REVIEW
Autoreceptors, membrane potential and
the regulation of transmitter release
Hanna Parnas, Lee Segel, Josef Dudel and Itzchak Parnas
It has been suggested that depolarization per se can control neurotransmitter release, in addition
to its role in promoting Ca21 influx.The ‘Ca21 hypothesis’ has provided an essential framework for
understanding how Ca21 entry and accumulation in nerve terminals controls transmitter release.
Yet, increases in intracellular Ca21 levels alone cannot account for the initiation and termination
of release; some additional mechanism is needed. Several experiments from various laboratories
indicate that membrane potential has a decisive role in controlling this release. For example,
depolarization causes release when Ca21 entry is blocked and intracellular Ca21 levels are held at
an elevated level.The key molecules that link membrane potential with release control have not yet
been identified:likely candidates are presynaptic autoreceptors and perhaps the Ca21 channel itself.
Trends Neurosci. (2000) 23, 60–68
I
N FAST SYNAPSES, at room temperature, release of
neurotransmitter starts about 0.5 ms after the arrival
of the action potential at the axon terminal. The process
of release terminates within 3–5 ms (Refs 1–3). Such
remarkable speed implies that there are abundant vesicles that are ‘ready to go’ and lack only a triggering signal. Depolarization provides such a signal, the nature
of which will be discussed in this article.
The Ca21 hypothesis
Hanna Parnas and
Itzchak Parnas are
at The Otto Loewi
Minerva Center for
Cellular and
Molecular
Neurobiology, Dept
of Neurobiology,
Alexander
Silberman Institute
of Life Sciences,
The Hebrew
University,
Jerusalem 91904,
Israel,
Lee Segel is at the
Dept of Computer
Science and Applied
Mathematics,
The Weizmann
Institute of Science,
Rehovot 76100,
Israel, and
Josef Dudel is at
the Physiologisches
Institut der
Technischen,
Universität
München,
D-80302 München,
Germany.
60
The experimental evidence for the role of Ca21 in the
release of neurotransmitter is overwhelming and dates
as far back as 1954 (Refs 4,5). The classical ‘Ca21 hypothesis’ for fast release of neurotransmitter is based on this
evidence. According to this hypothesis, depolarization
opens Ca21 channels, Ca21 enters the cell and quickly
reaches a concentration that is sufficiently high to
trigger the release process. The termination of release
occurs because Ca21 concentration falls below a
threshold value (see Refs 6–11 for reviews).
The classical Ca21 hypothesis accounts for major
experimental findings. It describes the quantitative
relationship between release of neurotransmitter and
extracellular Ca21 concentration12, or Ca21 current13. It
further accounts for facilitation14 and for other types
of synaptic modulation. However, the Ca21 hypothesis
cannot fully account for the timecourse of this release.
According to this hypothesis, any mechanism that
promotes faster accumulation of Ca21 near the release
sites should provide an earlier onset of transmitter
release, while slow accumulation should delay this
onset. Similarly, the termination of release should be
affected by processes that govern Ca21 removal from
the vicinity of the release sites. However, it has been
shown (for example, see Refs 2,15–17) that although the
amount of release (the quantal content) is modified as
expected by changes in Ca21 concentration, the probability of release as a function of time (the ‘timecourse
of release’1) is insensitive to treatments that are known
to affect Ca21 entry or its removal.
In recent years, the Ca21 hypothesis has been refined
by several authors into what can be termed the ‘Ca21
microdomain hypothesis’. According to this hypothesis,
TINS Vol. 23, No. 2, 2000
the depolarization-triggered opening of Ca21 channels
results in a very high Ca21 concentration (a few hundred mM) in tiny microdomains both below each Ca21
channel and in their close proximity. Such a high concentration is required to activate the release system that
produces release, and it is assumed that this is because
the putative Ca21 receptor has a low affinity for Ca21.
Owing to diffusion and buffering, the high Ca21 concentration falls rapidly when the channels close (in response
to repolarization), within about 500 ms, and release stops
soon after. It has been implied that the observed invariance of the timecourse of release can be explained
by the Ca21 microdomain hypothesis (see Refs 18–20
and the review in Ref. 10).
Several experiments lend support to the two fundamental assumptions of the Ca21 microdomain hypothesis: high local Ca21 concentrations are needed for initiating release, and release is controlled by low-affinity
Ca21-binding receptors. In the ribbon synapse of goldfish
bipolar cells, it was found that, under conditions where
depolarization was not applied, a Ca21 concentration as
high as 200 mM was needed to produce fast release21. By
using a low-affinity Ca21 indicator, aequorin, it was also
found that after high-frequency stimulation the intracellular Ca21 concentration reached levels of 300 µM in
restricted submembraneous domains22,23. A detailed
review has summarized evidence that synaptotagmin I
is the Ca21 sensor involved in triggering release from
nerve terminals, and that the intrinsic affinities of its
two binding sites are low (~60 mM and 300–400 mM)24.
Several observations cast doubt on the support of the
above experiments for the Ca21 microdomain hypothesis:
(1) The experiments of Heidelberger et al.21 were conducted without depolarizing pulses. For reasons outlined
below, it is possible that had depolarization been administered, much lower concentrations of Ca21 would have
been required to produce release.
(2) Llinás et al. found high local Ca21 concentrations22,23, but it is not clear that high-frequency
stimuli reproduce conditions that are typical of a single pulse. Moreover, no demonstration was made that
the high concentrations of Ca21 were required to trigger
release.
0166-2236/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0166-2236(99)01498-8
REVIEW
H. Parnas et al. – Membrane potential and transmitter release
(b)
(c)
0.5
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trends in Neurosciences
Fig. 1. Simulated timecourses of neurotransmitter release. (a) Timecourse of release, illustrated using the model for release in (b). Release is
normalized to peak release. Solid and broken lines correspond to solid and broken lines of Ca21 concentration (insert). The insert shows temporal
distributions of intracellular Ca21 levels, 50 nm away from a Ca21 channel, after a depolarizing pulse of 1 ms. The broken line represents release
after depolarization of 230 mV and the solid line represents release after depolarization to 0 mV. Extracellular Ca21 concentration (10 mM) is the
same in both cases. (b) Model for release of Yamada and Zucker19. C is the Ca21 concentration at the release sites. X and Y are Ca21 binding sites
with affinities of 200 mM and 15 mM, respectively. The first two lines of the kinetic scheme represent, respectively, the successive binding of C to n
and m sites on X and Y. The last line represents the assumption that all sites on X and Y must be bound to induce release, and that, once started,
release ceases after a time of the order of 1/k3. The values of the rate constants (taken from Yamada and Zucker19) are k1x 5 0.5 ms21mM21;
k2x 5 100 ms21; k1y 5 0.01 ms21mM21; k2y 5 0.15 ms21; k2 5 1 ms21mM21; k3 5 10 ms21; n 5 4 and m 5 1. (c) Experimental timecourse of
release at two levels of extracellular Ca21, 0.5 mM (filled circles) and 1 mM (open circles). The thick line denotes action potential. (a) Reproduced,
with permission, from Ref. 27. (c) Reproduced, with permission, from Ref. 2.
(3) Although the experiments described by Südhof and
Rizo report low intrinsic affinities for the putatative Ca21
sensor24, it should be remembered that the ‘intrinsic’
affinity might not be relevant. Muscarinic ACh receptors,
for example, exhibit different affinities under different
conditions. Membrane potential is one of the parameters
that affects the affinity of these receptors, which exhibit
high affinity (in the nM range) at resting potential and
shift to a low-affinity state (in the range of several tens of
mM) after membrane depolarization (see Refs 25,26).
Can the Ca21 microdomain hypothesis account for the
observed invariance of the timecourse of release when
changes occur in Ca21 concentration? In order to investigate this, Aharon et al. simulated the spatio–temporal
distribution of intracellular Ca21 concentration after
brief (1 ms) depolarizing pulses of various magnitudes
and at various levels of extracellular Ca21 (Ref. 27). They
showed that with an accurately dimensioned Ca21 channel and with continuous adjustment of the Ca21 current,
to take account of accumulated intracellular Ca21, Ca21
‘domains’ with concentrations of Ca21 in the millimolar
range are indeed obtained, but only directly below the
center of the channel mouth. A few nanometers away,
the Ca21 concentration drops sharply: 10 nm from the
center of the channel, the concentration is only 2% of
the value at the center; and 30 nm away from the center,
the value is 0.3% of the value at the center (see Ref. 27).
Thus, for high extracellular Ca21 concentration (10 mM),
the peak concentration of Ca21 expected at a distance of
approximately 10 nm from the channel is 20 mM during
the pulse and 50 mM during the tail current.
On the assumption that Ca21 domains do exist,
Aharon et al. further investigated whether these domains
were significant for determining the timecourse of
release. In order to do this, they incorporated their calculated Ca21 domains into the release model of Yamada
and Zucker19, an embodiment of the Ca21 microdomain
hypothesis. In this model, there are two Ca21 binding
receptors (Fig. 1b) with Kd values of 200 mM and 15 mM,
and the rate-limiting step occurs after Ca21 binding.
Figure 1 illustrates that for this model of the Ca21 microdomain hypothesis, the timecourse of release responds
sensitively to changes in intracellular Ca21 levels27.
At the time when Aharon et al. made their calculations, the release site was considered to be situated at
a distance of about 50 nm from the Ca21 channel, and
this was therefore the distance that they assumed. Now
estimates of this distance have decreased and could even
be as little as 10 nm. An important point to remember
is that the relationship between Ca21 profiles and the
timecourse of release is not expected to be affected by
the exact location of the release site. At any given time,
the concentration at the release site depends on the
distance of that site from the channel, but the value of
the concentration is not important. What matters for
release kinetics, according to the original Ca21 hypothesis, is the timescale for increase and decrease of Ca21
concentration, and these are not affected by how far the
site is separated from the channel. (For additional discussion of the relationship between the Ca21 hypothesis and
various aspects of release see Refs 28–30.)
When considering the exact location of the release
sites, one should take account of the dimensions of the
various proteins that are involved in release control,
and of the vesicle itself. It is, thus, rather an oversimplification to employ a ‘point’ release site; the Ca21 concentration that the exocytotic machinery will encounter
is probably an average in a few cubic nanomoles.
Aharon et al.31 obtained a value of about 20 mM when
they calculated the average Ca21 concentration within
a box whose sides were 40 nm long, and within which
four Ca21 channels opened simultaneously as a result
of a brief, strong, depolarizing pulse.
A study by Ravin et al. of neurotransmitter release in
crayfish release boutons is of considerable relevance to
the debate on the concentration of Ca21 at the release
sites and on the affinity of the putative Ca21 receptor32.
In this preparation, release produced by a brief depolarizing pulse is very sensitive to the steady-state level of
Ca21, [Ca21]ss (up to 4 mM when measured with fura-2).
Changes in [Ca21]ss of only hundreds of nanomolars
affected release significantly. This implies that at the
release sites, the Ca21 concentration is the sum of [Ca21]ss
and the Ca21 that entered and reached the release sites
(Y). As small changes in [Ca21]ss can have such marked
effects, the conclusion seems inescapable that, whether
TINS Vol. 23, No. 2, 2000
61
REVIEW
H. Parnas et al. – Membrane potential and transmitter release
or not microdomains exist, near release sites, the Ca21
entry augments [Ca21]ss transiently by, at most, 10 mM.
Indeed, the authors have developed a combined experimental and theoretical approach wherein [Ca21]ss is
measured and Y is calculated to provide a fit with
experimental data. They conclude that Ca21 influx
produces concentrations of Y of between less than 1 mM
up to a few mM, depending on the concentration of extracellular Ca21. It has been further found that the putative
Ca21 receptor associated with elicited release has a
high affinity for Ca21, in the range of 5 mM.
Much can be explained by the conventional view that
neurotransmitter release is due entirely to a single factor:
the brief transient high concentration (microdomain)
of Ca21 that collapses rapidly when Ca21 channels close.
Nevertheless, it has been shown that this single-factor
Ca21 hypothesis seems unable to account for the observed invariance of the timecourse of neurotransmitter
release. It is, thus, reasonable to entertain the possibility
that an additional factor is involved. Accordingly, some
years ago, the ‘Ca21 voltage hypothesis’ was formulated
to explain the initiation and termination of neurotransmitter release in fast synapses3,16. The essence of
this hypothesis is that the natural stimulus, depolarization, alters two factors that are necessary for release.
Depolarization leads to an increase of the Ca21 concentration near the release sites, as in the original Ca21
hypothesis and the Ca21 microdomain hypothesis. In
parallel, and independently, depolarization shifts an
inactive molecule (or a complex) T to its active form S,
which renders it ready to elicit release by some action
in tandem with Ca21. At rest, the concentration of both
Ca21 and S are low. After stimulation, both rise, but it is
the depolarization-dependent transition of the release
machinery from its inactive state, T, to its active state, S,
that initiates release, and the reverse transition from S
to T that terminates release. Consequently, according to
the Ca21 voltage hypothesis, the quantal content is determined by both the level of intracellular Ca21 and that
of S. The timecourse of release, however, is determined
mainly by the temporal behavior of S, and, hence, is
insensitive to the spatio–temporal distribution of intracellular Ca21. The phenomenological Ca21-voltage
hypothesis was given a mathematical form33 and was
shown to agree with several experimental findings that
could not be explained by the Ca21 hypothesis, in particular those findings that relate the independence of
the timecourse of release to changes in intracellular Ca21
concentration.
A central feature of the Ca21 voltage hypothesis is the
role of membrane potential in directly controlling
release, in addition to the indirect role of membrane
potential in opening and closing Ca21 channels. There
is evidence to suggest that membrane potential indeed
has such a direct role.
Membrane potential and release
Silinsky et al. demonstrated a direct role of membrane
depolarization in initiating release in frog neuromuscular
junction34. In these experiments the intracellular Ca21
concentration was elevated by means of Ca21-loaded
liposomes. The extracellular solution contained no
added Ca21 and 1 mM Mg21 (‘Ca21-free’ solution), as well
as the Ca21-channel blocker, Co21 (1 mM), and the Ca21
chelator ethylene glycol-bis(b-aminoethyl ether)N,N,N9,N9-tetraacetic acid (EGTA) (2 mM). In spite of the
elevated level of intracellular Ca21, release did not occur
62
TINS Vol. 23, No. 2, 2000
until the motor nerve was stimulated. The release that
then took place, in the absence of Ca21 influx, exhibited
a normal timecourse, reflected by the normal shape of
the end plate potentials (EPPs). The authors carefully
ensured that this release was not obtained by leakage
of Ca21 from the liposomes into the extracellular fluid
and a consequent depolarization-induced influx of Ca21
through the voltage-dependent Ca21 channels. It was
concluded that the ACh release produced in the frog
could occur in the absence of Ca21 entry through Ca21channels, providing that intracellular Ca21 was elevated
(see also Ref. 35).
Release termination is also influenced by changes in
membrane potential, as demonstrated by experiments
where rapid hyperpolarization of the presynaptic membrane was applied immediately after the depolarizing
pulse that initiated release3,36. These experiments demonstrate that the duration of release is shortened, and
quantal content is diminished by post-pulse hyperpolarization. One’s first reaction to these results is that
they are an expected consequence of the reduction of
Ca21 influx by the post-pulse hyperpolarization, and
indeed such a reduction is found37. The question is
whether this reduction is sufficient to explain the results, or whether the post-pulse hyperpolarization has
an additional role. In support of the latter, elevation of
the extracellular Mg21 levels does not cause premature
release termination, even though it reduces Ca21 influx
to a similar level to that produced by the post-pulse
hyperpolarization. It could be argued that Mg21 does
not affect the timecourse of release because it does not
alter the kinetics of Ca21 influx; it reduces Ca21 influx
evenly. The post-pulse, by contrast, modifies the kinetics
of Ca21 influx: it affects only the tail currents because of
the faster closing of Ca21 channels, and consequently
shortens the duration of release. However, the timecourse of release is not at all affected by changes in the
temporal distribution of intracellular Ca21 (Refs 2,15–17).
In particular, even when Ca21 is removed faster, by
injection of a fast Ca21 chelator, the quantal content is
reduced as expected, but the timecourse of release is
not altered17.
Another finding that supports the conclusion that
post-pulse hyperpolarization exerts an additional effect
besides reduction of Ca21 influx is that post-pulse hyperpolarization decouples both elicited and (surprisingly)
asynchronous release from intracellular Ca21 concentration: the sensitivity of release to changes in intracellular Ca21 greatly diminishes37. Such loss of sensitivity
is not observed when Ca21 influx is reduced by increasing the extracellular Mg21 concentration. Therefore, it
appears that it is not the reduction in Ca21 influx by
the post-pulse that causes faster termination of release.
Rather, the faster termination of release is primarily
caused by the hyperpolarizing effect of the post-pulse,
presumably by the partial decoupling between release
and intracellular Ca21. The Ca21 channel might well be
involved in this additional effect of the hyperpolarizing
post-pulse but as a ‘voltage sensor’ (see, for example,
our later discussion of Ref. 38) and not as a modulator
of Ca21 influx.
A final piece of evidence that membrane potential is
directly involved in release comes from recent experiments in which Mochida et al. demonstrated that
depolarization has a Ca21-independent enhancing effect
on transmitter release38. Release of ACh from cultured
superior cervical ganglion neurons from week-old rats
REVIEW
H. Parnas et al. – Membrane potential and transmitter release
(a)
(b)
Vesicle
Repolarization
Inactive
release machinery
Active
release machinery
Vesicle
V
M
Ca2+
CS
CS
Depolarization
Depolarization
CC
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(d)
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CC
Vesicle
V
V
M
CC
V
M
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T
M
Depolarization
CC
A
T
CC
trends in Neurosciences
Fig. 2. Mechanisms by which membrane potential can control release directly. (a) Representation of the central claim reviewed in this article: membrane potential
controls fast neurotransmitter release by shifting release machinery between its inactive and active states. [(b)–(d) show possible mechanisms for (a)]. (b) Depolarization
might shift a Ca21-binding protein to high affinity, which allows Ca21 to be concentrated at strategic intracellular regions at the active zone7. (c) Depolarization might
activate the interaction between the N-type Ca21 channel (open) and the SNARE proteins38. (d) Depolarization shifts the autoreceptor from high to low affinity, thereby
leading to dissociation of transmitter from the autoreceptor. The unbound autoreceptor no longer associates with the exocytotic machinery; the free machinery can
now be engaged in release (see Box 1). Abbreviations: A, transmitter autoreceptor; CC, Ca21 channel; CS, Ca21 sensor; M, membrane exocytic proteins; T, transmitter;
V, vesicular exocytic protein.
was measured. Stimulation was triggered by focal application of a hyperosmotic sucrose solution in Ca21-free
medium (no Ca21 added, 10 mM Mg21). Thus, entry of
Ca21 through voltage-dependent Ca21 channels was prevented. Tetanic stimulation by a train of action potentials increased neurotransmitter release even when
changes in intracellular Ca21 (which might be freed from
internal stores) were prevented by injection of a fast Ca21
chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9tetraacetate (BAPTA) or by thapsigargin-mediated
depletion of these stores.
In summary, evidence has been provided that membrane potential per se has a role in triggering and terminating neurotransmitter release. This alone confirms
the main assertion of the Ca21 voltage hypothesis, that
membrane potential directly controls release via a
voltage-dependent process other than influx of Ca21.
Membrane potential and control of release:
possible mechanisms
By what direct mechanism could membrane potential
exert release control, in addition to its indirect effect of
enabling Ca21 influx through voltage-gated channels?
The fast timecourse of neurotransmitter release implies
that the vesicles are primed and ‘ready to go’. It would
appear that the action potential causes only conformational changes in one or more proteins (rather than
inducing enzymatic reactions) and consequently that
protein–protein interactions are affected. Three possible
ways of achieving this will be discussed.
One possibility was suggested in 1985 by Silinsky,
who hypothesized that there is a Ca21-binding protein
sensitive to membrane potential that is decisive in the
control of neurotransmitter release. Depolarization activates this protein; as a result it binds Ca21 and release
commences. Upon membrane repolarization, this Ca21
receptor resumes its inactive state and release terminates7. Another way for membrane potential to control
release directly stems from the previously mentioned
finding of Mochida et al. that depolarization enhances
release in a Ca21-independent manner38. These authors
found that this enhancement was decreased reversibly
by introduction, into the presynaptic neurons, of a peptide from the synaptic protein interaction (synprint)
site on N-type Ca21 channels with SNARE proteins
[syntaxin, 25 kDa synaptosomal-associated protein
(SNAP25), and a vesicle-associated membrane protein
(VAMP/synaptobrevin)]. It was concluded that the
activated state of the N-type Ca21 channel had a direct
stimulatory effect on the actions of the SNARE proteins
in neurotransmitter release. Mochida et al. regarded this
stimulatory effect as one that could supplement the
action of Ca21 in the processes leading to release. However, we can extrapolate from their findings, and suggest
that membrane potential might control release in the
following manner. Release is initiated by depolarization, owing to its stimulatory effect on the interaction
between the (open) Ca21 channel and SNARE proteins.
This activated release machinery interacts with the Ca21
that has entered the presynaptic nerve terminal and
release commences. Termination of release occurs upon
membrane repolarization, owing to an interaction between the (closed) Ca21 channel and SNARE proteins
that induces the latter to adopt an inactive state.
Figure 2a presents the idea of direct control of release
by membrane potential: depolarization activates release
machinery and repolarization inactivates it. The depolarization-activated Ca21-receptor7 is shown in Fig. 2b, and
the interaction between the depolarization-activated
Ca21 channel and the SNARE proteins38 is depicted in
Fig. 2c. Figure 2d shows the view of this article, that
presynaptic inhibitory autoreceptors are the vehicle by
which membrane potential controls release. The central
hypothesis in this article is summarized in Box 1, and
is detailed in the following sections.
In seeking for molecules that are involved in the
depolarization-dependent activation of the release
machinery (the first step in the cascade of reactions
leading to release), it is helpful to consider the analogous
question with respect to enzymatic reactions. Suppose
that one wanted to identify the first step, A→B, in a
chain of reactions that produced a product P. If P inhibits
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H. Parnas et al. – Membrane potential and transmitter release
Box 1. How membrane potential can control
release via autoreceptors
(1) Presynaptic, autoreceptors interact physically with the core exocytotic
machinery: the proteins that have been implicated in the control of fast
Ca21-regulated exocytosis of neurotransmitter (SNARE proteins, synaptotagmin and the Ca21-channel) and are therefore likely to be involved in
the initiation (and termination) of elicited release.
(2) At rest conditions (resting potential and resting levels of transmitter concentration), the core exocytotic machinery is under tonic block. This tonic
block is achieved by the association of autoreceptors with exocytotic
machinery.
(3) Initiation of release is achieved by a depolarization-induced relief from
the tonic block.
(4) The mechanism that implements the voltage-dependent protein–protein
interaction by which depolarization exerts control over release is based
on the finding that the affinity of autoreceptors for transmitter depends
on membrane potential. Under resting conditions autoreceptors are in
a high-affinity state and, accordingly, are bound by the low, tonic concentrations of transmitter. Only in their bound state do autoreceptors
interact with the exocytotic machinery and thereby block exocytosis.
(5) Depolarization initiates release by inducing a fast transition of the autoreceptors to a low-affinity state. Then, transmitter dissociates immediately
from autoreceptor, and the free autoreceptor rapidly detaches from the
exocytotic machinery. The unblocked exocytotic machinery interacts
with the Ca21 that has entered and release commences. The voltage
sensor need not be the autoreceptor; for example, the Ca21 channel
itself could serve as the voltage sensor that induces the conformational
change in the autoreceptor.
(6) Upon repolarization, release terminates because autoreceptors rapidly
switch back to their high-affinity state. Now, even the low, tonic concentration of transmitter is sufficient to ensure rapid binding of transmitter
to autoreceptor, and hence to return the release machinery to a blocked
state because of its association with bound autoreceptor.
its own formation (feedback inhibition), it is often the
case that this inhibition acts on the first step of the reaction; thus, determining the locus of inhibition would
allow one to find a likely candidate for the reaction A→B.
There is ‘end-product inhibition’ (feedback) in neurotransmitter release: it is documented that transmitters
inhibit their own release by binding to autoreceptors39.
The autoreceptors that are relevant are the inhibitory
autoreceptors, irrespective of whether they are ionotropic or metabotropic, and these will be the focus of
this hypothesis.
It can be tested whether such end-product inhibition
indeed acts on the ‘first step’ of the chain of events that
induce neurotransmitter release (release initiation), by
establishing whether or not the inhibition is voltage
dependent. Irrespective of the mechanism (whether it
is depolarization-dependent influx of Ca21 or depolarization-dependent activation of the release machinery,
or both), release increases as depolarization rises. (At
even higher depolarizations, release declines, owing to
a reduced Ca21 current, but this range will not be considered.) As release initiation is voltage dependent, then
‘first-step’ inhibition of release must also be voltage
dependent. Khanin et al. gave a formal presentation of
this argument40 and further showed that if inhibition
was exerted at a later, voltage-independent step, then
inhibition would also be voltage independent.
It has been shown that the feedback inhibition of
release is voltage dependent. In experiments that have
used the macropatch electrode technique, a single release bouton in the crayfish neuromuscular junction41
or a small section of the presynaptic terminal of the frog
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neuromuscular junction42 were depolarized to different
levels by brief pulses (1 ms). At each level of depolarization, a control quantal content was established. Then
a fixed concentration of transmitter or its agonist (glutamate or NMDA for the crayfish, and muscarine, an
agonist of muscarinic ACh receptors, for the frog) was
added and release was measured again at various levels
of depolarization. In both cases, it was found that a
fixed concentration of transmitter inhibited release in a
voltage-dependent manner. Inhibition was maximal at
low depolarization and it became weaker as depolarization increased41,42. (At even higher depolarizations,
muscarine and glutamate, but not NMDA, enhanced
release. The enhancement of release was studied by the
respective authors but it will not be discussed in this
article.) Similar findings were also obtained using brain
synaptosomes, where depolarization was achieved by
changing KCl concentration43.
These findings support the conclusion that release
autoinhibition acts on the ‘first step’; but how relevant
are these findings to our attempt to identify the molecule (or molecules) that are primarily responsible for
direct control of release by membrane potential? A
second analogy with a sequence of enzyme reactions
is helpful. In cases of first-step inhibition for enzyme
reactions, the protein (enzyme) that binds the end
product is the protein that catalyzes the first step in the
sequence of reactions. This suggests that in the present
example, the protein that binds the end product
(transmitter), namely the autoreceptor, is likely to be
the molecule that is sought. [Readers should not be
confused by the fact that feedback inhibition is characterized by a slow time constant (min) while initiation
(and termination) of release is fast, in the millisecond
range. As will become evident later, different mechanisms underly the two processes (control of release and
feedback inhibition), even though the inhibitory autoreceptor has a central role in both mechanisms.]
All this motivating argument leads to a candidate
needle amidst a haystack of possible molecules that
might engender release control by membrane potential
(reviewed in Ref. 29). This ‘needle’ is the autoreceptor. In
Box 1, hypotheses are outlined to explain how the autoreceptor might provide the vehicle by which membrane
potential controls release (see also Fig. 2d). Whether or
not one is comfortable with the heuristic path to the
autoreceptor is irrelevant; the question is, will one be
convinced by the following point-by-point discussion
of autoreceptor action?
Autoreceptors interact physically with the core
exocytotic machinery
In order to exert a controlling influence, the autoreceptor presumably interacts physically, possibly indirectly, with one or more of the core proteins that have
already been implicated in release of neurotransmitter.
In an attempt to ascertain whether or not such an interaction occurs, the autoreceptor selected for examination
was the M2 muscarinic ACh receptor (mAChR), the subtype known to be involved in feedback inhibition42. It
was shown, using rat brain synaptosomes, that the M2
receptor interacted with syntaxin, SNAP25, VAMP and
synaptotagmin. In situ interaction was demonstrated
using both immunoprecipitation (see Fig. 3b) and
crosslinking experiments44. If inhibitory autoreceptors
(muscarinic receptors for the cholinergic system and glutamate receptors for the glutamatergic system) are the
REVIEW
H. Parnas et al. – Membrane potential and transmitter release
240
160
80
0
–0.8
–1.0
–1.2
–1.4
SV2
65
Synaptotagmin
38
Synaptophysin
35
Syntaxin 1A
25
SNAP25
19
VAMP
Amplitude (µA)
[3H]QNB (d.p.m./100µg)
kDa
85
320
(c)
l
ta
To
ACh release (% control)
(b)
Ab
hR
b
AC
m oA
N
(a)
1400
1200
1000
800
600
0
15
30
45
60
90
KCI (mM)
trends in Neurosciences
Fig. 3. Experimental results that provide the basis for the control of neurotransmitter release by membrane potential. (a) Effect of methoctramine
(1 mM), a selective antagonist for the M2 muscarinic ACh receptor (mAChR) subtype, on ACh release at different pulse amplitudes [the release is
expressed as a percentage of control (100%), broken line]. (b) The interaction of mAChRs with SNARE proteins and synaptotagmin44. A mixture of
polyclonal antibodies against various mAChRs (raised against M1–M5 receptors) was used for immunoprecipitation experiments. The co-precipitated
proteins were detected by the antibodies indicated on the right-hand side. (c) Depolarization-dependent interaction of syntaxin with mAChRs.
mAChRs were labelled under physiological conditions with [3H]QNB {[3H]guinuclidinyl benzilate} and the labeled synaptosomes were subjected to
varying depolarization levels (by changing KCl concentration and maintaining the ionic strength). This was followed by immunoprecipitation using
syntaxin 1A antibodies. The degree of mAChR–syntaxin interaction is reflected by the amount of [3H]QNB labeling that co-precipitated with
syntaxin. Abbreviations: SNAP25, 25 kDa synaptosomal-associated protein; SV2, synaptic vesicle protein 2; VAMP, vesicle-associated membrane
protein. (a) Data taken, with permission, from Ref. 42. Data in (b) and (c) are taken, with permission, from Ref. 44.
vehicle for release control, then the relevant autoreceptor
must be present in release sites. Such localization has
been demonstrated in crayfish for the NMDA autoreceptor (which was shown to be involved in feedback
inhibition, see Ref. 41) by staining with polyclonal
and monoclonal antibodies45.
The release machinery is under tonic block
The following experiments indicate that basal concentrations of transmitter block release. It was found
that electrically elicited release of ACh was enhanced
by antagonists (hyoscine, scopolamine or atropine) of
M2 receptors in guinea-pig ileum46,47, rat urinary bladder48, rat or mouse phrenic nerve (reviewed in Ref. 49)
and, with methoctramine, in frog neuromuscular junction42 (see Fig. 3a). The same effect was found in a glutamatergic system, using APV [(6)-2-amino-5-phosphonovaleric acid] as an antagonist in crayfish41.
The tonic block of neurotransmitter release that a
transmitter mediates (under rest conditions) via its
autoreceptor could be implemented via the Ca21 channels or by some other means. Evidence that the tonic
block is not achieved by block of Ca21 channels comes
from experiments showing that low and moderate
transmitter concentrations do not reduce Ca21 influx.
As mentioned before, in crayfish neuromuscular junction, it was found that on application of brief depolarizing pulses, glutamate (at concentration of between
5 3 1027 M and 1024 M) or NMDA (1027 M to 1024 M)
inhibit release in a voltage-dependent manner41. With
the aid of fura-2 imaging, it was further shown that, at
these concentrations of glutamate and NMDA, the inhibition was not accompanied by reduction in Ca21 influx
or by a change in presynaptic membrane conductance50.
Similarly, in the frog neuromuscular junction it was
shown that the voltage-dependent inhibition produced
by low and moderate (up to 10 mM) concentrations of
muscarine was not accompanied by reduction in Ca21
currents42. Finally, a voltage-dependent inhibition produced by changes in KCl concentration was also seen in
brain synaptosomes. This inhibition was independent
of the level of extracellular Ca21, indicating once again
that inhibition did not involve reduction in Ca21
influx43. In addition, when tonic inhibition of Ca21
channels, via a G protein, does exist, it is not mediated
by the autoreceptor (in this study, the M2 muscarinic
receptor)51.
In contrast to the findings cited above, there are
reports that, in fast systems, inhibition is voltage dependent but achieved through a reduction in Ca21 currents51–54. However, the concentration of transmitter
reported in those studies was higher than those used by
Slutsky et al.42 It, therefore, seems that, at least at low
and moderate transmitter concentrations, the voltagedependent inhibition is not accomplished by reduction
of Ca21 influx.
By what means does the autoreceptor mediate the
tonic block? Given the existence of physical interaction between the autoreceptor and the core exocytotic
machinery44, it is likely that this interaction blocks
release.
Initiation of release: a depolarization-induced relief
from a tonic block
If the tonic block of release occurs because of an association between a bound autoreceptor and the release
machinery, then the initiation of release by depolarization should occur by abolition of this block. If so, it
can be expected that the interaction between autoreceptor and exocytotic machinery will be strong at
resting potential (when block occurs) and will weaken
in response to membrane depolarization. Indeed, the
interaction (measured by immunoprecipitation) between
mAChRs and syntaxin was found to be voltage dependent (depolarization was induced by raising KCl concentration while maintaining the ionic strength); interaction was strong at resting potential (5 mM KCl) and
weakened as depolarization increased up to 90 mM KCl
(Fig. 3c). A possible role for Ca21 influx in these interactions is unlikely, as the experiments were conducted in
Ca21-depleted solutions (no Ca21 added, 2 mM EGTA)44.
Other relevant experiments, which were cited previously, are those in which neurotransmitter antagonists
were added at rest conditions (resting concentration
TINS Vol. 23, No. 2, 2000
65
REVIEW
H. Parnas et al. – Membrane potential and transmitter release
of transmitter and resting potential) and a brief depolarizing pulse elicited more release than control (no antagonist added). This instantanous enhancement of release
by a depolarizing pulse is not achieved by removing an
auto-inhibitory block of Ca21 currents, as these currents
are not blocked by low and moderate concentrations
of transmitter (see earlier discussion).
As has been mentioned, release autoinhibition is
observed if transmitter concentration is elevated above
its rest value. It takes minutes for relief of inhibition to
be achieved by removal of the transmitter. This, therefore, raises doubt regarding the feasibility of relief from
a tonic block as a mechanism for initiation of release.
However, the relief that is of relevance for release
initiation does not occur via neurotransmitter removal
but by depolarization. If a brief strong depolarizing pulse
is administered to the inhibited system, the relief from
block is instantanous41.
Depolarization shifts autoreceptor affinity
from high to low
It has been shown that there is a voltage-dependent
interaction between autoreceptor and release machinery, and it has been suggested that this interaction
underlies the control of release. How can such an interaction be implemented? Evidence will be presented to
show that depolarization shifts autoreceptor affinity
from high to low. This shift provides a mechanism by
which depolarization can alter the interaction between
the autoreceptor and the release machinery.
Ilouz et al. found that in rat brain synaptosomes, in
Ca21-depleted solutions, mAChRs, specifically M2
receptors, bind [3H]ACh (in the range of 5–150 nM) in
a voltage-dependent manner26. Binding was measured
using fresh synaptosomes that were depolarized to
various levels (by exposure to varying concentrations
of KCl while the ionic strength was maintained). For
each experiment, the synaptosomal membrane potential was determined by the level of [3H]TPP1 (tetraphenylphosphonium bromide) in the synaptosomes.
It was found that binding was maximal at resting potential (5 mM KCl) and decreased as depolarization was
increased (up to 60 mM KCl). Lysed synaptosomes, which
had been shown to be insensitive to changes in KCl
concentration, did not exhibit this voltage-dependent
binding. Displacement of the labeled antagonist
[3H]NMS {N-[3H]methyl-scopolamine} with carbachol
(an ACh-receptor agonist) under various KCl concentrations, showed that depolarization shifts the
mAChRs from a high-affinity state (Kd of about 20 nM)
to a low-affinity state (Kd of about 20 mM)26.
Additional experiments were carried out to determine whether the level of the receptor occupancy
affected its interaction with the exocytotic machinery.
Syntaxin was selected as a representative protein of
the exocytotic machinery. Co-immunoprecipitation of
M2 receptors (using an M2-receptor-specific antibody)
with syntaxin was measured after treatment with two
concentrations of muscarine (0.1 mM and 10 mM) and at
three levels of depolarization (5 mM, 30 mM and 60 mM
KCl). It was shown that the magnitude of M2-receptor–
syntaxin interaction decreased as depolarization increased, at both concentrations of muscarine. Furthermore, at the resting potential, 0.1 mM muscarine
enhanced the M2-receptor–syntaxin interaction by
about threefold, and higher muscarine concentrations
(10 mM) had no additional effect. At high depolariz66
TINS Vol. 23, No. 2, 2000
ation (60 mM KCl), 0.1 mM muscarine strengthened the
interaction only very slightly compared with control,
but 10 mM muscarine significantly strengthened the
M2-receptor–syntaxin interaction26.
It can be inferred from observations of feedback
inhibition in concentrations of glutamate (or NMDA) as
low as as 1027 M (Ref. 41) or in 1027 M muscarine42, that
presynaptic autoreceptors exhibit high affinity at rest
conditions. This means that the tonic concentration
of transmitter is lower than 1027 M and that this low
concentration produces tonic inhibition.
Concluding remarks
The summary of the experimental findings that
support the ideas proposed in Box 1 is now complete.
A formal embodiment of the ideas discussed was provided by Yusim et al., who constructed and analyzed an
appropriate kinetic and mathematical model55. With
this model, Yusim et al. showed that the fast timecourse
of release was guaranteed by the membrane-potentialmediated change in affinity of the autoreceptor. Release
initiation is governed by depolarization-induced transition of the receptor to a low-affinity state, and the
consequent fast dissociation of the transmitter from
the autoreceptor. How fast is this dissociation? The lowaffinity Kd is in the range of 20 mM (Ref. 26). Assuming
that on change in Kd it is the ON rate constant that is
altered, Yusim et al. estimated the rate constant of transmitter dissociation from the receptor to be 2 ms21, which
allows fast release in the millisecond range. Termination
of release is governed by the association of transmitter to
the high-affinity receptor, its Kd being around 20 nM.
Retention of the rate of dissociation at 2 ms21, as for
the low-affinity receptor, yields an ON rate constant of
108 M21ms21. This enables an effective rate constant for
transmitter association in the range of 10 ms21 even with
the low tonic concentration of transmitter (below 1027 M).
This model of Yusim et al. provides a detailed molecular mechanism for the Ca21 voltage hypothesis. As
was stated previously, this hypothesis postulates an inactive state, T, of a controlling entity that predominates
at resting potential and that shifts to an active state, S,
upon depolarization. In the model of Yusim et al., T corresponds to exocytotic machinery that is associated with
autoreceptor (and hence blocked), while S corresponds
to free (and hence active) exocytotic machinery. The proposals of Silinsky7 and of Mochida et al.38 (Fig. 2b,c) also
fit within the framework of the Ca21 voltage hypothesis.
For the former and latter proposals, respectively, T would
be the inactive Ca21 sensor or an inactive complex of
Ca21 channel and SNARE proteins, whereas S would
correspond to the active Ca21 sensor or to the active
complex of the Ca21 channel and SNARE proteins.
The data reviewed in this article that concern the
crucial importance of autoreceptors in mediating control of release by membrane potential, suggest that autoreceptors should be added to the list of core exocytotic
proteins. It is natural to ask whether autoreceptors themselves can sense changes in membrane potential, and,
indeed, this is the case56. Another possibility is suggested
by findings of Cohen-Armon et al., that postsynaptic
muscarinic receptors are shifted by depolarization from
a high-affinity state to a low-affinity state, and that the
voltage sensor in this case is the Na1 channel25. By analogy, it is possible that the shift in affinity of the presynaptic mAChR is effected via interaction with the
Ca21 channel.
REVIEW
H. Parnas et al. – Membrane potential and transmitter release
Knowledge concerning feedback autoinhibition was
used as a heuristic guide to pinpoint autoreceptors as
the agent for voltage-dependent release control. It must
be emphasized that the release control and the autoinhibition act by different mechanisms, although these
autoreceptors have a key role in both mechanisms.
Evidence has been presented that initiation of release
occurs by direct depolarization-induced abrogation of
an association between autoreceptors and exocytotic
machinery. Release termination results when repolarization induces reconstitution of this association directly.
By contrast, feedback inhibition that occurs upon elevation of transmitter concentration presumably involves
the action of a second messenger. The exact mechanism
of this action is not currently known; however, the
slow timecourse of feedback inhibition is presumably
governed by slow accumulation and decay of the second
messenger involved.
What further experiments can challenge the idea that
autoreceptors mediate direct release control by membrane potential? It would be illuminating to examine
how the timecourse of release is affected by the addition
of autoreceptor antagonists. One could conjecture that
the timecourse would not be strongly affected, for no
effect seems to have been reported in experiments in
which quantal content is increased by the addition of
antagonists to the rest concentration of transmitter41,42.
Although these experiments have not yet been carried
out, the model of Yusim et al.55 is capable of explaining
invariance of the timecourse of release to a range of
antagonist concentrations, because during the time of
release the average concentration of transmitter in the
synaptic cleft rises briefly to a high value. Such transient
elevations in transmitter concentration do not cause
feedback inhibition, as the transients are too brief.
However, elevations persist long enough to ‘evict’ the
added antagonist temporarily. When the average transmitter concentration returns to a level that is close
to its tonic concentration (following termination of
release), the antagonists are no longer efficient because
by that time release has practically terminated (K. Yusim,
H. Parnas and L. Segel, unpublished observations).
In conclusion, one more matter should be considered.
Because of its intrinsic importance, slow transmitter
release has been the subject of a large literature. Are
the various findings discussed in this article applicable
to slow release? It is unlikely, as it can be expected that
special mechanisms are required to ensure that the
duration of release is in the millisecond range.
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TINS Vol. 23, No. 2, 2000
Acknowledgements
The authors thank
J.G. Nicholls for his
very constructive
comments and
S. Fliegelmann for
superb wordprocessing. The
authors’ research
was supported by
Sonderforschungsbereich 191 der
Deutschen
Forschungsgemeinschaft to J.D., I.P.
and H.P.
I.P is grateful to
the Anna Lea
foundation for their
continued support.
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419, 665–687
Gap junctions, synchrony and seizures
José Luis Perez Velazquez and Peter L. Carlen
The old concept that the direct intercellular cytoplasmic connections between neurones
participate in the coordination of neuronal activity has gained new relevance, owing to recent
theoretical and experimental evidence, particularly with regard to neuronal synchronization and
epileptogenesis. Computer simulations demonstrating that neurones synchronize and alter their
firing patterns depending on gap-junctional communication, have provided insights into the
interactions between electrotonic coupling and cellular and synaptic characteristics. Experimental
manipulations of gap-junctional communication support its role in the generation and maintenance
of synchronized neuronal firing and seizures. Hence, in addition to chemical transmission, direct
electrotonic coupling might contribute to normal and abnormal physiological brain rhythms.
Trends Neurosci. (2000) 23, 68–74
D
José Luis Perez
Velazquez and
Peter L. Carlen are
at the Playfair
Neuroscience Unit,
Bloorview Epilepsy
Programme, Depts
of Medicine
(Neurology) and
Physiology,
University of
Toronto, Toronto
Western Hospital,
Toronto, Ontario,
Canada M5T 2S8.
68
URING an epileptic seizure, there is synchronous
activity of many thousands of cellular elements.
This pathological synchrony is the hallmark of a
seizure, but how it occurs is still not clearly defined.
Experimental and theoretical evidence suggests that
the old concept of direct electrotonic communication
between neurones via intercellular connections, now
known as gap junctions, is an important synchronizing
mechanism that, in combination with field effects, and
synaptic and ionic mechanisms, contributes to the
generation and maintenance of seizures.
The reticulum theory of brain-cell communication
In the 19th century it was thought that the brain was
composed of cells that were connected to each other directly to form a ‘reticulum’. Around 1900, experimental
observations revealed the brain structure as a network of
cells, called neurones1, and the multitudinous connections between them as synapses2. The later discovery of
chemical synaptic transmission3 led to a complete shift
in emphasis towards the subtleties and variety of the
modulatory actions of this most-common form of neuronal communication in the CNS. Seizure generation was
examined mostly from the perspective of chemical synaptic mechanisms. However, recent experimental and
TINS Vol. 23, No. 2, 2000
theoretical findings have revitalized our appreciation of
the role of gap-junctional communication (GJC) in neuronal firing synchrony and seizure generation, resurrecting
the old reticulum theory with new meaning.
The relevance of GJC to brain function is evident in
early brain development. Many studies have shown an
abundance of GJC in immature brain tissue (when compared with mature brain), which declines rapidly as
maturation progresses4. The role of GJC in the immature
brain is apparently related to the organization of cellular networks, which it achieves by coordinating electrical and biochemical activity5,6. In the immature brain,
nonsynaptic epileptiform activity is more common7,
which could be a consequence of this extensive neuronal coupling. These observations have promoted the
concept that GJC in the mature CNS represents the
remnant of what was once a crucial feature required
for early brain development8. The important issue is not
how much GJC is left, but the functional role of whatever is present. Whether it is needed or not, GJC between
mature neurones has been established experimentally,
and converging evidence indicates that its functional
manifestations participate in neuronal firing synchrony that can lead to low- and high-frequency brain
oscillations and seizures.
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