibition occurred
with no apparent effect on kinetics. Recovery upon washout was not observed in whole cell recordings. Similar
inhibition was observed at the single-channel level, although in these experiments washout was observed. Single-channel current amplitude was not affected, suggesting that inhibition was due to a reduction in the
probability of opening (Po).
Dopamine (10 nM) inhibited T-type currents of rat
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Among the first criteria used to distinguish LVA from
HVA channels was their resistance to rundown in solutions that lack cAMP, ATP, and Mg2⫹, suggesting that LVA
channels were not phosphorylated by cAMP-dependent
protein kinase (115). Consistent with this hypothesis, hormones that stimulate adenylyl cyclase, such as the ␤-adrenergic agonist isoproterenol, had no effect on T-type
currents recorded from either canine atrial myocytes (32),
rabbit sinoatrial nodal cells (152), canine Purkinje cells
(166, 410), guinea pig ventricular myocytes (415), rabbit
ear artery (35), or guinea pig hippocampal CA3 neurons
(117). Stimulation of L-type currents via cAMP-dependent
pathways provided a positive control for most of these
studies. Although some studies have reported a small
stimulation of T-type currents by isoproterenol (9, 283),
this may be due to either incomplete separation of T- from
L-type currents (as evidenced by BAY K 8644 stimulation)
or due to secondary regulation induced by changes in
internal Ca2⫹ concentrations (411). In contrast to the
apparent lack of protein kinase A (PKA) regulation, hormonal regulation of T-type currents has been reported in
many systems. Both inhibition and stimulation have been
reported, and in some cases the same agonist did both.
For example, the GABAB agonist baclofen has been reported to cause stimulation at 2 ␮M but inhibition at 100
␮M (361).
anterior pituitary lactotrophs by 25– 40% (239, 240). Dopamine shifted the h⬁ curve from ⫺63 to ⫺77 mV (5 Ca)
and accelerated current decay during the pulse. It had no
effect on voltage dependence of activation. Dopamine’s
effects were reversible, mimicked by the dopamine agonist (D2 class) bromocryptine (10 nM), and prevented by
the D2 antagonist sulpiride (100 nM). Inhibitory regulation
could be abolished by omitting GTP from the intracellular
solution or by pretreatment with pertussis toxin. Inhibitory modulation could also be disrupted by inclusion of
antibodies that specifically recognize the ␣-subunit of Go,
while antibodies against Gi subunits had no effect (239).
Parallel studies showed that D2 receptors coupled to potassium channels via a different G protein, Gi-3␣.
Dopamine inhibited rat adrenal glomerulosa T-type
current by 33% (103). Inhibition was mediated by the D1
receptor class as evidenced by 1) inhibition with the D1
agonist SKF 82958; 2) block by the D1 antagonist SCH
23390; 3) but not by the D2 antagonist spiperone. Dopamine-mediated inhibition was blocked by agents that disrupt signaling by 1) G proteins [2 mM guanosine 5⬘-O-(2thiodiphosphate) (GDP␤S)], 2) adenylyl cyclase (100 ␮M
2⬘,3⬘-dideoxyadenosine), and 3) protein kinase activity
(10 ␮M H-89). Consistent with an action through D1 receptors, dopamine caused a stimulation of cAMP formation. Somewhat surprisingly, bath application of 8-bromocAMP had no effect on basal T-type currents but could
block the effects of dopamine. Addition of 100 nM G␤␥
subunits to the pipette solution did not affect T-type
currents. However, G␤␥ inhibition was observed after
addition of 1 mM 8-bromo-cAMP to the bath. These results suggested that G␤␥ subunits might bind directly to
the T-type channel.
Somatostatin (10 nM) was found to inhibit T-type
currents by 50% in rat somatotrophs (72). Somatostatin
caused the currents to decay faster during a sustained
pulse and shifted the midpoint of the steady-state inactivation curve (h⬁) curve from ⫺52 to ⫺63 mV (charge
carrier was 5 mM Ca2⫹). This regulation was abolished by
pertussis toxin. Neurotensin (200 nM) and substance P
(200 nM) inhibited by 25% the T-type currents from rat
cholinergic neurons (263). The ␮-opioid agonist Tyr-D-AlaGly-Met-Phe-Gly-ol (DAGO) was found to inhibit DRG
currents by 50% (360). Enkephalin was found to inhibit
T-type currents 20 – 40% in the neuroblastoma cell line
NG108 –15 (196). In these cells there was no effect of
either the phorbol ester 4␤-phorbol 12-myristate 13-acetate (PMA), arachidonic acid (10 ␮M), or forskolin. Bradykinin (0.1 ␮M) inhibited 57% of the T-type current from
ND7–23 cells, a cell line derived from DRG (213). This
study also showed that (⫺)-baclofen (1 ␮M) could inhibit
currents by 56%, whereas guanosine 5⬘-O-(3-thiotriphosphate) (GTP␥S) inhibited by 20%. Pertussis toxin treatment did not block baclofen inhibition. Baclofen (20 ␮M)
T-TYPE CALCIUM CHANNELS
B. Hormonal Stimulation
T-type currents can also be stimulated acutely by a
variety of hormones. One of the first studies showed that
10 ␮M serotonin could stimulate rat spinal motoneuron
T-type currents by 66% in a slice preparation (36). Also in
a slice preparation, dorsal horn neurons were found to be
stimulated by substance P (1 ␮M), although some neurons
were inhibited (347). Cholinergic agonists also stimulate
T-type currents, and this was demonstrated at the singlechannel level. Carbachol and muscarine doubled the number of openings of single T-type channels in adult guinea
pig hippocampal CA3 neurons (117). This stimulation was
blocked by 0.1 ␮M atropine. The modulation was likely
Physiol Rev • VOL
due to an increase in the probability of opening of each
channel, as there was no change in the single-channel
conductance, which was 7.1 pS in a solution containing 95
mM Ba2⫹. Under similar conditions, carbachol inhibited
single L-type currents. Carbachol (50 ␮M) and serotonin
(30 ␮M) were also shown to stimulate hippocampal Ttype currents 54 and 61%, respectively (126). These effects
were blocked by the appropriate receptor antagonists and
were quickly reversible. Serotonin appeared to shift the
I-V to the left (peak shifted from ⫺25 to ⫺30 mV), but this
effect was not quantitated (126). Erythropoietin increased
T-type currents 20% in the human neuroblastoma cell line
SK-N-MC (18). It also stimulated increases in intracellular
Ca2⫹ that was dependent on external Ca2⫹ but not internal stores. Norepinephrine acting via ␣-adrenoceptors
was reported to increase T-type currents in canine Purkinje cells by 69% (410).
Endothelin has been reported to stimulate T-type
currents from both cardiac and smooth muscle myocytes
(129, 181). In neonatal rat ventricular myocytes, endothelin-1 displayed an EC50 of 1.3 nM and a maximal stimulation of 54% (129). Heat inactivation of the endothelin, or
omission of GTP from the pipette, occluded the effects.
The stimulation induced by 10 nM endothelin was
blocked by 0.2 ␮M staurosporine or 20 ␮M H-7. Protein
kinase C (PKC) agonists stimulated the currents directly,
and this could be blocked by H-7 as well. In guinea pig
smooth muscle myocytes, endothelin stimulated the activity of a 12-pS channel, that may or may not be carried
through T-type channels (181). Endothelin has been reported to have no effect on T-type currents recorded from
smooth muscle cells from rat renal resistance arteries
(143).
Acetylcholine stimulated T-type currents in NIH 3T3
cells that had been transfected with recombinant m3 and
m5 muscarinic receptors (322). Stimulation was modest
(25%) and was accompanied by a leftward shift in the I-V
relation. Parallel assays demonstrated that cAMP levels
were increased. T-type currents were also stimulated by
treatments designed to stimulate cAMP-mediated signaling, such as 500 ␮M 8-bromo-cAMP or 10 ␮M forskolin.
Likewise, the PKA inhibitor 10 ␮M Rp-adenosine 3⬘,5⬘cyclic monophosphothionate (RpcAMPS) inhibited basal
activity and occluded the response to acetylcholine.
These results suggested that T-type channels might be
directly phosphorylated by PKA. An apparent contradiction to this hypothesis was that T-type currents were not
regulated in cells transfected with the m1 muscarinic
receptor, although these cells showed robust increases in
cAMP. The authors speculated that m1 receptors might
simultaneously activate inhibitory PKC pathways that obscured the PKA-mediated stimulation. First they showed
that the PKC activator phorbol 12,13-dibutyrate (PDBu;
0.5 ␮M) directly inhibited T-type currents as observed in
other cells types. Next they showed that preincubation
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has also been reported to inhibit the T-type currents in
hippocampal neurons by 53% (126).
Muscarinic inhibition of T-type currents from hen
granulosa cells has also been reported (425). In contrast
to most studies that report ⬃40% inhibition, this study
reported 90% inhibition. Part of this difference may be due
to their use of the perforated patch technique, which is
expected to preserve the integrity of the intracellular
milieu. The effect of carbachol was blocked by atropine.
In contrast, muscarinic agonists have been reported to
have no effect on LVA channels in cholinergic neurons (5).
ANP (3 nM) inhibited T-type currents of bovine adrenal glomerulosa cells by 28% (274). Inhibition was dependent on the holding potential, with little block when
membrane potential (VM) was ⫺90 mV, but near complete
block when VM was ⫺45 mV. This voltage dependence of
block also caused a ⫺10 mV shift of the h⬁ curve to more
negative potentials. In contrast, ANP did not affect the
voltage dependence of activation as measured using tail
currents (26). The effects of ANP were also studied at the
single-channel level. Single T-type channels were identified by their 8.3-pS conductance. Inhibition was accompanied by a decrease in the number of active sweeps, with
no change in the amplitude of single openings. The ability
of bath-applied ANP to modulate channels recorded in the
cell-attached mode indicated that ANP induced the production of a soluble second messenger.
ANG II and the AT2 selective ligand CGP-42112 inhibited by 25% the T-type current in NG108 –15 cells (58, 59).
Inhibition was associated with a ⫺5-mV shift in the I-V but
no effect on the h⬁ curve. Inclusion of 3 mM GDP␤S in the
pipette blocked the ANG II effect. In contrast, 3 mM
GTP␥S did not block the ANG II inhibition. This result is
surprising since this concentration of GTP␥S should have
maximally stimulated G protein signaling. Washout was
not reported. A role for tyrosine phosphorylation in the
signaling pathway was suggested by the ability of 50 ␮M
sodium orthovanadate to disrupt ANG II modulation of
the T-type current.
135
136
EDWARD PEREZ-REYES
up to 87% (361). Inhibition was accompanied by an acceleration of inactivation, such that the ␶ decreased from 22
to 18 ms. Neither cholera toxin pretreatment nor photorelease of caged GDP␤S had any effect. Pertussis toxin
pretreatment blocked GTP␥S inhibition, but not stimulation. GTP␥S has also been shown to inhibit T-type currents (40%) of rat nodose ganglion neurons (148). This
effect was blocked by pretreatment with pertussis toxin.
Somewhat surprisingly, G proteins do not appear to
mediate the regulation of T-type currents by serotonin
and nociceptin. Serotonin inhibition (25%) of T-type currents has been observed in Xenopus sensory neurons
(383, 384). Inhibition was not observed when the T-type
channels were recorded in the cell-attached patch configuration, indicating that its effects were membrane delimited. Inhibitory regulation was not blocked by pertussis
toxin pretreatment or inclusion in the pipette of GDP␤S,
GTP␥S, or 5⬘-guanylyl-imidodiphosphate (GMP-PNP). The
ability of these manipulations to block the regulation of
HVA currents in the same neurons provided a convenient
positive control. It was concluded that G proteins are not
involved in the serotonin inhibition of T-type channels. A
similar conclusion was reached in a study on the nociceptin inhibition of DRG T-type currents (1). Nociceptin inhibited currents with an IC50 of 0.1 ␮M, and in 22 of 77
neurons it blocked 100% of the current at 1 ␮M. Inclusion
in the pipette of GTP␥S, GDP␤S, or AlF3 did not alter
response, leading the authors to conclude that a G protein
is not involved. It should be noted that nociceptin had no
effect on T-type currents from small nociceptive trigeminal ganglion neurons (51). The endogenous cannabinoid
anandamide also blocks T-type currents independently of
G proteins and receptors (71).
The ability of pertussis toxin treatment to block regulation indicates that Gi/Go proteins are involved. Support
for this hypothesis comes from studies that used specific
antibodies in the patch pipette to block regulation (239,
251). Although the ␤␥-subunits of G proteins have been
suggested to interact directly with T-type channels (103),
more studies are required to establish this point.
C. Guanine Nucleotides
D. Protein Kinases
As noted above, T-type currents are regulated by G
protein-coupled receptors. Therefore, this regulation
should require the presence of GTP and should be modified by activators (GTP␥S) and inhibitors (GDP␤S) of G
proteins. Accordingly, no hormonal regulation was observed when GTP was omitted from the patch pipette
(129, 239) or when it was replaced with GDP␤S (59, 103).
The effects of GTP␥S are difficult to interpret because it can activate all G proteins. With the use of
photoactivation of caged GTP␥S in DRG neurons, low
concentrations were observed to stimulate T-type currents by 54%, while higher concentrations inhibited them
Physiol Rev • VOL
With the notable exception provided by studies on
fibroblasts (322), T-type currents appear not to be regulated by PKA. In contrast, T-type currents appear to be
inhibited by members of the PKC family and stimulated by
tyrosine kinases and CaMKII.
The effects of PKC have been evaluated by activating
the kinase directly with compounds such as diacylglycerol 1-oleolyl-2-acetylglycerol (OAG), or various phorbol
esters: PMA, 12-O-tetradecanoylphorbol-13-acetate (TPA),
or PDBu. Although many studies have found no effect of
these compounds (310), both stimulation and inhibition of
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with the PKC inhibitor calphostin C (0.5 ␮M) allowed
m1-mediated stimulation of the T-type current. Basal Ttype currents were not regulated by acetylcholine in cells
transfected with either m2 or m4. However, inhibition of
T-type currents, as well as inhibition of cAMP formation,
could be observed in these cells after stimulation of adenylyl cyclase by 10 ␮M forskolin. These results indicate
that hormonal regulation of T-type channels may depend
on both basal phosphorylation levels as well as cross-talk
between PKA and PKC pathways.
Barrett and colleagues (82) have extensively studied
the stimulation of adrenal glomerulosa T-type Ca2⫹ channels by ANG II. Initial studies established the presence of
T-type channels in these cells through the use of tail
current analysis (82). Slowly deactivating currents gated
with the voltage dependence expected of T-type channels,
while fast-deactivating currents behaved like HVA, L-type
channels. ANG II increased the T-type tail currents by 50%
(82). The effects of ANG II were also studied at the
single-channel level (273). ANG II increased both the
number of active sweeps and increased the number of
openings per sweep, with no appreciable change in the
amplitude of single openings. The ability of bath-applied
ANG II to modulate channels recorded in the cell-attached
mode indicated that ANG II induced the production of a
soluble second messenger. The effects of ANG II were
blocked by the ANG II receptor antagonist saralasin (1
␮M). ANG II also shifted the voltage dependence of activation by 10 mV to more negative potentials, with no
change in the h⬁ curve. Pretreatment of the cells with
pertussis toxin abolished these effects, indicating that
ANG II receptors couple through G proteins, possibly Gi
(251). The final step in this signal transduction pathway
appears to be phosphorylation of the T-type channel by
calmodulin-dependent protein kinase II (CaMKII; see below) (27, 252). However, not all studies have found an
ANG II stimulation of adrenal glomerulosa currents, with
reports of either no effect (250) or frank inhibition (103,
344).
T-TYPE CALCIUM CHANNELS
Physiol Rev • VOL
also blocked regulation. As observed with native channels, stimulation was accompanied by an 11-mV hyperpolarizing shift in the voltage dependence of activation, with
no shift in the h⬁ curve. In contrast, the activity of human
Cav3.1 was not regulated under similar conditions. These
studies strongly suggest that the modulatory phosphorylation site(s) reside directly on the ␣1-subunit of T-type
channels and that these sites are subtype specific, in this
case only found on Cav3.2. Although CaMKII regulation of
T-type currents has not been reported in other systems, it
might have mediated the 30% stimulation observed in
cardiac myocytes (411).
E. Voltage
An intriguing property of many HVA Ca2⫹ currents is
that their activity can be increased, or “facilitated,” by
strong depolarizing pulses. These prepulses can induce
channel conformations that are 1) more susceptible to
phosphorylation, 2) have a lower affinity for divalent
cations in the pore, or 3) have a lower affinity for G
protein ␤␥-subunits. The prepulse can also directly induce
high activity gating modes (mode 2) of cardiac L-type
channels. Both native (below) and recombinant (140)
T-type currents have also been reported to be facilitated
by prepulses.
The T-type currents of guinea pig coronary smooth
muscle myocytes were stimulated twofold by 200-ms prepulses to voltages above ⫺30 mV (131). Studies where the
prepulse potential was varied indicated that saturation
occurs at ⫺10 mV. Use of longer prepulses (10 s) shifted
this voltage dependency to the left, such that saturation
occurred at ⫺60 mV. Changing the duration of the prepulse showed that the peak effect occurred between 160
and 320 ms, which then decayed and was gone by 5 s. This
potentiation was greater when the interpulse voltage was
⫺100 compared with ⫺80 mV, and at 36 versus 24°C.
Potentiated currents inactivated faster (␶inact 5.6 vs.
14.5 ms).
Facilitation has also been observed in bone marrow
cells, where T-type currents were stimulated twofold by a
750-ms prepulse to ⫹150 mV (330). Compared with the
results obtained in smooth muscle, stronger depolarizations were required; stimulation was not observed with
prepulses lower than ⫺30 mV, and saturated at ⫹100 mV.
Changing the duration of prepulse (⫹10-mV pulse)
showed that half-maximal stimulation occurred at ⬃250
ms, and saturated by 1,000 ms. Changing the interval
between the prepulse and the test pulse showed that
potentiation peaked after 1 s and decayed in a biexponential manner: 9.4 and 43.5 s. Potentiation was not affected
by omission of ATP or GTP or bath application of nonspecific protein kinase inhibitors (e.g., 100 ␮M H-7). Potentiated currents decayed slightly faster (␶inact decreased
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activity have been reported. Studies on rat DRG neurons
found that 10 nM PMA inhibited the T-type current by
27%. Likewise, the inactive analog 4␣-phorbol had no
effect. Of note, the PMA effect was not observed at room
temperature, requiring preincubation at 29°C or higher
(359). Similarly, 100 nM TPA was found to inhibit the
T-type current by 26% in ventricular myocytes and Purkinje cells. A phorbol ester analog that does not activate
PKC had no effect, while the protein kinase inhibitor H-8
prevented TPA inhibition (411). In contrast, 50 ␮M H-7
was not able to block the inhibition of hippocampal Ttype currents by 10 ␮M TPA or 5 ␮M OAG (407). In
addition, the TPA and OAG effects occurred faster than
would be expected (200 ms), leading Toselli and Lux
(407) to conclude that the compounds were blocking the
channel directly. PKC may in part mediate the inhibitory
effects of arachidonic acid (459).
PKC-mediated stimulation of T-type currents has also
been reported (129). Currents in rat neonatal ventricular
myocytes were stimulated 30% by either 0.2 ␮M PDBu or
PMA. The inactive analog 4␣-PDBu had no effect. Stimulation was blocked by inclusion of 20 ␮M H-7 in the
pipette.
The ability of CaMKII to stimulate adrenal T-type
currents has been studied at both the whole cell and
single-channel level, and recently with recombinant channels. Inclusion of Ca-CaM in the pipette shifted the voltage dependence of activation to more negative potentials
(252). The CaMKII inhibitor KN-62 or the synthetic peptide inhibitor that corresponds to residues 290 –309
blocked this effect. Stimulation was also demonstrated at
the single-channel level in both cell-attached and excised
patches (27). In cell-attached patches, manipulation of
intracellular Ca2⫹ led to an increase in channel activity
that could be blocked by KN-62, but not the inactive
analog KN-04. Channel activity could also be stimulated
when excised inside-out patches were exposed to calmodulin and appropriate concentrations of Ca2⫹. This
stimulation could be blocked with the autocamtide 2-related inhibitory peptide (AIP). Stimulation could also be
mimicked by the direct addition of a constitutively active
mutant of CaMKII. Heat-inactivated kinase had no effect.
As observed previously with ANG II (273), this regulation
appeared to be due to an increase in active channels.
These studies clearly establish that native bovine glomerulosa T-type channels are regulated by CaMKII. Adrenal cortical T-type channels are encoded by ␣1H, or Cav3.2
(358). Recent studies have shown that calcium/calmodulin can stimulate the activity of cloned human Cav3.2
channels when coexpressed with CaMKII␥c in HEK-293
cells (443). Stimulation could be blocked by CaMKII inhibitors added to either the bath (3 ␮M KN-62) or to the
intracellular pipette used for whole cell recording (AIP, 2
␮M). Replacement of pipette ATP with the nonhydrolyzable analog 5⬘-adenylyl-imidodiphosphate (AMP-PNP)
137
138
EDWARD PEREZ-REYES
IV. MOLECULAR CLONING
OF T-TYPE CHANNELS
A. Cloning
Cloning of high voltage-gated Ca2⫹ channels began
with purification and microsequencing of the skeletal
muscle dihydropyridine receptor (396). Low-stringency
screening of cDNA libraries with probes derived from the
␣1-subunit of skeletal muscle (␣1S, or Cav1.1) led to the
discovery of cardiac (␣1C, Cav1.2) and brain (␣1D, Cav1.3)
isoforms of the L-type family (Cav1; reviewed in Ref. 326).
The more distantly related members of the Cav2 family
were cloned during the “brain era” (Fig. 4A) using even
lower stringency hybridization (374). PCR provided an
alternative approach, and primers based on highly conserved regions (IIIS5 and IVS5) were used successfully to
isolate a fragment of Cav2.3 (357). Despite the efforts of
Physiol Rev • VOL
many groups, these approaches were not successful in
isolating cDNAs encoding T-type channels.
Progress in the sequencing of human, yeast, and C.
elegans genomes provided a novel library that could be
screened with a computer, or in silico (Fig. 4B). Screening
with the highly conserved P loop, or K⫹ channel signature
sequence, allowed Ketchum et al. (205) to identify a novel
K⫹ channel sequence from Saccharomyces cerevisiae. We
decided to try a similar approach using S5 segments. This
led to the identification of two Ca2⫹ channel-like proteins
that were predicted from the C. elegans genome (325).
These proteins were homologous to the skeletal muscle
Ca2⫹ channel, and this similarity was noted in the GenBank entry. We reasoned that there might be other sequences in the GenBank that were similarly labeled and
used a text-based search (Fig. 4B) to find sequences that
were “similar to a calcium channel.” This led to the identification of the human EST clone H06096 (324). Although
this clone is 1.4 kb long, only a part of the 5⬘-sequence was
in the database. This fragment had a very low sequence
identity to the S1 segment of other Ca2⫹ channels, precluding its direct identification by homology search. The
full sequence of H06096 showed that it might encode a
protein with many of the hallmarks of voltage-gated channels, notably the S4 voltage sensor sequence was well
conserved, and the pore loop sequence was highly homologous to other Ca2⫹ channels. Screening of the GenBank
with the H06096 sequence and the program BLAST identified C54d2.5 as a C. elegans homolog. Similar to in vitro
cloning where one “walks” through a cloning project using probes to the ends, we screened the GenBank with the
3⬘-end of C54d2. This led to the identification of the EST
clone H19230. In vitro screening of a rat brain cDNA
library with H06096 allowed the cloning of Cav3.1 (324),
whereas use of this clone to screen a human heart library
led to the cloning of Cav3.2 (90). Screening of a rat brain
library with H19230 under low-stringency conditions allowed us to identify not only rat versions of Cav3.1 and
Cav3.2, but also Cav3.3 (228). A similar approach was used
by Monteil et al. (288) to identify C54d2.5 and the H06096
and H19230 ESTs, which then allowed them to clone the
full-length human Cav3.1. Similar PCR approaches were
also used to clone a human Cav3.2 cDNA (438) and mouse
and rat versions of Cav3.1 (211, 463). PCR approaches
based on the C. elegans sequence C27f2.5 led to the
cloning of a distantly related channel gene whose function remains unknown (227).
Sequencing of the human genome also played an
important role in the cloning of T-type channels. The
Cav3.3 gene is located on chromosome 22, which was the
first chromosome to be completely sequenced (105).
BLAST search with rat Cav3.1 identified the coding regions of Cav3.3, which then allowed the design of PCR
primers to either clone its cDNA (228, 289) or to examine
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from 17 to 13 ms; currents isolated by subtraction). These
results suggest that voltage directly affects channel activity without the involvement of second messenger signals.
The opposite conclusion was reached in studies of bullfrog atrial cells (8). Amphibian channels are stimulated by
␤-adrenergic agonists, and a prepulse enhances this stimulation. These effects were enhanced by GTP␥S and
ATP␥S, but blocked by either GDP␤S or pertussis toxin
treatment. From these studies it was concluded that G
proteins are involved and that phosphorylation of G proteins may play a role as well.
Facilitation of T-type currents from mouse spermatogenic cells has also been reported (14). In this case,
prepulses could only stimulate activity 50% above control.
Prepulses to voltages greater than ⫺30 mV were required
to observe stimulation, and saturated around ⫹60 mV.
Short prepulses (⬎10 ms) were capable of stimulating
currents, and this effect wore off very slowly. Membranepermeable inhibitors of tyrosine kinases (tyrphostins A47
and A25) stimulated basal T-type currents but had no
effect on facilitated currents. This suggests that tyrosine
kinases may tonically inhibit activity in this system and
that a prepulse somehow reverses their activity. Consistent with this hypothesis, inhibitors of tyrosine kinase
phosphatases inhibited basal T-type currents and prevented voltage-induced potentiation.
In summary, T-type channels can be both stimulated
and inhibited by hormones and neurotransmitters that are
coupled to the Gq or Gi/Go families. Regulation appears to
be mediated by phosphorylation, with inhibition mediated
by PKC and stimulation by CaMKII. Additional studies are
required to establish the location of the modulatory phosphorylation site(s), whether G protein subunits are capable of interacting directly with the channel, and the mechanisms by which voltage regulates channel activity.
T-TYPE CALCIUM CHANNELS
139
the splicing patterns of its gene (285). Proper identification of the 5⬘- and 3⬘-ends requires traditional cDNA cloning or specially designed PCR protocols. Recent studies
indicate that the human Cav3.3 mRNA contains an additional exon that encodes 214 additional amino acids (140)
than previously recognized (289). Studies comparing the
full-length to truncated Cav3.3 channels revealed differences in channel expression and their ability to be modulated by a strong prepulse (140).
Genetic mutations in various Ca2⫹ channel subunit
genes have been associated with ataxic and epileptic
phenotypes (187). As a first step in exploring such a link,
we mapped the human and mouse genes for Cav3.1,
CACNA1G (324), and Cav3.2, CACNA1H (90). The genes
were mapped using the Cambridge 4 radiation hybrid
panel and fluorescent in situ hybridization. The locations
of the human genes are shown in Table 8. Mutations in
human Cav3 genes have yet to be described.
All three T-type channel genes are alternatively
spliced, producing variants that differ in their intracellular
loops (68, 284, 285, 463). Splicing produces Cav3.1 variants that differ in at least two locations: 1) the II-III loop,
where two variants are produced by insertion/deletion of
Physiol Rev • VOL
exon 16; and 2) the III-IV linker, where three (or actually
four, Ref. 229) variants are produced by a combination of
insertion/deletion of exon 26 and use of an alternate
5⬘-splice site in exon 25 (288). These variants differ in
their electrophysiological properties (68) and may account for the differences noted in the gating of T-type
channels between lateral geniculate (LGN) relay neurons
and interneurons (318). Splice variants of Cav3.3 have
been detected by PCR that differ in the I-II loop and
carboxy terminus (285). Similarly, PCR has identified
splice variation in the III-IV linker of Cav3.2 in both rat
(231) and human (182).
Cav3 cDNAs have been cloned from rat, mouse, and
human sources (Table 8). Although all three have been
cloned from rat and human, cloning from mouse is still
ongoing. Comparisons of their nucleotide sequences reveal a high level of conservation between species. Although species differences in the predicted amino acid
sequence have been noted, some of these changes can be
ascribed to either frameshifts in the reading, cloning artifacts, or differences in splicing. For example, it is likely
that a cloning artifact caused the deletion of three amino
acids from IIIS4 in a rat cDNA of Cav3.3 (279).
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2⫹
FIG. 4. A timeline of Ca
channel cloning. A: graph
illustrates the time of the first published reference to the
cloning of each of the 25 subunits. Skeletal muscle Ca2⫹
channel subunits were purified, microsequenced, then
cloned using degenerate oligonucleotide probes based on
the protein sequence. This initial period has been called
the Muscle Era. Low-stringency screening of brain cDNA
libraries with the skeletal muscle probes led to the cloning
of other ␣1- and ␤-subunits during the Brain Era. In the
final epoch, or In Silico Era, subunits were first identified
by database searching programs (BLAST) of Human and
Caenorhabditis elegans genome sequencing projects, then
cloned by standard in vitro methods. (Adapted from Randall A and Benham C. Recent advances in the molecular
understanding of voltage-gated Ca2⫹ channels. Mol Cell
Neurosci 14: 255–272, 1999.) B: flow diagrams describing
two ways of cloning in silico.
140
TABLE
EDWARD PEREZ-REYES
8. Comparison of the three cloned T-type channels
Cav3.1
Alias
Gene name
Chromosomal location
Database entries*
Human
Rat
Mouse
Tissue expression
Cav3.3
␣1G, CavT.1
CACNA1G
17q22
␣1H, CavT.2
CACNA1H
16p13.3
␣1l
CACNA1l
22q12.3–13.2
NP_061496, O43497
AAC67372, O54898
NP_033913, Q9WUB8
Brain, ovary, placenta, heart
AAC67239, O95180
AAG35187, Q9EQ60
NP_67390
Kidney, liver, adrenal cortex,
brain, heart
XP_001125, Q9UNE6
NP_064469, Q970Y8
⫺70
⫺30
1
11
3.0
⫺72
117
7.5
⫺70
⫺30
2
16
2.2
⫺72
395
9
250
1.2
0.27
12
1.1
0.14
Brain
Reference
Nos.
249
90, 228, 324
90, 228, 288,
289, 324
⫺70
⫺30
7
69
1.1
⫺72
352
11
216
1.5
209
209
209
209
209
209
209
228, 324, 438
230
267
267
* Accession numbers are for a representative GenBank entry followed by the SWISS-PROT entry.
† Results for conductance were obtained
in 110 mM Ba2⫹, while all other electrophysiological measurements were made in 1.25 mM Ca2⫹. Activation and inactivation kinetics were measured
with exponential fits to the currents elicited by test potentials to ⫺10 mV. Deactivation kinetics were measured at ⫺90 mV. Pharmacology was
measured in 10 mM BaCl2, or as noted, in 2 mM CaCl2 solutions.
B. Structure
Conservation of amino acid sequence (Figs. 1 and 5)
and predicted secondary structure (Fig. 6) indicates that
T-type channels are evolutionarily related to the ␣-subunits of K⫹, Na⫹, and HVA Ca2⫹ channels (184). Although
little is known about the structure of Na⫹ and Ca2⫹
channels, recent advances with K⫹ channels provide a
framework to interpret their structures. The most significant advance was the determination of the KcsA K⫹
channel structure by X-ray crystallography (102). This
channel contains two transmembrane segments separated by a pore loop (Fig. 6). The second transmembrane
segment forms the barrel walls of the pore, while the
selectivity filter is formed by the pore loops that dip
partially into the barrel. In addition to this fundamental
motif, voltage-gated channels also contain four additional
transmembrane segments, with one (S4) acting as a voltage sensor. In K⫹ channels, four proteins assemble to
form one channel, while in Na⫹ and Ca2⫹ channels a
single protein contains four of these modules, or repeats.
Each transmembrane segment can be identified by a number to indicate the repeat it is in (traditionally a Roman
numeral) followed by its position within a repeat (Fig. 6).
For example, the third transmembrane segment of the
third repeat can be abbreviated IIIS3. Among voltagegated ion channels, the most highly conserved sequence
belongs to the S4 segments, where every third residue is
positively charged. This region is thought to move outPhysiol Rev • VOL
ward when the plasma membrane is depolarized. Little is
known about how this movement is transduced into channel opening. The pore loops are also well conserved,
especially within the members of a particular ion channel
class. In voltage-gated Ca2⫹ channels, the pore loops all
contain the following signature sequence: T-x-E/D-x-W. In
HVA channels, all four repeats have a glutamate residue in
the pore, leading to the abbreviation EEEE. In repeats III
and IV of Cav3 channels, the glutamate has been replaced
by an aspartic acid residue, or EEDD. The importance of
these residues in Ca2⫹ permeation has been extensively
established in HVA channels (449), and recently with
Cav3.1 (391).
Site-directed mutagenesis studies on the S6 region of
Cav3.1 suggest that these segments play a similar structural role in these channels as in KcsA (266). This provides a theoretical basis for the use of the KcsA crystal
structure to guide studies of drug binding pockets, which
in the case of Cav1.2 appear to be located within the
channel walls on the cytoplasmic side of the pore loops
(174).
As shown in Figure 6, most of the channel is thought
to be intracellular. The extracellular loops are predicted
to be quite short, with the notable exception of the loop
connecting IS5 to the pore loop (98 –103 amino acids).
This extracellular loop contains six cysteine residues that
are conserved in all three Cav3 channels and a number of
possible sites for glycosylation. Neuraminidase treatment
of cardiac myocytes stimulates T-type (but not L-type)
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Electrophysiology†
Threshold, mV
Peak of I–V, mV
Activation kinetics, ms
Inactivation kinetics, ms
Deactivation kinetics, ms
h⬁, mV
Recovery from inactivation, ms
Conductance, pS; Ba
Pharmacology
Nickel (IC50, ␮M)
Mibefradil (IC50, ␮M) in 10 Ba2⫹
Mibefradil (IC50, ␮M) in 2 Ca2⫹
Cav3.2
T-TYPE CALCIUM CHANNELS
C. Distribution
The expression patterns of these genes in peripheral
tissues and brain have been studied using Northern blots,
RNA dot blots, and in situ hybridization (Table 8). Cav3.1
mRNA is primarily expressed in human brain, but also in
ovary, placenta, and heart (288, 324). A wider distribution
was noted in dot blots prepared from human fetal tissues,
with strong expression noted in kidney and lung (288).
Cav3.2 mRNA is primarily expressed in kidney and liver,
but also in heart, brain, pancreas, placenta, lung, skeletal
muscle, and adrenal cortex (90, 438). Cav3.3 mRNA is
almost exclusively expressed in brain (228); the only peripheral tissues that showed expression on dot blots were
adrenal (but see Ref. 358) and thyroid (289).
The distribution of all three Cav3 channel transcripts
in rat brain has been studied in exquisite detail using in
situ hybridization (393), and similar patterns of expression have been obtained using Northern blots of human
brain mRNA (288, 289, 438). Their patterns of expression
are complementary in many regions, with most brain
regions expressing more than one isoform. In fact, some
neurons may express all three genes as suggested by the
labeling of olfactory granule cells and hippocampal pyramidal neurons. Many brain regions showed heavy expression of Cav3.1 mRNA, including thalamic relay nuclei,
olfactory bulb, amygdala, cerebral cortex, hippocampus,
hypothalamus, cerebellum, and brain stem. Two separate
studies have confirmed this pattern of mRNA expression
and extended the findings by showing a similar distribution of Cav3.1 protein (88, 197). Cav3.2 mRNA expression
was detected in olfactory bulb, striatum, cerebral cortex,
hippocampus, and reticular thalamic nucleus. Cav3.3
mRNA expression is high in olfactory bulb, striatum, cePhysiol Rev • VOL
rebral cortex, hippocampus, reticular nucleus, lateral habenula, and cerebellum. DRG neurons express both
Cav3.2 and Cav3.3 mRNA (393). One study found that this
expression was restricted to small and medium-sized neurons (393), while a second study reported that Cav3.3
transcripts were equally abundant in large DRG neurons
(454).
D. Electrophysiology of Recombinant Channels
Expression of recombinant Cav3 channels leads to
the appearance of robust currents in a variety of heterologous expression systems. Currents mediated by the
cloned ␣1-subunit are nearly identical to those recorded
from native channels: they both open and inactivate at
potentials near the typical resting membrane potential,
recover rapidly from inactivation, and close slowly producing prominent tail currents (Fig. 7).
The currents mediated by rat and human isoforms of
the three Cav3 channels have been compared under a
variety of experimental conditions (209, 218, 228, 279). In
general these results agree quite well with each other,
with the differences most likely due to recording conditions or sequence variability. For example, Cav3.1 and
Cav3.2 currents inactivate (⬃1.7-fold) faster with Ba2⫹
than Ca2⫹ (209, 211). Similarly, recording conditions such
as choice of charge carrier or length of depolarizing
pulses can affect estimates of both deactivation kinetics
(211, 216, 430) and recovery from inactivation (430).
Therefore, comparisons under one set of conditions are
the most relevant; Table 8 presents the results obtained in
1.25 mM Ca2⫹ (209), whereas Figure 7 results were obtained in 5 mM Ca2⫹.
All three Cav3 clones form LVA channels. Depolarization of the membrane to ⫺70 mV is sufficient to trigger
channel opening, and the I-V curves for all three channels
peak around ⫺30 mV (Fig. 7B). Some studies have found
that Cav3.3 currents activate at slightly more positive
potentials than either Cav3.1 or Cav3.2 (127, 289). Part of
this difference can be ascribed to the methods used to
estimate the voltage dependence of activation. Of note,
not all the recombinant channels within a given preparation gate at the same low potentials, with ⬃40% of the
channels requiring much stronger depolarizations (⫹10 to
⫹50 mV) for opening (288, 438). Activation curves constructed using tail currents that detect both channels
show a lower slope and higher midpoint of activation than
other methods.
The kinetics of the currents are very voltage dependent between ⫺70 and ⫺20 mV, producing a typical crisscrossing pattern of traces (Fig. 7A; Ref. 334). Therefore, it
is useful to compare the kinetics of the three channels at
⫺10 mV or above (Table 8). Cav3.1 and Cav3.2 both activate relatively quickly at this potential (␶ ⫽ 1–2 ms) and
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currents threefold, suggesting that glycosylation of the
channel inhibits its activity (116, 451). Despite the fact
that the sequences of intracellular loops differ considerably across Na⫹ and Ca2⫹ channels, they have a similar
overall size: long loops connect repeats 1–2 and 2–3
(range 207–375 amino acids for Cav3 channels), while
repeats 3– 4 are connected by short loops (55– 62 amino
acids). Similarly, the amino termini are usually short
(⬍100 amino acids), whereas the carboxy termini are
quite long (433– 493 amino acids). The sequence of these
loops is poorly conserved, with the exception of sequences close to the end of the S6 segments (Fig. 5). In
HVA channels these loops are important for binding auxiliary subunits such as the ␤-subunit, binding to regulatory
proteins such as calmodulin or the G protein ␤␥-complex,
and are sites for protein phosphorylation (424). Although
the roles of these loops in T-type channels are presently
unknown, it is likely they contain important regulatory
sites as well.
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FIG. 5. Alignment of the predicted amino acid sequence of human Cav3.1, 3.2, and 3.3 channels. Residues conserved
in all three sequences are shown in the consensus sequence (CON). The approximate location of the membrane-spanning
regions is indicated above the sequence. ␤-Subunits bind to the I-II loop of HVA channels through a sequence termed the
␣-interaction domain (AID). The consensus sequence of the AID is shown above the homologous region in the Cav3
channels. The residues are colored according to the structural properties of the individual amino acids: red, positively
charged; green, negatively charged; blue, polar; yellow, nonpolar.
Physiol Rev • VOL
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T-TYPE CALCIUM CHANNELS
143
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FIG.
5—Continued
inactivate 10-fold slower (␶ ⫽ 11–16 ms). In stark contrast, Cav3.3 currents activate and inactivate very much
slower. This difference is even greater when comparisons
Physiol Rev • VOL
are made using Xenopus oocytes (228), and it remains a
mystery why Cav3.3 channels behave so differently in
amphibian eggs than in mammalian cells.
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144
EDWARD PEREZ-REYES
Recombinant Cav3 channels, like their native counterparts, close slowly producing prominent tail currents.
The voltage protocol used to measure tail currents includes a short pulse to open channels, followed by repolarization to a voltage where they close (Fig. 7C). Although all three Cav3 channels show slow tail currents
upon repolarization to ⫺90 mV, Cav3.1 are the slowest
(␶ ⫽ 3 ms), while Cav3.3 are the fastest (␶ ⫽ 1 ms). In
contrast, HVA channels close 10-fold faster.
Despite large differences in the rate at which they
inactivate, the voltage dependence of steady-state inactivation of the three recombinant Cav3 channels is remarkPhysiol Rev • VOL
ably similar. The midpoint of the h⬁ curves are ⫺72 mV
(Table 8). Native T-type currents inactivate over a similar
range. These studies indicate that T-type channels, as
observed with other channels, can reach inactivated
states without passing through open states (364). In fact,
during a depolarizing pulse up to 30% of Cav3.3, channels
inactivate without opening (127).
Cav3 channels recover rapidly from short-term inactivation. Cav3.1 channels recover fastest, displaying a
monoexponential recovery with a time constant of ⬃100
ms (Fig. 7, Table 8). In contrast, Cav3.3 channels recover
threefold more slowly, while Cav3.2 channels recover
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FIG. 6. Structural models. A: hydropathy plots of the KcsA and Shaker K⫹
channels and repeat III of Cav3.1. The
hydropathy index varies from 3 to ⫺1.
Letters above the plots indicate the relative position of the putative transmembrane segments (S1–S6) and pore loops
(P). B: transmembrane segments are also
typically displayed in two dimensions as
snake diagrams, wherein the protein is
shown to snake its way in and out of the
membrane. C: a model of the three-dimensional structure of four-repeat ion
channels. The repeats are assumed to be
oriented in a clockwise manner based on
work with Na⫹ channels. The S5-P-S6
segments are thought to form a similar
structure as the S1-P-S2 segments of
KcsA, with the S6 segment forming the
walls of the channel. The S4 segment is
highly charged, so it is likely to be surrounded by the other segments with water filling the cracks. The S1–S3 segments
are shown to form the exterior of the
channel. The transmembrane segments
are most likely composed of ␣-helices,
but we have no clue to their orientation.
The intra- and extracellular loops are not
shown for clarity. D: a snake diagram
where each amino acid is represented by
a circle. Residues that are conserved in
all three Cav3 channels are shown with a
solid circle. (Adapted from Perez-Reyes
E. Three for T: molecular analysis of the
low voltage-activated calcium channel
family. Cell Mol Life Sci 56: 660 – 669,
1999.)
T-TYPE CALCIUM CHANNELS
145
even more slowly (209). The channels also differ in their
recovery from steady-state inactivation, and again, Cav3.2
was found to recover the slowest (209). These results
suggest the existence of multiple inactivated states, as
noted previously for T-type currents from sensory neurons (53). These results also suggest that recovery kinetics can be used to differentiate currents carried by native
Cav3.1 from Cav3.2 (351). Fast recovery from inactivation
is a critical property of native T-type channels that allows
them to participate in rebound burst depolarizations.
The ability of T-type channels to open at similar
potentials at which they inactivate suggests that they
might generate current under steady-state conditions.
This is commonly referred to as a window current and is
Physiol Rev • VOL
operationally defined as the overlap region between the
activation and steady-state inactivation curves (Fig. 7, G
and H). All three Cav3 channels are predicted to generate
window currents supported by ⬍1% of the channels. Recording conditions and methods of analysis have a dramatic effect on estimates of the window current, so these
estimates must be interpreted with caution. For example,
analysis of Cav3.3 results obtained in Xenopus oocytes
indicates that up to 2% of the channels can be open in the
steady-state. Evidence for window currents have also
been obtained in thalamacortical neurons, where they
play an important role in signal amplification (441). Window currents also appear to be important in determining
intracellular Ca2⫹ concentrations (18, 69, 264). Expres-
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FIG. 7. Electrophysiological properties of recombinant Cav3 channels. Recombinant channels were expressed in
human embryonic kidney cells (293) and recorded with the ruptured patch method using 5 mM Ca2⫹ as the charge
carrier. A: the current-voltage (I-V) relationship is typically measured using a series of step depolarizations where the test
potential (⫺80 to ⫺10 mV) is increased 10 mV between runs. After each run the cell is held at ⫺100 mV for at least 10 s
to allow recovery. Recordings were made using a stably transfected cell line expressing Cav3.2. B: I-V curve is a plot of
the current as a function of test potential. In this example the currents for each cell were normalized to the peak current
observed, and then averaged. Typical I-V relations are shown for human Cav3.1 (‚), 3.2 (ƒ), and 3.3 (E; same symbols for
all panels). The data were fit with an equation: G ⫽ Gmax ⫻ (Vm ⫺ Vrev)/[1 ⫹ exp(V1/2 ⫺ Vm)/k], where G is conductance,
Vm is the test potential, Vrev is the apparent reversal potential, V1/2 is the midpoint of activation, and k is the slope factor.
C: tail currents are measured with a two-step protocol where the first step is designed to open channels (e.g., 2 ms to
⫹60 mV), and the second step measures how fast they close at different repolarization potentials. Traces were obtained
with Cav3.1 and Cav3.3 after repolarization to ⫺90 mV. Deactivation kinetics are measured by fitting tail currents with
exponential functions. D: deactivation kinetics vary with the repolarization potential. E: recovery from inactivation can
be measured using a three-step protocol where step 1 is a long depolarizing pulse used to inactivate channels, step 2 is
a repolarizing step to voltages where channels recover, and step 3 is a test pulse to measure the fraction of channels that
recovered. The duration of step 2 is increased in subsequent runs. Recovery is defined as the ratio of the peak current
in step 3 divided by the current in step 1. F: recovery time courses of the three human Cav3 channels. Data were fit with
an exponential to calculate the rate of recovery. Cav3.1 channels recover fastest with a ␶ of ⬍100 ms, Cav3.3 are
intermediate (250 –300 ms), while Cav3.2 recover slowest (⬎400 ms). G: steady-state inactivation curves of the three
human Cav3 channels estimated with 15-s prepulses. Smooth curves represent fits to the data with a Boltzmann equation.
The foot of the activation curve is also shown. There is a small voltage range where these two curves overlap, leading
to the prediction that channels can produce steady-state (window) currents. H: fraction of channels open in the
steady-state can be estimated by multiplying the Boltzmann functions that describe activation and inactivation.
146
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Physiol Rev • VOL
Studies using mock action potentials suggest that
most of the Ca2⫹ influx through T-type channels occurs
after the peak voltage and corresponds to the tail current
(218, 276, 334). This Ca2⫹ influx might play a role in spike
repolarization and afterhyperpolarization mediated by
apamin-sensitive Ca2⫹-dependent K⫹ channels (440). Due
to differences in their rates of activation and inactivation,
the three T-type channels respond quite differently to
trains of mock action potentials (218). Both Cav3.1 and
Cav3.2 channels inactivate rapidly during trains delivered
at ⬎20 Hz. In contrast, Cav3.3 continues to gate during
100-Hz train frequencies. These differences suggest that
the three channels play distinct roles in determining neuronal firing patterns and signal integration. Cav3.1 and
Cav3.2 channels are likely involved in short burst firing as
seen in thalamacortical neurons, whereas Cav3.3 channels
are likely involved in sustained burst firing as seen in
thalamic reticular neurons (70).
E. Auxiliary Subunits?
Auxiliary subunits of HVA Ca2⫹ channels were first
identified in purified preparations of skeletal muscle
channels (see Ref. 326 for review). This channel complex
was found to contain at least four subunits: ␣1 (Cav1.1),
␣2␦ (␣2␦1), ␤ (␤1), and ␥ (␥1). Purified cardiac L-type and
brain N-type channels have a similar ␣1␣2␦␤ structure, but
with distinct ␣1- and ␤-subtypes. Additional members of
each subunit class have been cloned (Fig. 4), such that
there are now seven HVA ␣1-, four ␤-, three ␣2␦-, and eight
␥-like subunits (reviewed in Ref. 171). The ␤-subunits play
a critical role in channel function, controlling both channel expression at the plasma membrane, voltage dependence, and pharmacology. The ␣2␦- and ␥-subunits also
play a role in expression of functional channels. Mutations in Ca2⫹ channel genes, or calcium channelopathies,
have been linked to a variety of disease phenotypes in
both mice and humans (reviewed in Ref. 248).
Native T-type channels have yet to be purified, so
their subunit structure is unknown. Two strategies have
been used to infer its structure: to assay whether antisense oligonucleotides directed against HVA ␤-subunits
can alter native T-type channel function, and to test
whether the activity of the cloned T-type ␣1-subunit is
affected by coexpression with cloned HVA subunits. Antisense depletion of ␤-subunits causes profound reductions in HVA channel density; however, this treatment has
no effect on T-type channels recorded from the same
neurons (226, 234). Similarly, coexpression of cloned
␤-subunits has little or no effect on cloned T-type channel
activity (100). In contrast, coexpression with either ␣2␦1
or ␣2␦2 was found to double the size of Cav3.1-mediated
currents (100, 132). Concomitant immunolocalization
studies supported the contention that this rise in currents
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sion of recombinant Cav3.1 or Cav3.2 was found to increase basal Ca2⫹ in HEK-293 cells, and this increase was
blocked by appropriate concentrations of either mibefradil or nickel. Similarly, basal Ca2⫹ is increased after
differentiation of the human prostatic cancer cell line
LNCaP, and this correlates with the expression of Cav3.2
mRNA and currents (264). Hormonal regulation of the
T-type window current provides cells with another mechanism to regulate intracellular Ca2⫹. In adrenal glomerulosa cells, ANG II increases T-type window currents by
selectively shifting activation to more negative voltages
(273, 443), an effect mediated by CaMKII phosphorylation
(discussed in sect. IIID).
Another distinguishing property of T-type channels is
that they have a lower Ba2⫹ conductance than HVA channels. Therefore, studies of cloned T-type channels have
focused on the amplitude of single-channel currents in
isotonic barium solutions. All three recombinant channels
were found to have small single-channel currents, corresponding to slope conductances in the 7–11 pS range
(Table 8). In contrast, Cav1.2 channels display slope conductances of 20 –30 pS, while Cav2 channels are in the
13–16 pS range. Measurement of the single-channel amplitude is complicated by the presence of subconductance
activity (228). Failure to account for these subconductance openings would result in lower estimates, and this
may explain why Cav3.2 was reported to have a conductance of 5.3 pS (90). Subconductance activity has also
been observed with native T-type channels (64).
The effect of changing external pH has been studied
with Cav3.1 (391) and Cav3.2 (95). In contrast to native
channels, whose activity is affected by small deviations
from pH 7.2, the recombinant channels are largely unaffected by pH changes in the 8.2 to 6.9 range. Further
acidification of the external solution to pH 5.5 decreases
the currents measured during standard test pulses. Mutation of the aspartate in the dmain III pore loop to glutamate (EEDD to EEED) shifted the apparent pKa of Cav3.1
from 6.0 to 7.0 (391). In addition,this mutation increased
the sensitivity to Cd2⫹ block. Therefore, each domain
contributes a negatively charged residue to form a highaffinity binding site for Ca2⫹ (and Cd2⫹) in Cav3 channels
(EEDD) as shown previously for Cav1 and Cav2 (both
EEEE) channels (75, 207, 319, 449). In addition to affecting permeation, shifting the pH from 8.2 to 5.5 produced a
dramatic 40-mV shift in the voltage dependence of activation of Cav3.2, lowered its slope factor, and produced an
11-mV shift in the h⬁ curve (95). These results suggested
that protonation of the channel affected the voltage dependence of transitions between deep closed states (R to
C3 transitions in Fig. 3). Although it is unknown where on
the channel the affected residues lie, they may very well
be in the pore. Consistent with this hypothesis, mutations
in the Cav3.1 pore also lowered its voltage dependence
and shifted activation to more positive potentials (391).
T-TYPE CALCIUM CHANNELS
V. PHARMACOLOGY
Cardiovascular L-type calcium channels are an established drug target in the control of blood pressure and
cardiac rhythm. Selective Cav1 blockers have been developed from over three drug classes, including dihydropyridines, phenylalkylamines, and benzothiazepines. In contrast, there are no highly selective drugs or peptide toxins
(369) that selectively block Cav3 channels. A comprehensive review of drugs tested on native T-type channels was
recently published (158); therefore, this review focuses
on drugs whose mechanism may involve block of T-type
channels.
Physiol Rev • VOL
A. Antihypertensives
Marketing of mibefradil (Posicor, Roche) as the first
selective T-type channel blocker stimulated considerable
interest in the pharmacology and physiology of these
channels (412). Mibefradil was approved for use in essential hypertension and stable angina pectoris until it was
withdrawn from the market due to drug-drug interactions
leading to irregular heart rhythms (219). Submicromolar
concentrations of mibefradil, formerly Ro-40 –5967, preferentially block T-type channels, while higher concentrations are required to block cardiovascular L-type channels
(282). Its 10- to 30-fold selectivity for T- over L-type channels has been confirmed in a number of preparations,
including recombinant channels expressed in mammalian
cells (267, 323). The average IC50 from 11 studies for
mibefradil block of native T-type channels was 1.5 ␮M
(see Table 1 in Ref. 267). Mibefradil potency is affected by
the concentration, and choice, of divalent cation, with
less block in Ba2⫹ than in Ca2⫹ solutions (267). In 2 mM
Ca2⫹ solutions, mibefradil blocked recombinant Cav3.2
channels with an IC50 of 0.14 ␮M, whereas slightly higher
concentrations were required for block of Cav3.1 and
Cav3.3 (267). Mibefradil block of both HVA and LVA channels is state dependent (41, 278). Single-channel measurements indicate the presence of open channel block, and in
addition, a longer lasting block that results in a reduction
in active sweeps (280). Whole cell measurements indicate
that inhibition is enhanced by holding the membrane at
potentials where T-type channels inactivate, suggesting
the drug has higher affinity for inactivated states. The
apparent affinity for these states was estimated to be 83
nM using native T-type currents (278), and similar results
were obtained with recombinant channels (267). This provides another mechanism for selectivity, since T-type
channels inactivate near the resting membrane potential
of most cells, while HVA channels require depolarization.
A similar mechanism has been invoked to explain the
selectivity of dihydropyridines for L-type channels in depolarized vascular beds over L-type channels in heart.
These results suggest that mibefradil selectivity in vivo
was probably greater than estimated in vitro since most
studies used Ba2⫹ solutions, and a significant fraction of
T-type channels is inactivated in vivo. Therapeutic plasma
concentrations of mibefradil were found to be in the 0.5–1
␮M range, with 99.5% bound to plasma proteins such as
␣1-acid glycoprotein (433). Therefore, a significant fraction of T-type channels in vascular smooth muscle myocytes would have been blocked during treatment. Subsequent studies have established that micromolar
concentrations of mibefradil are capable of blocking a
wide range of ion channels, including those for Na⫹ (106),
K⫹ (138), and Cl⫺ (304). It remains to be determined
whether block of these other channels contributed to its
antihypertensive activity. This ability to block other ion
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was due to increased expression of ␣1-␣2␦1 complexes at
the plasma membrane (100). Similar results were obtained by coexpression of a ␣2␦2 with mouse Cav3.1 (169),
although the effects of ␣2␦1 failed to reach statistical
significance (224). Coexpression with ␣2␦1 causes no detectable changes in the biophysical properties of Cav3.1
channels, while ␣2␦2 has been reported to have minor
effects on inactivation (169). Mutations in the murine ␣2␦2
gene have been linked to an absence of epilepsy phenotype in the mouse strain ducky (23). Recordings from
Purkinje neurons isolated from ducky cerebella showed
half the P-type current density observed in controls.
These results, in combination with coexpression studies,
suggest that ␣2␦2 plays a greater role in HVA than LVA
channel expression.
Identification of the mutation in the mouse strain
stargazer, which also exhibits an absence epilepsy phenotype, led to the cloning of ␥2 (233). In silico cloning has
extended the ␥-like family to eight members (78). The
␥2-protein is only 25% identical to skeletal muscle ␥ and
may play different physiological roles. Immunoprecipitation studies have shown that ␥2 may be a subunit of both
HVA Cav2.2 Ca2⫹ channels and glutamate receptors of the
AMPA class (74, 190, 366). Coexpression of ␥2 inhibits the
expression of Cav2.1 and Cav2.2 channels in the presence
of ␣2␦1 and has minor effects on their biophysical properties (190, 346). In contrast, ␥2 had little effect on Cav3.3
expression, although it could slow the decay of the tail
current (145). The related isoforms, ␥3 and ␥4, have been
reported to have no effect on Cav3.3 currents (145). In
contrast, ␥2, ␥4, and ␥5 have been reported to accelerate
inactivation of Cav3.1 currents and shift steady-state inactivation by ⫺4 mV (210). To reiterate, the electrophysiological activity of T-type ␣1-subunits expressed alone is
very similar to that observed with native channels. This is
not the case for HVA channels, which require ␤-subunits
for proper gating (223), and ␣2␦1- and ␥1-subunits for
proper pharmacological activity (381). In conclusion,
these studies indicate that HVA auxiliary subunits can
interact with Cav3 ␣1-subunits, but additional biochemical
studies are required to establish this interaction in vivo.
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Physiol Rev • VOL
B. Antiepileptics
Generalized, or petit mal, absence epilepsies are
characterized by periods of synchronized neuronal activity, generating a 3- to 4-Hz spike-wave pattern on the
electroencephalogram. Considerable evidence suggests
that spike-wave discharges are produced by synchronized
oscillations of cortical, reticular thalamic, and corticothalamic neurons (92, 136, 378). Spike-wave discharges of
corticothalamic neurons are in part mediated by T-type
channels that produce LTS (93, 277, 377, 387). Ethosuximide is a first-line drug in the treatment of absence
epilepsy (193). In vitro studies found that ethosuximide
can block T-type channels with little effect of HVA channels in thalamic neurons (86). Because block occurred at
therapeutically relevant concentrations (0.3 mM), it was
concluded that T-type channel block might explain its
mechanism of action. Support for this hypothesis came
from the following studies with related analogs: 1) T-type
current block was also observed at therapeutically relevant concentrations of methyl-phenylsuccinimide (MPS),
the active metabolite of the antiepileptic drug methsuximide, and 2) no block was observed using either the
inactive analog succinimide or the convulsant analog
tetra-methylsuccinimide (87). With one exception (215),
most studies have failed to confirm ethosuximide block of
T-type currents at therapeutically relevant concentrations
(403), leading to the suggestion that block of other ionic
channels may be more relevant (232).
Ethosuximide and MPS are capable of blocking all
three recombinant T-type Ca2⫹ channels (137). As observed with mibefradil, block is enhanced up to 10-fold by
holding the membrane at potentials that partially inactivate T-type channels. In addition, ethosuximide has
greater affinity for open than rested channels, suggesting
that it is both a gating modifier and an open channel
blocker. The effect of ethosuximide on other recombinant
Ca2⫹ channels has not been rigorously tested, although
block of Cav2.3 (IC50 ⫽ 20 mM) has been reported (295).
In particular, its affinity for partially inactivated HVA
channels has not been studied, so its selectivity is unknown. Studies with MPS suggest that it is only about
twofold selective for LVA over HVA channels in thalamic
neurons (87). The affinity (Ki) of ethosuximide for inactivated states was estimated to be 2 mM. Because therapeutic plasma concentrations are close to 0.5 mM (57),
only a fraction of channels is expected to be inhibited
during therapy. This partial block may be relevant due to
“pharmacological amplification” (297). This is due to the
role of thalamic T-type channels in gating Na⫹ channels,
such that nominal block of the T-type channel is sufficient
to block all-or-none action potentials (178).
T-type channels are also blocked by antiepileptics
that are thought to work by blocking Na⫹ channels. Perhaps the best studied is phenytoin, which clearly blocks
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channels complicates the interpretation of mibefradil effects, and additional studies are required to establish the
role of T-type channels in smooth muscle proliferation (355),
cardiac remodeling (113, 422), and renal protection (28).
Akaike et al. (4) have extensively studied the sensitivity of neuronal T-type currents to calcium channel
blockers. These studies demonstrated that many drugs
blocked neuronal T-type currents at micromolar concentrations. The order of potency (IC50 in parentheses, in
␮M) was flunarizine (0.7) ⬎ nicardipine (3.5) ⬎ nifedipine
(5) ⬎ nimodipine (7) ⬎ methoxyverapamil (50) and diltiazem (70). A similar rank order was found in a study of
septal neurons, except nicardipine was found to be more
potent (IC50 ⫽ 0. 77 ␮M; Ref. 6). Because early studies
using 0.1–1 ␮M dihydropyridines had shown selective
block of L-type channels with little block of T-type channels, these authors concluded that there was a subset of
dihydropyridine-sensitive T-type channels. A similar conclusion was reached from a study of thalamic and mesenteric SMC T-type currents (246, 398). Felodipine is also
a potent T-type blocker, displaying an apparent affinity for
inactivated channels of atrial myocytes of 13 nM, but only
700 nM for those of pituitary GH3 cells (83). Such highaffinity block led these authors to conclude that block of
T-type channels might contribute to felodipine’s antihypertensive properties. Amlodipine (Norvasc, Pfizer),
which is currently the most widely prescribed dihydropyridine, is ⬃12-fold selective for cardiac L-type channels
(IC50 ⫽ 0.5 ␮M), blocking T-type channels with an apparent IC50 of 5.7 ␮M (323). Recombinant Cav3.2 channels
were slightly less sensitive (IC50 ⫽ 31 ␮M). Because amlodipine serum concentrations are ⬃14 nM (432), at least
3% of the T-type channels will be blocked during therapy,
and this fraction might be higher in depolarized cells.
The dihydropyridine structure-activity relationships
of T- and L-type channels are different. This is most
clearly demonstrated by the stereoisomers of BAY K 8644;
T-type channels are weakly blocked by micromolar concentrations of both (237), while for L-type channels the
(⫺)-isomer is an agonist (198). Stimulation of LVA currents by BAY K 8644 is difficult to interpret because this
compound shifts gating of L-type channels to more negative potentials. In contrast, the stereoselectivity and potency of niguldipine were similar for both L- and T-type
channels; the racemic mixture blocked T-type currents in
guinea pig atrial myocytes with an IC50 of 0.18 ␮M (341).
Although the binding site for dihydropyridines is different
between L- and T-type channels, the mechanism of block
appears similar. Dihydropyridines appear to stabilize inactivated states of both channels, causing a negative shift
in the steady-state availability curve (31, 83, 221, 336). In
conclusion, dihydropyridines are selective for L-type
channels, and some analogs are capable of blocking Ttype channels in the 1–10 ␮M range.
T-TYPE CALCIUM CHANNELS
C. Anesthetics
Although the precise mechanism of action of general
anesthetics is unknown, many studies have demonstrated
that they are capable of modulating the activity of ion
channels, including ligand-gated GABA and glycine channels. A newly described mechanism for inhalation anesthetics is that they hyperpolarize neurons by potentiating
the activity of leak K⫹ currents carried by the two-pore
domain channels such as TASK-1 (371). Isoflurane, haloPhysiol Rev • VOL
thane, and nitrous oxide block DRG T-type currents at
therapeutically relevant concentrations of anesthetics
(401, 403). Isoflurane block of either DRG or recombinant
Cav3.1 occurs at concentrations (271–303 ␮M) that are
lower than those achieved during anesthesia (404). Block
is not totally selective, as block of HVA currents by halothane occurs at similar millimolar concentrations (160,
390). In contrast, nitrous oxide selectively blocked DRG
LVA currents with no effect on HVA currents (401). In
addition, nitrous oxide was found to be a selective
blocker of Cav3.2 currents, with little effect on Cav3.1
currents.
Octanol (20 ␮M), an aliphatic alcohol, was reported
to block LVA currents totally from inferior olivary neurons (245). Subsequent studies required much higher concentrations for block (IC50 ⬃200 ␮M; reviewed in Ref.
158). Recombinant T-type channels can be totally blocked
by octanol, with 50% inhibition occurring at 160 ␮M for
Cav3.1 and 219 ␮M for Cav3.2 (404). Studies on hippocampal CA1 pyramidal neurons indicated that octanol is nonselective, blocking T-, N-, and L-type channels to a similar
extent. In conclusion, T-type currents are blocked at therapeutically relevant concentrations of some anesthetics,
and this block may contribute to their anesthetic and
analgesic properties.
D. Antipsychotics
Antipsychotics are used in the treatment of psychotic
disorders such as schizophrenia, the main phase of manicdepressive illness, and other acute idiopathic psychotic
illnesses (20). Most antipsychotics (neuroleptics) act by
blocking D2 class dopaminergic receptors, although drugs
of diphenylbutylpiperidine class can also block T-type
channels.
Among diphenylbutylpiperidines, penfluridol was the
most effective at blocking T-type currents of human medullary thyroid cancer (TT) cells (108). Penfluridol blocked
with an IC50 of 224 nM, and similar results were obtained
with adrenal zona fasciculata cells (109). It was also
⬃10-fold more selective for LVA currents over HVA currents as 500 nM penfluridol inhibited 82% of the LVA
current but only 20% of the HVA. Block of other channels
has not been studied in detail, although it should be noted
that these drugs are also potent blockers of K⫹ channels
(139) and bind with subnanomolar affinity to L-type channels (208). Despite strikingly different structures, thioridazine, clozapine, and haloperidol have comparable activity in TT cells, but are still much less active than
fluspiriline or penfluridol (108). Similar results have been
obtained using recombinant rat Cav3 channels, although
the apparent affinity was higher than observed with native
channels (349). Cav3.1 channels were blocked with the
following order of potency (IC50 in parentheses, in nM):
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both channels at therapeutic concentrations (413). Phenytoin appears to be selective for LVA over HVA currents
(413). The mechanisms of block are also similar, with
higher affinity for inactivated states as evidenced by its
ability to shift the h⬁ curve (413). From this shift it can be
calculated that phenytoin blocks inactivated channels
with a Ki of 7 ␮M. Studies using recombinant T-type
channels indicated that Cav3.2 was the most sensitive,
although block was variable even in a clonal cell line,
suggesting that additional factors may be important for
block (404). Zonisamide has also been reported to partially block T-type channels (60% max, IC50 ⬃70 ␮M) with
little effect on HVA currents (386).
Additional support for the hypothesis that an increase in T-type currents might underlie epilepsy comes
from animal models. One extensively studied model is the
genetic absence epilepsy rats from Strasbourg (GAERS;
Ref. 419), whose T-type currents were larger than those
recorded from a seizure-free rat strain (409). Consistent
with the enhanced currents, enhanced expression of
Cav3.2 was detected in the reticular thalamic nuclei, although the changes in current density (50%) were larger
than the changes in channel mRNA (20%; Ref. 394). In
addition, a small increase in Cav3.1 expression in the
ventrobasal nucleus of thalamus was detected at the message level, but not with currents (151). Another interesting result of this study was that T-type channel mRNA was
very similar, and in fact rose slightly, between juvenile (15
days old) and adult (⬎40 days old) rats. Enhanced expression of Cav3.2 mRNA was noted at both time points.
Because the absence epilepsy phenotype of these animals
is not manifested until after 30 days, these data suggest
that maturation of the thalamocortical circuit plays a
more important role in determining the onset than the
intrinsic properties of these neurons (419, 431). Increased
expression of T-type currents has also been found in
hippocampal CA1 pyramidal neurons in three different
models: a kindling model of focal epilepsy (112), a pilocarpine model of temporal lobe epilepsy (380), and an in
vitro model (301). These currents, as well as intrinsic
bursting of these neurons, could be blocked by 100 ␮M
Ni2⫹ (380), suggesting the involvement of Cav3.2 channels
(230).
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VI. CONCLUSIONS
A. Physiological Roles
The expression of T-type channels in diverse cell
types suggests that they play a role in various physiological functions. The voltage dependence of activation and
inactivation provides clues to these roles and places constraints on when these channels will be active. In neurons
where the resting membrane potential is in the ⫺90 to
⫺70 mV range, T-type channels can play a secondary
pacemaker role; an excitatory postsynaptic potential
(EPSP) opens T-type channels and generates a LTS,
which in turn activates Na⫹-dependent action potentials
and HVA Ca2⫹ channels. Therefore, T-type channels play
an important role in the genesis of burst firing. In neurons
where the resting membrane potential is relatively depolarized (greater than ⫺70 mV), T-type channels are inactivated and an EPSP directly activates Na⫹ channels. Due
to their fast recovery from inactivation, T-type channels
can produce a rebound burst in depolarized neurons following an IPSP. Electrophysiological recordings indicate
that these channels are preferentially localized to dendrites, and hence play a role in signal amplification (97,
256, 265, 328). Large T-type currents have been found in
thalamic neurons, where they play an important role in
oscillatory behavior. The thalamus acts as a gateway to
the cerebral cortex, and inappropriate oscillations of
these circuits, or thalamocortical dysrhythmias, have
been implicated in a wide range of neurological disorders
(242). T-type currents also appear to play a role in olfaction, vision, and pain reception (201, 317, 402).
Calcium influx not only depolarizes the plasma membrane but also acts as a second messenger, leading to the
activation of a plethora of enzyme and channel activities.
T-type channels can cause robust increases in intracellular Ca2⫹, especially in proximal dendrites (294, 460). Calcium and voltage synergistically open Ca2⫹-activated K⫹
channels, which contribute to spike repolarization and
Physiol Rev • VOL
afterhyperpolarizations (40, 244, 416). In addition to transient increases in intracellular Ca2⫹, T-type window currents may play an important role in slower increases in
basal Ca2⫹. This property appears to be important for
hormone secretion from adrenal cortex and pituitary,
myoblast fusion (45), and possibly smooth muscle contraction.
B. Future Directions
Cloning of the T-type channel ␣1-subunits has provided tools with which to study the distribution of these
channels. As expected, mRNA for these channels was
found to be distributed throughout the central nervous
system; however, it was very surprising to find expression
in organs such as liver and kidney. Future studies are
required to discern their role in these tissues. Such studies would be aided by the development of high-affinity
antibodies. Antibodies would also be useful in studying
the distribution of these channels within neurons and in
their purification. HVA channels are multisubunit complexes; therefore, it seems likely that T-type channels will
also have auxiliary subunits. These hypothetical subunits
may regulate surface expression or might be involved in
hormonal regulation of channel activity.
Because mutations in many ion channel genes have
been linked to inherited diseases, it seems likely that
mutations in T-type channel genes would lead to a phenotype. Mutations that lead to enhanced expression of
channels, or that reduce inactivation, might tip the balance to overexcitability. Enhanced expression of T-type
channels have been observed in animal models of epilepsy (409), neuronal injury (79), cardiac hypertrophy
(308), and heart failure (362). The opposite approach is
also being tested by creating transgenic mice that lack
expression of Cav3 genes. Studies with the Cav3.1 knockout mouse have established the central role this isoform
plays in generating burst firing and thalamic spike-wave
discharges (206). What will be the phenotype of other
Cav3 knock-out mice?
Somewhat surprisingly the transcriptional regulation
of Ca2⫹ channel subunits has received scant attention.
Preliminary studies indicate that the Cav3.1 promoter is
methylated in some tumors, which presumably leads to
decreased expression (408). It has been noted that the
Cav3.2 gene contains a region with high homology to the
consensus sequence of neural restrictive silencer elements, providing another avenue for future research
(342). Transcriptional regulation also appears to be important for hormonal upregulation of T channels (22, 447).
Studies on gene regulation may also provide insights into
the control of T-channel expression during the cell cycle
(220).
The pharmaceutical industry is using combinatorial
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pimozide (43) ⬎ penfluridol (110) ⬎ flunarizine (530) ⬎
haloperidol (1,163). Similar results were obtained with
Cav3.2 and Cav3.3, although flunarizine block of Cav3.2
required sevenfold higher concentrations. These compounds all shifted the steady-state inactivation curve to
more negative potentials, indicating that they bind preferentially to the inactivated state. Use-dependent block
has been noted previously in studies of native channels,
suggesting open channels may also have a higher affinity
than rested channels (15, 108). Pimozide binding to D2
receptors occurs over the same concentration range
(KD ⫽ 29 nM) as block of the recombinant Cav3 channels,
suggesting that T-type channels may be blocked during
therapy (349).
T-TYPE CALCIUM CHANNELS
libraries and high-throughput screening to search for new
drugs, and hopefully one of these will be a highly selective
T-type channel blocker. Such a blocker might be useful in
the control of blood pressure, as evidenced by the utility
of mibefradil. If the drug penetrates the central nervous
system, it might be useful in a variety of disorders that
involve thalamocortical dysrhythmias, such as epilepsy
and neuropathic pain. Clearly, a highly selective blocker
would also be extremely useful in research and would
lead to a greater understanding of the physiological roles
of voltage-gated Ca2⫹ channels.
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I thank my current and former fellows Drs. Jung-Ha Lee,
Juan Carlos Gomora, and Janet Murbartián for help with recordings of recombinant channels and proofreading. I thank Jennifer
DeLisle for countless trips to the library to photocopy articles. I
thank Drs. John Huguenard, David McCormick, Thierry Bal,
Hee-Sup Shin, Andy Randall, and Chris Benham for permission
to use figures. I also acknowledge the valuable critiques and
suggestions provided by my collaborator Dr. Paula Barrett.
This work was supported in part by National Institutes of
Health Grants HL-58728 and NS-38691 and an Established Investigator Award from the American Heart Association.
Address for reprint requests and other correspondence: E.
Perez-Reyes, Associate Professor of Pharmacology, Univ. of
Virginia, 1300 Jefferson Park Ave., Charlottesville, VA 229080735 (E-mail: [email protected]).
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