Physiol Rev 94: 303–326, 2014
doi:10.1152/physrev.00016.2013
L-TYPE CaV1.2 CALCIUM CHANNELS: FROM IN
VITRO FINDINGS TO IN VIVO FUNCTION
Franz Hofmann, Veit Flockerzi, Sabine Kahl, and Jörg W. Wegener
FOR923, Institut für Pharmakologie und Toxikologie, Technische Universität München, Germany; and
Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, Homburg, Germany
L
I.
II.
III.
IV.
V.
VI.
VII.
VIII.
IX.
X.
XI.
XII.
XIII.
INTRODUCTION
STRUCTURE AND SUBUNITS OF THE...
SPLICE VARIANTS AND DISTRIBUTION
ELECTROPHYSIOLOGY OF L-TYPE...
REGULATION OF THE CARDIAC...
Ca2ⴙ-DEPENDENT INACTIVATION...
VOLTAGE- AND...
MUTATION IN GENES CODING...
CaV1.2 AND THE BRAIN
CaV1.2 AND SMOOTH MUSCLE
CaV␤2 AND CaV␤3 KNOCKOUT MICE
CaV1.2 AND SECRETION
CONCLUSION
303
304
305
309
309
313
313
314
315
315
316
317
317
I. INTRODUCTION
The voltage-gated L-type calcium channel CaV1.2 is an integral cell membrane protein complex that mediates the
influx of Ca2⫹ into the cell in response to membrane depolarization. The entered Ca2⫹ serves as an essential intracellular messenger that regulates a variety of cellular processes
including muscle contraction, hormone secretion, neuronal
transmission, and gene expression. These physiological
processes depend partially on the activity of the CaV1.2
channel and have been characterized as excitation-contraction, excitation-secretion, or excitation-transcription coupling (49, 184, 204).
Voltage-gated calcium channels consist of at least four subunits: the ␣1 subunit shapes the Ca2⫹ selective pore, contains
the voltage sensor and the binding sites for most regulatory
modulators and drugs, whereas the accessory subunits ␣2␦, ␤,
and ␥ are involved in anchorage, trafficking, and regulatory
functions (48, 132, 204).
CaV1.2 channels typically require a strong depolarization for
activation, show long-lasting activity, and are substantially
blocked by low micromolar concentrations of organic L-type
calcium channel antagonists including dihydropyridines
(DHP), phenylalkylamines, and benzothiazepines (243, 245,
292). These properties classify the CaV1.2 channel as a member of the high voltage-gated, DHP-sensitive, L-type calcium
channels. This group contains four members named according
to their ␣1 subunits CaV1.1, CaV1.2, CaV1.3, and CaV1.4 (79).
The other subfamilies of voltage-gated calcium channels, discriminated by their electrophysiological and pharmacological
properties, are likewise named according to their ␣1 subunits
and include CaV2.1 (P/Q-type Ca2⫹ current), CaV2.2 (N-type
Ca2⫹ current), CaV2.3 (R-type Ca2⫹ current), and CaV3.1–
3.3 (T-type Ca2⫹ current) (79).
CaV1.2 channels are multisubunit protein complexes composed of the three subunits, designated ␣1, ␣2␦, and ␤ (50, 69,
70, 205). CaV1.1 is additionally associated with the ␥1subunit. Similarly, N-type Ca2⫹ channels (195, 309), P/Q-type
Ca2⫹ channels (190), and the R-type Ca2⫹ channels (308) are
also built of these three subunits ␣1, ␣2␦, and ␤. In contrast,
0031-9333/14 Copyright © 2014 the American Physiological Society
303
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Hofmann F, Flockerzi V, Kahl S, Wegener JW. L-Type CaV1.2 Calcium Channels:
From In Vitro Findings to In Vivo Function. Physiol Rev 94: 303–326, 2014;
doi:10.1152/physrev.00016.2013.—The L-type Cav1.2 calcium channel is present
throughout the animal kingdom and is essential for some aspects of CNS function, cardiac
and smooth muscle contractility, neuroendocrine regulation, and multiple other processes.
The L-type CaV1.2 channel is built by up to four subunits; all subunits exist in various splice variants that
potentially affect the biophysical and biological functions of the channel. Many of the CaV1.2 channel
properties have been analyzed in heterologous expression systems including regulation of the L-type
CaV1.2 channel by Ca2⫹ itself and protein kinases. However, targeted mutations of the calcium channel
genes confirmed only some of these in vitro findings. Substitution of the respective serines by alanine
showed that ␤-adrenergic upregulation of the cardiac CaV1.2 channel did not depend on the phosphorylation of the in vitro specified amino acids. Moreover, well-established in vitro phosphorylation sites of
the CaV␤2 subunit of the cardiac L-type CaV1.2 channel were found to be irrelevant for the in vivo
regulation of the channel. However, the molecular basis of some kinetic properties, such as Ca2⫹dependent inactivation and facilitation, has been approved by in vivo mutagenesis of the CaV1.2␣1 gene.
This article summarizes recent findings on the in vivo relevance of well-established in vitro results.
HOFMANN ET AL.
T-type channels seem to consist only of an ␣1 subunit (226), since
blocking expression of all CaV␤ gene products by anti-CaV␤ antisense oligonucleotides in ganglion neurons (170) or absence of
CaV␤1 or CaV␥1 (279) did not affect T-type currents.
II. STRUCTURE AND SUBUNITS OF THE
VOLTAGE-GATED L-TYPE CaV1.2 Ca2ⴙ
CHANNEL
Subunit
␣1
␣2␦
␤
␥
Forms
Genes
␣1S (CaV1.1␣1)
␣1C (CaV1.2␣1)
␣1D (CaV1.3␣1)
␣1F (CaV1.4␣1)
␣1A (CaV2.1␣1)
␣1B (CaV2.2␣1)
␣1E (CaV2.3␣1)
␣1G (CaV3.1␣1)
␣1H (CaV3.2␣1)
␣1I (CaV3.3␣1)
␣2␦-1 (CaV␣2␦-1)
␣2␦-2 (CaV␣2␦-2)
␣2␦-3 (CaV␣2␦-3)
␣2␦-4 (CaV␣2␦-4)
␤1 (CaV␤1)
␤2 (CaV␤2)
␤3 (CaV␤3)
␤4 (CaV␤4)
␥1 (CaV␥1)
␥2
␥3
␥4
␥5
␥6 (CaV␥6)
␥7
␥8
cacna1 s
cacna1c
cacna1d
cacna1f
cacna1a
cacna1b
cacna1e
cacna1g
cacna1h
cacna1i
cacna2d1
cacna2d2
cacna2d3
cacna2d4
cacnb1
cacnb2
cacnb3
cacnb4
cacng1
cacng2
cacng3
cacng4
cacng5
cacng6
cacng7
cacng8
Reference Nos.
79
59, 162
39, 137
42, 52, 161
(Please note, only ␥1
and ␥6 are
considered to be a
subunit of a
voltage-gated
calcium channel)
52
The voltage-gated L-type Ca2⫹ channels were first purified
from skeletal muscle membranes using their extensive binding
of radioactive labeled Ca2⫹ channel blockers of the DHP type
(48, 87, 91, 93, 107, 108). The purified DHP receptor complex was built up of five proteins which were named ␣1
(⬃170 –240 kDa), ␣2 (⬃150 kDa), ␤ (⬃54 kDa), ␦ (17–25
kDa), and ␥ (⬃32 kDa). Although the ␣2 and ␦ proteins were
initially described as single subunits, a follow-up study revealed that a single ␣2/␦ mRNA encodes a precursor protein
that is proteolysed into the ␦ and ␣2 subunits linked by disulfide bonds (63) probably involving Cys404 and Cys1047 (46).
Purification of these skeletal muscle proteins allowed the cloning of the respective genes (33, 76, 144, 248, 283) and by
homology screening the identification of the other highvoltage-activated Ca2⫹ channels (for review, see Refs. 48, 70,
133).
units (47, 137, 227, 248). For the ␥ subunit, the first subunit
was cloned from skeletal muscle (33, 144). Successively, seven
additional genes have been found to code for CaV␥ subunits
and termed cacng1– 8 (42, 52, 161). Only CaV␥1 and CaV␥6
are considered to be a subunit of a voltage-gated calcium channel (52). Deletion of CaV␥1 affected the properties of the skeletal muscle CaV1.1 channel (94). The mRNA of CaV␥6 has
been identified in cardiac muscle. Coexpression of this subunit
with CaV3.1 in HEK cells increased the expressed current
(122). The other subunits, CaV␥2, 3, 4, 7, and 8, have been
identified as transmembrane AMPA receptor regulatory proteins (TARPs) that affect the trafficking and gating of AMPA
receptors (276). Although a number of studies reported interaction of the other CaV␥ subunits with CaV channels in expression systems (154, 316), it remains to be shown that these
subunits interact with the voltage-gated calcium channel in
native tissue.
Ten mammalian ␣1 subunit cDNAs were cloned (TABLE 1). All
10 subunits can be divided into three structurally related subfamilies, namely, CaV1, CaV2, and CaV3 (79). For the ␣2␦
subunit, four mammalian genes have been identified which are
named cacna2d1, cacna2d2, cacna2d3, and cacna2d4 and encode CaV␣2␦-1, CaV␣2␦-2, CaV␣2␦-3, and CaV␣2␦-4, respectively (59, 162). Likewise, four mammalian genes (cacnb1– 4)
have been described to encode for the diverse CaV␤1– 4 sub-
In recent times, it has become evident that with the exception
of the CaV␣1 subunit, the other auxiliary subunits may associate also with different channels or proteins. The CaV␤3 subunit has been found to affect proteins not related to voltagegated Ca2⫹ channels (23, 321). Several studies have revealed
that CaV␤3 and CaV␤4 have roles in gene transcription. CaV␤3
was reported to bind to a novel short splice isoform of the
transcription factor PAX6S, which was revealed by a yeast
304
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A depth of information was obtained by electrophysiological,
pharmacological, and physiological studies on the distribution
and physiological function of the various voltage-gated calcium channels. These experiments were performed mainly in
expression systems and isolated tissues and have been extensively reviewed (49, 133, 204). While the current knowledge
about voltage-gated calcium channels is mainly based on these
in vitro studies, the physiological characteristics of these channels in native tissue, i.e., in vivo, and the contribution of the
channels to broad scope function are revealed only just now.
In recent years, the in vivo role and regulation of the CaV1.2
channel has been investigated by overexpressing, knocking
out, or site-directed mutagenesis of the CaV1.2␣1 and the
CaV␤ genes in transgenic mice. This approach confirmed or
challenged the established facts regarding Ca2⫹ channel regulation and the control of Ca2⫹-dependent cellular processes.
This review provides an overview of properties of CaV1.2
channel in expression studies and in studies using subunitspecific mutations of the CaV1.2 channel complex in mice. In
addition, spontaneously occurring monogenic defects in Ca2⫹
channel subunit genes will also be covered.
Table 1. Subunits of the voltage-gated calcium channel complex
IN VIVO FUNCTION OF L-TYPE CaV1.2 CALCIUM CHANNELS
two-hybrid approach (321). CaV␤4 acts as a nuclear repressor
recruiting platform to control neuronal gene expression in cerebellar Purkinje neurons (280). The truncated chicken CaV␤4c
subunit interacts directly with a nuclear protein (HP1) (130).
The CaV␣2␦ subunits have been reported to be involved in
synaptogenesis (78, 167). The CaV␣2␦-1 and the CaV␣2␦-2
subunits act as receptor for the drugs gabapentin and pregabalin that are used in the therapy of neuropathic pain (103,
110). As stated above, the CaV␥2, 3, 4, 7, and 8 subunits
mainly act as TARPs (52, 156, 276).
III. SPLICE VARIANTS AND
DISTRIBUTION
A. The CaV1.2␣1 Channel Subunit
Alternative splicing contributes to both gene regulation and
protein diversity. The human CaV1.2␣1 gene contains 50 exons (FIGURE 1, A AND B) (268), whereas the CaV1.2␣1 gene
from mouse (181) and rat (305) was reported to miss exon 45,
which results in 49 exons. At least 20 exons undergo alternative splicing (1, 54, 268).
There are at least 13 alternative splicing loci that generate
different variants. Alternative splicing occurs in the NH2 terminus, in the DI-II and DII-DIII linkers, between S5 and S6 in
DI, between S2 and S3 in DIV and between S3 and S4 in DIV,
in DI-S6, in DII-S2, in DIV-S3, and in the COOH terminus
(284) (FIGURE 1). Changes in the expression of the CaV1.2␣1
subunit have been reported during development of cardiomyocytes (CM), suggesting a differential role of fetal and mature isoforms in juvenile cells (116). Some of these splice variants have been associated with a diversity of biophysical properties: 1) a depolarizing shift of activation for alternatively
spliced exons 31 to 33 encoding the DIV S3-S4 region (284); 2)
faster inactivation kinetics, strong voltage dependence, and
variable Ca2⫹-dependent inactivation for exon 41 to 42 variants in the COOH terminus (269, 270, 327); and 3) a higher
DHP sensitivity for exon 8a in DI-S6 (168, 304). The exclusive
alternatively spliced exons 8 and 8a of human cacna1c are
mutated in two different types of the Timothy syndrome. Be-
The relative abundance of CaV1.2␣1 splice variants shows
disease-specific variability (297). The splice variant containing
exon 31 is more abundant in normal human hearts, whereas a
splice variant with a switch to the exon 32 variant was found
in failing hearts (297). An increase in the number of nonfunctional CaV1.2␣1 splice variants was found in hearts from spontaneously hypertensive rats compared with normotensive
Wistar-Kyoto rats (285). An isoform with preferential deletion
of exon 9A and inclusion of exon 33 has been observed in rats
with myocardial infarction (179). The wild-type isoform
Delta9A/33 (deletion of exon 9A and inclusion of exon 33)
channel was reduced greatly in the scar region. A novel isoform 9A/Delta33 (inclusion of exon 9A and deletion of exon
33) channel was detected in the scar region (179). As noted
above, exon 9A/(synonymous 9*) follows exon 9 and is an
independent additional exon not related to 9. In contrast, exon
33* is still exon 33, but differentially spliced resulting in elongation of the common amino acid sequence encoded by exon
33. The significance of most splice variants is not known. For
example, 12 of 42 splice variants have been found exclusively
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The CaV␣1 protein was predicted by hydropathical analysis to
consist of four homologous domains (DI-DIV), each composed of six membrane-spanning ␣-helices (termed S1 to S6)
that are linked by variable cytoplasmic loops (linkers) and
ended by NH2- and COOH-terminal intracellular segments,
similar to the predicted structure of the voltage-gated Na⫹ and
K⫹ channels (73, 283) (see FIGURE 1). Expression of the
CaV1.2␣1 subunit together with the auxiliary subunits CaV␤2
and CaV␣2␦ was sufficient to produce functional Ca2⫹ channels with their characteristic key features such as DHP binding, gating, ion selectivity, and permeation (27, 201, 263). The
human gene for the CaV1.2 ␣1 subunit is localized at chromosome 12p13 (257). A structural model of the L-type Ca2⫹
channel pore has been suggested (274).
cause these exons are mutually exclusive in humans, the effect
of a gain-of-function mutation in one exon might be mitigated
to some degree by the activity of the alternative wild-type exon
in some tissues (273). The switch from exon 8 to exon 8acontaining CaV1.2 mRNAs has been shown to be controlled
by the polypyrimidine tract-binding protein (PTB) and its neuronal homolog (nPTB) (286) and to be repressed during neuronal development in mouse brain (286). Exon 9 contains the
binding domain for CaV␤-subunits, whereas the adjacent exon
9A (FIGURE 1), if present, affects channel gating (180) probably by differentially modulating the interaction with CaV␤
isoforms (220, 275). Exon 9A, sometimes called exon 9*, is
not an extension of exon 9 or alternatively used for exon 9 but
an exon in its own right (285, 290). Fox proteins, another
family of splicing factors implicated in the control of alternative splicing during development, were recently shown to repress exon 9A and promote inclusion of exon 33 into
CaV1.2␣1 mRNAs expressed in the cortex (287). 4) Three
NH2 termini have been reported for CaV1.2␣1 (28, 54, 201)
(see FIGURE 2, A – C). Exon 1a (the long NH2 terminus) is
expressed in cardiac myocytes (201); exon 1b (the short NH2
terminus) is expressed in smooth muscle and brain (27, 267,
304); and exon 1c (even shorter than exon 1b) is present in
resistance cerebral artery (54). The 1b and the 1c isoforms
have been detected in arterial smooth muscle from rat and
human by Western blot analysis (15). The “smooth muscle”
splice variant CaV1.2b (28) is more sensitive to inhibition by
DHPs than the heart variant CaV1.2a, because it contains the
DHP-sensitive exon 8B (304). In vitro and in vivo studies suggest that the CaV1.2 channel containing exon 1b or 1c is better
expressed than the channel containing exon 1a (67, 71, 95,
304). The reason for this difference is presently unknown.
Exon 1a is crucial for protein kinase C (PKC)-dependent upregulation of the cardiac CaV1.2 current, although this sequence is not phosphorylated by PKC (31).
HOFMANN ET AL.
A
I
II
7
3
2
5
14
8A/B
12
9
or
9 + 9A
26
24
21/22
15
IV
36
27
28
30
33/33*
37
20
6
4
III
35
16
11 13
19
23
25
29
31/32
17/17*
39
42
18
40
41
10
1A/B/C
38
34
43
44
47
46
50
1A
1B
1C
2
3
4
5
6
7
8A
8B
9
9A
10
11
12
13
14
15
16
17
17
*
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
33
*
34
35
36
37
38
39
40
41
42
43
44
46
47
48
49
50
B
Cav1.2 a
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
a
a
a
a
b
b
b
b
b
b
b
b
b
b
b
b
b
c
C
Mouse
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
(14)
(15)
(16)
(17)
(18)
(19)
306
48
Rat
Rabbit
GenBank
Ref
GenBank
Ref
GenBank
Ref
FM872409
FM872410
FM872411
(181)
(181)
NM_012517.2
(266)
X15539
(201)
DQ538522
M67515
(54)
(267)
X55763
(28)
AY074797
(54)
(181)
NM_009781
FM872412
FM872413
FM872414
AY728090
L01776
(181)
(181)
(181)
(181)
(181)
(181)
(181)
(181)
(181)
(126)
(184)
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49
IN VIVO FUNCTION OF L-TYPE CaV1.2 CALCIUM CHANNELS
A
Gene (kbp)
1
139
602
Position
88393420
Exon
B
Cav1.2
Mouse chromosome 6
1A
1B
1C
2
MIR
MVN
MLC
GSN
3
a
c
C
Cav1.2
49 50
a
b
c
in hypertensive rat hearts (285). At present, we do not know
whether or not the detected CaV1.2␣1 splicing variants are
disease specifically regulated or represent just individual splice
variations. It is also possible that some splice variations are
related to the change from adult to fetal gene expression program observed in cardiac remodeling (106).
The four members of the L-type Ca2⫹ channels, namely,
CaV1.1 to CaV1.4, have a distinct tissue distribution. CaV1.1
has been found exclusively in skeletal muscle, but a report
suggested that human CaV1.1 was also coexpressed with ryanodine receptors (RYRs) in GABAergic neurons (281).
CaV1.2 is expressed in various tissues including heart (32, 50,
314), smooth muscle (131, 206), pancreas (256), adrenal
gland, and brain (124). The CaV1.2 channel has been assumed
to be the major L-type channel involved in cardiac excitationcontraction coupling. CaV1.3 is mainly found in brain, albeit
at lower levels than CaV1.2 (124), but also in pancreas, kidney, ovary, and cochlea. CaV1.3 transcripts have also been
detected in the sinoatrial node (32, 234) and in both atria and
ventricles at fetal and neonatal stages but not in adult ventricles (241). CaV1.4 expression seems so far to be restricted to
the retina (20).
B. The CaV␣2␦ Channel Subunit
Several splice variants of the CaV␣2␦ channel subunit have
been reported. The subunit found in rat skeletal muscle has
been described to differ from that found in rat brain containing an insertion of 7 amino acid residues and a deletion of a
19-amino acid segment between putative transmembrane domains 1 and 2 (158). Subsequent analysis in the mouse revealed the presence of at least five different isoforms that arose
from various combinations of three alternatively spliced regions (7). Different combinations of these alternatively spliced
regions were found in mouse brain and skeletal muscle,
whereas all five isoforms were reported to be present in the
cardiovascular system (7). A number of splice variants of the
human CaV␣2␦-2 and CaV␣2␦-4 subunit have been also described (17, 240). However, up to now, it is not known
whether the different splice variants contribute to different
properties of native CaV1.2 channels (for review, see Refs. 7,
69, 162). Recently, a gene-disrupting mutation in cacna2d3
(CaV␣2␦-3) was found in an exome sequencing study on families with children affected by autism spectrum disorders
(142).
FIGURE 1. Composition of the mRNA for CaV1.2 (cacna1c) channel. CaV1.2 variants from mouse, rat, and rabbit. A: CaV1.2 with exon numbering
according to the nomenclature used for the human CaV1.2 gene (268). Note that the rabbit, rat, and mouse CaV1.2 cDNAs lack the exon 45 DNA
described in the human gene. Alternative exons indicated as A, B, and C are shown in red. Exon 9A is an independent exon which, if expressed, is
always coupled with exon 9 (shown in green). Exons 17* and 33* (shown in green) represent elongated variants of exons 17 and 33 and are caused
by use of different splice donor/acceptor sites. B: CaV1.2 variants in the order of starting exon 1A (Cav1.2a), B (Cav1.2b), and C (Cav1.2c). Note that
most mouse variants (1 to 4 and 6 to 15) (181) and the rat variants 16 and 19 (54) have been obtained as full-length cDNAs by single step RT-PCR,
respectively, followed by subcloning and sequencing. Other cDNAs were constructed based on partial cDNA clones. C: accession numbers and
references of clones shown in B. [Adapted from Link et al. (181).]
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b
87791081
FIGURE 2. NH2 terminus of the CaV1.2
(cacna1c) gene 21. Exons of the mouse
cacna1c gene (NT_039353.8) located on
chromosome 6 coding for the N-termini a,
b, and c (A and B). The alternative mouse
protein-coding exons 1A (position
88393420
to
88393280),
1B
(88305164 to 88305117), and 1C
(88276861 to 88276837) are shown,
which are spliced to exon 2 (GNSY . . . ,
88254705 to 88254382), respectively,
to yield Cav1.2a, Cav1.2b, and Cav1.2c
variants. C: corresponding mouse sequences. Note that the corresponding sequences of exon 1A and exon 1B have been
identified in mouse, rat, rabbit, and human
(see FIGURE 1); the 1C sequence has been
identified in rats (see FIGURE 1), and the
mouse sequence shown is predicted based
on its similarity with the rat sequence.
HOFMANN ET AL.
C. The CaV␤ Channel Subunit
A
Gene (kbp)
1
Exon
1B
1A
(mouse chromosome 2)
2A
2B
2C
2D
MVQ MDQ SYG MQC MLD MKA
B
270
3 4 5 6
383
7
8
9 10 11 12 13 14
GSA
β2- N1
655 aa
N2
632 aa
N3
604 aa
N4
605 aa
N5
611 aa
C
a)
b)
c)
d)
mouse
cacnb2
exon
mCavβ2
N-terminus
1A + 2A
N1
1B + 2A
N2
2C
N3
2D
N4
N4c)
N4
2ba)
2E
N5
N5
N5
2ea)
N-terminal protein
sequence from mouse
mouse
heart
human
heart
human
heart
rat
brain
rat
heart
rabbit
heart
(181)
(92)
(128)
(57, 227)
(57, 227)
(137, 239)
N1
N1
2da)
N2
2ca)
2aa)
2b
2ab)
According to prediction of the human cacn2b gene on chromosome 10 (57)
Shown not to be present in rat (57, 227) and rabbit heart (137, 239)
34 from 60 independent full-length Cavβ2 cDNA clones from a mouse cardiac cDNA library (181)
6 from nine independent full-length Cavβ2 cDNA clones from a rabbit cardiac cDNA library (137)
FIGURE 3. NH2-terminal splice variants and expression of the CaV␤2 (cacnb2) gene.
308
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The amino acid sequences of the Cav␤ subunits are encoded
by four genes and differ considerably. The differences are located within their NH2 termini (called the variable region 1 or
V1), within the linker between their SH3 domains and GK
domains (variable region 2 or V2) and within their COOH
termini (V3), whereas their SH3 and GK domains are very
similar (39, 75). The CaV1.2␣1 subunit most probably is associated with either the CaV␤2 or the CaV␤3 subunit (181, 183,
216) and in brain also with the CaV␤4 subunit (183, 230).
Only a single variant of the CaV␤3 subunit has been reported
to be present in human myocardium (136). Splicing of the 5’
exons of the CaV␤2 gene results in at least five variants of the
CaV␤2 subunit that differ in the NH2 terminus (FIGURE 3). The
CaV␤2 cDNA was detected by two groups that screened
heart and brain libraries (137, 227) but identified two
different splice variants; both were called CaV␤2a. The
brain CaV␤2a subunit (CaV␤2-N3, see FIGURE 3 for nomenclature and NH2 terminus) contains two cysteine residues at positions 3 and 4 that are palmitoylated (239).
This splice variation confers distinguishing features to
the CaV1.2␣1 subunit (239) and has not been found in
rabbit, rat, or murine heart (57, 136, 181, 239). Because
knockouts of single CaV␤2 splice variants are not available, the delineation of specific functions to Cavb2-N3 or
of other Cavb2 splice variants in vivo remains to be
shown. The major splice variant expressed in adult heart and
originally also called CaV␤2a is CaV␤2-N4 (FIGURE 3). We
suggest renaming the CaV␤2 subunits according to the
expressed NH2 terminus (FIGURE 3), because that will
prevent further confusion on the used CaV␤2 subunit.
IN VIVO FUNCTION OF L-TYPE CaV1.2 CALCIUM CHANNELS
IV. ELECTROPHYSIOLOGY OF L-TYPE Ca2ⴙ
CHANNELS IN HETEROLOGOUS
EXPRESSION SYSTEMS
The CaV␣2␦, CaV␤, and CaV␥1 subunits have pronounced
effects on the characteristics of CaV1.2 currents. For example,
coexpression of the CaV␣2/␦ and CaV␤ subunits enhanced the
amplitude of the current in Xenopus oocytes (263). Potentiation of CaV1.2 currents by a DHP agonist was differentially
modulated by the CaV␣2/␦ and CaV␤ subunits. The combined
expression of CaV1.2 ␣1, CaV␣2/␦, and CaV␤ was the least
sensitive combination (263). Furthermore, VDA and VDI was
accelerated and delayed by CaV␣2/␦ and CaV␤, respectively
(263). From these findings, it seems that single subunits modulate specific functions of CaV1.2 currents, whereas other
properties of the current appear to be modulated by the different subunits acting in concert (39, 263). Coexpression of the
CaV␣2␦-1 and CaV␤ subunits increased IBa,L most likely by
facilitating transfer from the Golgi apparatus to the membrane
(69). VDA is mediated by membrane depolarization that shifts
the four S4 segments (“the voltage sensor”) of DI through DIV
within the membrane and opens the channel gate at the inner
side of the membrane (25). Experiments with chimeras between CaV1.2 and CaV1.1 channels revealed that the DI-S3
segment and the linker connecting DI-S3 and DI-S4 is responsible for the fast VDA of CaV1.2 channels (214, 282). Other
molecular determinants of VDA include 1) the external pore
and the ion selectivity filter of CaV1 channels (166, 251, 315),
2) the inner pore of CaV1 channels (322), and 3) the activation
gate (312).
Inactivation of CaV1.2 currents during depolarization consists
of at least two processes in CMs with time constants ␶ of 10
and 90 ms (see Ref. 291). These time constants represent VDI
and CDI. Both processes are distinguished experimentally by
the use of high intracellular concentrations of the fast Ca2⫹
chelator BAPTA or of Ba2⫹ as the charge carrier.
Activation of CaV1.2 channel currents using Ba2⫹ as the
charge carrier results in inward Ba2⫹ currents that activate
rapidly and inactivate slowly revealing VDI (193). In CMs,
VDI with Ba2⫹ as the charge carrier could be obtained more
clearly if longer depolarization pulses were applied (i.e., ⬎2 s)
VDI is further affected by the CaV␤ subunit. The CaV␤ subunit
interacts at the CaV1.2 ␣1 subunit with a conserved 18-amino
acid section present in the DI-DII intracellular loop. This side
has been named AID (␣1 interacting domain) (236). Crystallographic studies have shown that the AID ␣-helix binds to the
hydrophobic groove present in the CaV␤ subunit GuK (guanylate kinase) domain (53, 223, 295). Binding of the CaV␤
subunit to AID induced a conformational change in the DIS6
and the DI-DII linker that modifies VDA and VDI. The DI-DII
linker is suggested to be the particle that occludes the channel
pore during inactivation under VDI and CDI conditions (3,
39, 159). Alternative mechanisms for VDI include the participation of the CaV␤ subunit and/or the NH2 and COOH terminus of Cav1.2 ␣1 (reviewed in Ref. 275). CDI is a Ca2⫹triggered inactivation process that requires an intact ␣1C
COOH terminus IQ domain (327). The IQ domain binds
Ca2⫹-CaM and is the critical sequence for CDI (229, 325).
Mutation of the isoleucine in the IQ motif lowers the affinity
for Ca2⫹-CaM and abolishes CDI (232, 326). The same region
is involved in CDF (325). CDF requires also Ca2⫹/calmodulin
kinase II (CaMKII) (74) and phosphorylation of residues in the
CaV1.2 ␣1 (175) or the CaV␤ subunit sequence (113).
V. REGULATION OF THE CARDIAC CaV1.2
CHANNEL COMPLEX IN EXPRESSION
SYSTEMS AND IN VIVO
L-type Ca2⫹ channels are regulated by a change in membrane
potential. Their activity is modulated further by hormones.
Reuter (244) showed early that the positive inotropic effect of
norepinephrine on the heart muscle increased the cardiac
ICa,L. Later, it was then confirmed that norepinephrine binding to the ␤-adrenergic receptor increased intracellular cAMP
levels and activated cAMP-dependent protein kinase (PKA).
Elegant experiments showed that the catalytic subunit of PKA
increased the duration of the action potential plateau (224)
and ICa,L (151) in guinea pig cardiomyocytes. Ten years later,
it was reported that cGMP and cGMP-dependent protein kinase (PKG) decreased cardiac L-type current (199). However,
later experiments suggested that NO/cGMP affects multiple
cardiac ion channels by different mechanisms (reviewed in
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Initial experiments showed that the cardiac CaV1.2 ␣1 cDNA
could be functionally expressed in Xenopus oocytes (201).
Coexpression of the CaV␣2␦-1 and CaV␤2 subunit increased
L-type Ba2⫹ current (IBa,L) in CHO cells (303). Identical results were obtained when the CaV1.2a or CaV1.2b was expressed in HEK cells (28). The CaV1.2 ␣1 subunit mRNA
directed expression of functional calcium channels in Xenopus
oocytes (263). The classical characteristics of an L-type calcium channel are encoded by the CaV1.2 ␣1 subunit. These
characteristics include voltage-dependent activation and inactivation (VDA and VDI), voltage-dependent inhibition of the
current by DHPs, Ca2⫹-dependent inactivation (CDI) and facilitation (CDF).
(192, 197). The molecular determinants of VDI include the
cytosolic ends of the S6 segments, the I-II linker, considered to
be the inactivation gate, and the NH2 and COOH termini of
CaV1 (for review, see Refs. 127, 275). Interestingly, a single
point mutation (F1126E) eliminated the permeation and gating differences between Ca2⫹ and Ba2⫹ resulting in a Ca2⫹like current in the presence of Ba2⫹ which supports the idea of
interaction between ion occupancy in the outer vestibule of the
CaV1.2 channel pore and calcium channel gating (178, 261).
However, it should be kept in mind that Ba2⫹ has been considered as a weak agonist for CDI, because Ba2⫹ itself was
reported to induce CDI (85) and permeation of Na⫹ through
CaV1.2 channels decreased further the VDI time constant
(222).
HOFMANN ET AL.
Ref. 89). The mechanism(s) by which PKG modulates cardiac
CaV1.2 channels remains to be established.
Unfortunately, these initial successful reports (38, 101, 115,
259) were not repeated by other laboratories (200, 323). A
variety of explanations have been offered to explain these differences including the importance of the correct isoform of
auxiliary subunits and the need for targeting PKA to the channel by AKAPs (101). These inconsistencies may be also related
to differences in the expression systems used, differences in the
isoforms used for expression, and differences in the accessory
proteins coexpressed with the CaV1.2␣1 subunit. Xenopus
oocytes have endogenous CaV␤s that influence exogenously
expressed CaV␣1 (288). In addition, other enzymes such as
protein phosphatases may modify the basal levels of phosphorylation of the channel (157). This view comes from the
findings that inhibitors of PKA decreased channel activity in
expression systems, suggesting that the CaV1.2␣1 channels
were already endogenously phosphorylated in these systems
without stimulation (225, 228, 262).
In recent years, this analysis was refined by the use of mice
carrying mutations in the CaV1.2 channel complex. The
CaV␤2 subunit is phosphorylated in vitro by various protein
kinases (105) and may be crucial for adrenergic upregulation
310
As an alternative to the CaV␤2 subunit, phosphorylation of Ser
1928 in the COOH terminus of the CaV1.2 ␣1 subunit was
widely accepted as a mechanism for the upregulation of ICa,L
(58, 119, 139, 152). However, mutation of Ser 1928 to Ala in
the mouse generated viable animals with an unaltered fightor-flight reaction (176). Expression of a CaV1.2␣1 subunit
with a Ser1928Ala mutation in TsA-201 cells did not affect the
adrenergic upregulation of the IBa,L (98). Adenoviral expression of a CaV1.2 channel expressing the Ser 1928 Ala mutation in guinea pig CMs did not abolish ␤-adrenergic regulation
of the expressed channel (100). Additional attempts to support the importance of Ser 1928 phosphorylation were unsuccessful. Truncation of the CaV1.2 ␣1 gene at Asp 1904 (71) or
at Gly 1796 (95) removed the COOH-terminal part of the
COOH terminus, but resulted in mice that died during or after
birth. Further analysis suggested that the truncation of the
CaV1.2a splice variant prevented insertion of the channel in
the membrane, because the truncated protein was directed to
the proteasome (71). In contrast, the CaV1.2b channel was
normally expressed in non-CM cells, but not in the CMs (67,
71). Together, these results did not support the hypothesis that
phosphorylation of Ser 1928 mediates the ␤-adrenergic upregulation of ICa,L in the mouse heart.
Several groups suggested that reconstitution of the ␤-adrenergic upregulation of ICa,L required the presence of a PKA anchoring protein (AKAP) (101, 148). AKAPs are a family of
proteins that bind PKA regulatory subunit and phosphatases
and locate them to specific sites (258). AKAP 79/150 (101)
and AKAP 15/18 (112) have been proposed to be involved in
the regulation of the cardiac CaV1.2a. The COOH terminus of
Cav1.2a contains an AKAP binding site (138) that is distal of
the truncation site Asp 1904. Location of PKA close to the
CaV1.2 channel protein seems to be essential for some aspects
of the ␤-adrenergic regulation. However, AKAP7 (also called
AKAP15/18) is not required for regulation of Ca2⫹ handling
in mouse CMs, since AKAP7-KO CMs respond normally to
␤-adrenergic stimulation as determined by the stimulation of
ICa,L, [Ca2⫹]i, or Ca2⫹ reuptake (149). The differential compartmentalization of CaV1.2 channels in CMs may cause colocalization of distinct AKAPs (24). They are located either
together with the ␤2-adrenergic receptor at the caveolae or
with the ␤1-adrenergic receptor outside of the caveolae (12).
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These results stimulated a number of laboratories to unravel
the phosphorylation mechanism by expressing the channel in
various cultured cells. These in vitro studies suggested that
PKA can phosphorylate the CaV1.2␣1 and CaV␤ subunit. For
example, the CaV1.2␣1 and CaV␤2-N3 subunits expressed in
SF9 cells were phosphorylated by purified PKA and PKC in
vitro (237). PKA phosphorylated the CaV1.2 ␣1 and the
CaV␤2-N3 subunit independent of each other. Adrenergic
stimulation of intact canine myocardium led to PKA-mediated
phosphorylation of the CaV␤-subunit, whereas no phosphorylation of the CaV1.2␣1 subunit was detected (114). These
early studies were disputed by the finding that a part of the in
vitro isolated ␣1 subunits was proteolytically cleaved thereby
removing a significant part of the COOH-terminal tail (51, 64,
125, 169, 319). PKA-induced phosphorylation was observed
with the purified full-length 250-kDa form of the CaV1.2␣1
subunit but not in the truncated 200-kDa form (319). This
finding was confirmed when it was reported that Ser 1928
located at the COOH-terminal tail was in vitro a major site of
PKA-induced phosphorylation (62, 101, 118, 125, 139, 202,
225). In addition, Ser 1627 and Ser 1700 have been detected as
PKA site in partially purified, truncated bovine cardiac Ca2⫹
channels (172). Phosphorylation of Ser 1700 and Thr 1704
located in the proximal COOH-terminal domain of CaV1.2␣1
were implicated as the PKA phosphorylation sites (98). Several
phosphorylation sites were identified in vitro on the CaV␤2
subunit (101, 114, 237). Mutations of Ser 478 and Ser 479 to
Ala on the CaV␤2-N3 subunit were shown to abolish PKAinduced regulation of a truncated CaV1.2␣1 subunit expressed
in tsA-201 cells (38).
of ICa,L (38, 115). Truncation of the murine CaV␤2 gene at Pro
501 removes all predicted phosphorylation sites for PKA,
PKG, and CaMKII (35). The generated mouse is viable and the
␤-adrenergic stimulation of ICa,L was preserved in the intact
animal and in the isolated cardiomyocytes (35). These results
do not support a fundamental role for phosphorylation of the
cardiac CaV␤2 subunit in the fight-or-flight regulation (see also
TABLE 2). However, two additional residues are present that
are predicted to be phosphorylated by PKA, Ser 143 and Thr
165 (in CaV␤2-N4). Interestingly, the Ser 143 residue corresponds to the Ser 182 in rabbit CaV␤1a, which has been shown
to be phosphorylated in vivo (248).
IN VIVO FUNCTION OF L-TYPE CaV1.2 CALCIUM CHANNELS
Table 2. List of mouse strains carrying targeted or spontaneous mutations in the CaV1.2 channel genes
Genes (Subunit)
cacna1c (␣1C,
CaV1.2)
Tissue
Mutation, Cre Promotor
Effect
Reference Nos.
Global
Knockout (KO)
Embryonic lethal
260
Global
Heterozygous
Global
Smooth muscle (SM)
conditional
SM vascular
CaV1.2-T1066Y
SM-specific KO,
SM-22-Cre
SM-specific KO,
SM-22-Cre
Hypoactive, increased anxiety
Decreased cardiac current density
Decreased sensitivity to DHP
Lethal at 21st day after
Cre-induction
Reduced blood pressure and
myogenic tone
Loss of depolarization-induced
Ca2⫹ release
Reduced spark frequency
Reduced micturition and
contractility
Loss of PKC-mediated regulation
Loss of motility
1 phase insulin secretion reduced
10
111
264
206, 298
83
No obvious phenotype
Lethal around birth
Increased atrial CaV1.3
Lethal around birth
176
71
67
95
Loss of AKAP15 binding in neurons
Reduced cardiac CDF
189
30
Embryonic lethal
No CDF, Ca2⫹-independent CDI
29
29, 235
Lethal after 12days; DCM
Hypertrophy and heart failure
111
Unchanged cardiac ICa,L
246
Hypertrophy and death in 1 yr
212, 213
Decreased sensitivity to DHP
317
SM intestine
Pancreatic ␤-cells
conditional
Global
Beta-cell-specific KO,
Rip-Cre
CaV1.2-S1928A
CaV1.2-truncated at 1904
CaV1.2-truncated at
G1796
Heart conditional
Brain conditional
CaV1.2-S1512A and
S1570A
I/E mutation
I/E mutation, ␣MHC
(MerCreMer)
Heart-specific KO, ␣MHC
(MerCreMer)
Heart-specific
heterozygous mice
(␣MHC MerCreMer)
Overexpression, ␣-MHC
promotor
Heart specific
CaV1.2-T1066Y/Q1070M,
FLAG-epitope tagged,
␣MHC-doxycycline-inducible
promoter
Heart-specific CaV1.2S1700A-T1704A⌬1798NNAN1801,
␣MHC-doxycyclineinducible promoter
KO Nex-Cre; hippocampal
and cortex neurons
Nestin-Cre, global brain KO
CaMKII-Cre, cortex, and
hippocampus
Nestin-Cre, global brain KO
Brain
Nestin-Cre, global brain KO
81
141, 299
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SM urinary bladder
206
298
256
Unchanged ␤-adrenergic control of
cardiac ICa,L
Reduced L-LTP, reduced spatial
learning, reduced activation of
CREB
Reduced fear in the thalamusamygdala pathway
No effect on fear acquisition,
consolidation, and extinction
Impairment in spatial memory
Mediate cocaine-induced GluA1
trafficking
Increased axon outgrowth in DRGs
203
171
196, 307
252
77
Continued
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HOFMANN ET AL.
Table 2.—Continued
Genes (Subunit)
Tissue
Mutation, Cre Promotor
Nestin-Cre; global brain
I/E mutation
KO forebrain, CaMKII-Cre
N anterior cingulate
cortex, Cre injection
Global
cacna2d2
␣2␦-2
173
145
Neuropathic phenotype
177
Global
Overexpression, thy-1.2
promotor
Global
Premature death
143
Spontaneous mutation
Spontaneous mutation
Ducky phenotype
Entla phenotype: epileptic and
ataxic
16
37
Global
Decreased startle reflex;
increased aggression,
hyperactivity
Loss of retinal signaling
http://jaxmice.jax.org/
strain/005780.html
Cone-rod dysfunction
Embryonic lethal
Embryonic lethal
Small reduction in ICa
Normal ERG a-wave, distorted
b-wave
Increased glucose-stimulated
insulin secretion
Decreased P/Q-, N- and L-type
current; salt-sensitive
hypertension; decreased pain
perception
No obvious effect on contraction
of ileum
Defective survival of naive CD8⫹ T
lymphocytes
Cochlea, outer hair cells:
decreased Ca2⫹ current
Lethargic phenotype: lethargic
behavior, seizures, ataxia;
defective cell-mediated immune
responses
Required for normal TCR-mediated
calcium response, nuclear
factor of activated T cells (NFAT)
nuclear translocation, cytokine
production, but are unnecessary
for proliferation
Cochlea, inner hair cells:
decreased Ca2⫹ current
310
13, 302
302
198
13
Global
Spontaneous mutation
cacnb2-␤2
Global
Heart conditional
Heart conditional
Global except heart
(rescue)
Global
Mutation in humans
Global
MLC2a-Cre
␣MHC (MerCreMer)
␣MHC promotor ⫹
Cav⶿?⶿2-N3 cDNA
Global
l
Spontaneous mutation
Disruption of the AKAP5 gene (also called AKAP79/150)
abolished adrenergic stimulation of Ca2⫹ transients as well as
phosphorylation of the ryanodine receptor and of phospho-
312
150
Global
cacna2d4
␣2␦-4
cacnb4-␤4
Reduced neuronal CaV1.2; less
depressive behavior
Increased anxiety
Impaired observational fear
learning
Reduced behavioral pain
responses
Homo- and heterozygous lethal
Heterozygous with behavioral
abnormalities related to Timothy
syndrome
Decreased myocardial contractility
G406R, exon 8A
G406R with an inverted
neomycin cassette in
exon 8A
Global
cacna2d3
␣2␦-3
cacnb3-␤3
Reference Nos.
10
10
97
311
23
208, 210, 216
123
146
165
43
11
165
lamban. Surprisingly, isoproterenol stimulation of cardiac
CaV1.2 channels remained intact in the AKAP5 knockout
mice (218). At present, it is not clear why deletion of AKAP5
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cacna2d1
␣2␦-1
Effect
IN VIVO FUNCTION OF L-TYPE CaV1.2 CALCIUM CHANNELS
affected cardiac function in such a compartment-specific manner (reviewed in Ref. 24), but these findings support the importance of an AKAP protein for reconstitution of the in vivo
regulation of the CaV1.2 complex in cell systems.
Birnbaumers’s (300) and Hosey’s (104) group showed that
removal of part of the COOH terminus increased the ICa,L of
expressed CaV1.2 channels. It was then reported that the
cleaved COOH terminus (CCt) stays with the CaV1.2 channel
as an inhibitor at the membrane (140). The functional significance of CCt was then extended as an independent transcription factor that regulates the expression of a wide variety of
neuronal genes (109) and the transcription of the cardiac
cacna1c gene (255). The cardiac CCt migrates to the nucleus
and downregulates the CaV1.2 promotor (255). These results
were extended to the arterial smooth muscle (14). The results
suggest that cleavage of the COOH terminus disinhibits ICaL
only under specific conditions but limits the transcription of
the cacna1c gene. Cleavage must occur after translation of
mRNA at the membrane, because truncation of the Cav1.2
gene at Asp 1904 (71) or at Gly 1796 (95) results in death
around birth and proteasomal degradation of the truncated
protein. It is not clear which mechanism decides whether the
CCt stays at the membrane and inhibits ICa,L or moves to the
nucleus and decreases the transcription of the cacna1c gene.
Some groups have not observed the truncation of the cardiac
CaV1.2 protein (205).
VI. Ca2ⴙ-DEPENDENT INACTIVATION
CDI terminates Ca2⫹ entry after opening of the CaV1.2 channel to avoid Ca2⫹ overload and arrhythmias (5, 18). This
regulation involves direct association of the calmodulin (CaM)
to the CaV1.2 channel (5, 120, 229, 325). Overexpression of
mutant, Ca2⫹-insensitive CaM ablated CDI in a “dominantnegative” manner and demonstrated that CaM is constitu-
Mutation of isoleucine 1624 to glutamate of the murine
CaV1.2 gene is embryonic lethal (235). Mice with a conditional heart-specific I/E mutation in the CaV1.2␣1 channel died
within 3 wk after activation of the Cre recombinase, because
this mutation prevents membrane incorporation of adequate
amounts of CaV1.2␣1 protein. Further analysis indicated that
these mice develop dilated cardiomyopathy (DCM) accompanied by apoptosis of cardiac myocytes and fibrosis (29). Treatment with metoprolol and captopril reduced DCM at day 10.
However, cellular contractility and global Ca2⫹ transients remained unchanged in isolated CMs (29). Electrophysiological
analysis at day 10 after Cre induction revealed that the I/E
mutation blocks CaM -mediated regulation of the CaV1.2
channel in the heart and induces a channel phenotype with
permanent CDI characteristics (235). One explanation for this
phenomenon may be that the introduction of the charged glutamate into the IQ motif may mimic the presence of the naturally occurring charged Ca2⫹/CaM complex with respect to
protein folding during channel gating. In contrast to CDF, it is
not clear whether or not CDI requires an active CaMKII (see
below).
VII. VOLTAGE- AND Ca2ⴙ-DEPENDENT
FACILITATION
Voltage-dependent facilitation (VDF) of CaV1.2 channels has
been observed in native CMs and represents an increase in
current amplitude after the respective stimulation. VDF can be
observed during repetitive channel activation, i.e., frequencydependent VDF (174), or after a strong prepulse to positive
potentials, i.e., VDF (219). At the single-channel level, mode 2
gating (long openings) were shown to be strongly promoted
following large depolarization (231). VDF is also observed if
CaV1.2 subunits are expressed in heterologous expression systems (160, 259) but attenuated in Xenopus oocytes by coexpression of the CaV␣2␦ subunit (233) and not observed with
CaV2 subunits expressed in Xenopus oocytes (4, 34). VDF is
abolished by replacement of Ca2⫹ by Ba2⫹ (82) and blocked
by the fast Ca2⫹ buffer BAPTA but not the slower EGTA
(134), indicating that this process is mediated by Ca2⫹ entry
itself then by a change in the membrane potential. Thus it is
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Recently, the ␤-adrenergic regulation of the cardiac CaV1.2
channel was reconstituted in TsA-201 non-muscle cells by expression of a truncated CaV1.2 ⌬1800, its noncovalently associated distal COOH-terminal domain (aa 1801–2171), the
auxiliary ␣2␦1 and CaV␤2b (-N4) subunits, and AKAP 15.
These proteins formed an autoinhibited signaling complex
containing the proteolytic processed CaV1.2 channel (98). Isoproterenol, PKA, and/or PKC stimulated IBa,L through phosphorylation of Ser 1700 and Thr 1704 located at the COOH
terminus of the truncated CaV1.2 ␣1 protein. These results are
very encouraging and may solve the long-standing riddle on
the mechanism of the ␤-adrenergic regulation. However, adenoviral expression of a DHP-resistant CaV1.2 channels with
or without the mutation of Ser 1700 Ala and Thr 1704 Ala in
adult CMs indicated that ␤-adrenergic upregulation of ICa,L
was not affected by these mutations (317). Thus the final proof
is lacking that the above mechanism exists in an intact mouse
heart. For additional discussion, see review by Weiss et al.
(301).
tively tethered to the CaV1.2 channel complex (229). CaM
binds to the IQ motif of the CaV1.2 channel that is located at
amino acids 1624 –1635 (exon 42) of the CaV1.2␣1 COOH
terminus (120, 159, 294, 325). Disruption of this domain prevents CDI, whereas a putative Ca2⫹-binding EF hand motif in
the COOH terminus was not involved in this process (221,
238). CaM binding requires isoleucine 1624 of the IQ motif.
The mutation I1624E decreased the affinity of the IQ sequence
for CaM ⬃100-fold and abrogated CDI of L-type Ca2⫹ channels expressed in Xenopus oocytes (325, 326). Likewise, the
mutation I1624A abolished CDI of CaV1.2 channels expressed in oocytes (325), whereas the mutated I1624A CaV1.2
channels expressed in HEK cells showed essentially normal
CDI but an attenuated VDI (18).
HOFMANN ET AL.
(147). In the other model, the CaV␤2 gene was truncated at
Pro 501 (35). This truncation removes COOH-terminal phosphorylation sites for CaMKII, PKA, and PKG (35). The
CaV␤2-Stop mice showed normal basic behavior, normal
CDF, and normal adrenergic channel regulation, indicating
that the COOH-terminal phosphorylation sites of CaV␤2 are
not necessary for CDF or adrenergic regulation in the murine
heart.
CDF serves to potentiate Ca2⫹ influx through CaV1.2 channels during repeated activity which contributes to increased
force-frequency relationship of the heart muscle during exercise (186, 247). CaM is required for CDF. CDF is blocked by
Ca2⫹-insensitive CaM or by inhibition of CaMKII (229, 294,
325, 326). Single amino acid mutation of the IQ motif (I/E)
abrogated CDF of CaV1.2 expressed in Xenopus oocytes
(326). CMs from I/E mice lacked CDF (235). Facilitation was
completely abolished by dialysis with either of two inhibitory
peptides of CaMKII (320). Internal dialysis of rabbit myocytes
with synthetic inhibitory peptides derived from the pseudosubstrate peptide (amino acids 273–302) and CaM binding
peptide (amino acids 291–317) sequence blocked enhancement of CaV1.2 currents (6). CDF was also reduced in mice
deficient in CaMKII␦, which indicates that inhibition of physiological CaMKII activity in CMs may significantly affect
Ca2⫹ influx at fast heart rates by reduction of CDF (313).
In an elegant study, Oliveria et al. (222) suggested that, in
cultured hippocampal neurons, CDI of CaV1.2 currents is mediated by the CaM-dependent phosphatase calcineurin and
dephosphorylation of the CaV1.2 channel complex, whereas
CDF is caused by CaMKII-dependent phosphorylation of
the CaV1.2 channel complex. Calcineurin was scaffolded to
CaV1.2 by the AKAP 79/150. Interestingly, interfering with
calcineurin by addition of cyclosporin A inhibited CDI. A similar effect of cyclosporin was observed in an expressed channel
that contained the Timothy mutation at Ser 439 (80). Cyclosporin facilitated phosphorylation of Ser 1517 and mode 2
gating that is characteristic for CDF. Mutation of Ser 1517 to
alanine abolished this cyclosporine effect (80). Similar conclusions were published by Cohen-Kutner et al. (56). These authors found that VDI depended on calcineurin. Together,
these experiments support the notion that CaMKII contributes to CDF, whereas calcineurin contributes to CDI.
CaMKII induces a channel-gating mode that is characterized
by frequent, long openings of L-type Ca2⫹ channels (74). The
CaV1.2␣1 and the CaV␤2 subunit are substrates for CaMKII
(2, 113, 135, 175). CaV1.2␣1 was shown to be phosphorylated at S1512 and S1570 by coexpression of CaMKII in HEK
293 cells (175). CaMKII was further shown to bind to the
CaV␤2-N3 subunit and preferentially to phosphorylate Thr
498 (113). Viral-induced overexpression of the mutated
CaV␤2-N3 subunit (T498A) ablated CaMKII-mediated CDF
in adult rat CMs (113) and rabbit (163). In contrast, CDF was
still observed in HEK 293 cells coexpressing a truncated
CaV1.2␣1 subunit and the CaV␤2-N4 subunit with a mutation
of this putative phosphorylation site (T500A) (175).
VIII. MUTATION IN GENES CODING FOR
VOLTAGE-GATED CaV1.2 Ca2ⴙ
CHANNELS
The in vivo significance of the proposed CaMKII phosphorylation sites for CDF was examined in two mouse models (30,
35). In one model (SF mice), S1512 and S1570 of the
CaV1.2␣1 subunit were mutated to alanine (30). CDF was
reduced in CMs from SF mice in a CaMKII-dependent manner. In agreement with the anticipated effect of CDF, i.e., prolongation of the plateau phase of the action potential at high
frequency (186), QT time was reduced at high frequency (30).
However, CDF was not completely abolished, suggesting that
an additional mechanism may be required for CDF. This
mechanism requires the IQ motif, because the I/E mutated
channel did not show CDF anymore (235). It is possible that
the second mechanism requires inactive CaMKII as suggested
for CaV2.1 channels in adult rat hippocampal pyramidal neurons which may be modulated solely by binding of CaMKII
314
The analysis of human diseases has identified mutations in
voltage-gated Ca2⫹ channels as cause of incomplete X-linked
congenital night blindness (CaV1.4), familial hemiplegic migraine (CaV2.1), ataxia disorders (CaV2.1), various murine
disorders (CaV2.1, ␣2␦, ␤, and ␥2 subunits), and epilepsy susceptibility (CaV3.2) (reviewed in Refs. 26, 182, 277). Mutation of the CaV1.2 ␣1 subunit has been associated with the
Timothy syndrome, the Brugada syndrome 3, and early repolarization syndrome (reviewed in Ref. 217). Timothy syndrome has been associated with the mutation G406R in exon
8A of the CaV1.2 gene (273). Macroscopic analyses of CaV1.2
channels carrying Timothy syndrome mutations (G406R) revealed a delayed CDI resulting in sustained depolarization (18,
272, 273). Homo- and heterozygous mice with a G406R mutation did not survive, whereas heterozygous G406R mice
with an inverted neomycin cassette in exon 8A showed distinct
behavioral abnormalities, in line with the core aspects of autism and autism spectrum disorders (10). The neomycin cassette left in exon 8A was supposed to reduce expression of
mutant exon 8, most likely by transcriptional interference.
Interestingly, oligomerization of wild-type CaV1.2 channels
with G406R CaV1.2 channels was shown to increase Ca2⫹
currents and the frequency of arrhythmogenic Ca2⫹ fluctuations in transfected rat ventricular myocytes (68). The latter
findings support the hypothesis that functional CaV1.2 channels are clustered and open in a cooperative manner (65).
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likely that VDF represents a CDF. In Xenopus oocytes, CDF of
CaV1.2␣1 was reduced by disruption of the IS6-AID linker and
loss of CaV␤ binding supporting a requirement of CaV␤ binding to the AID and an intact IS6-AID ␣-helix for CDF (88).
Overall, facilitation of CaV1.2 channels is likely to involve
multiple processes including the voltage protocol, the amount
of Ca2⫹ entry, the CaV␤ association, and phosphorylation (4,
259).
IN VIVO FUNCTION OF L-TYPE CaV1.2 CALCIUM CHANNELS
Brugada syndrome has been associated with a loss-of-function
missense mutations in either the CaV1.2␣1 gene (A39V and
G490R, Brugada syndrome-3) or in the N4 encoding exon of
the CaV␤2 gene (S481L, Brugada syndrome-4) (8, 96). Recently, also mutations in the CaV␣2␦1 gene have been reported
in some Brugada patients (40). All these mutations have been
correlated with shorter QT intervals leading to sudden cardiac
death.
IX. CaV1.2 AND THE BRAIN
Neurotransmitter release, neuronal excitability and plasticity,
excitation-transcription coupling, synaptogenesis, and dendritic growth are regulated by Ca2⫹ influx via voltagedependent Ca2⫹ channels. LTP-induced changes probably
serve as cellular mechanism for learning and memory (153,
187, 191). Critical for the induction and maintenance of hippocampal LTP is a rise of the postsynaptic cytosolic Ca2⫹
concentration (22, 187, 324) usually caused by the opening of
NMDA receptor, a receptor-triggered Ca2⫹ channel. CaV1.2
channels were reported to be present on the nerve soma and
pre- and postsynaptic in rat hippocampus (289). CNS neurons
express two L-type channels, the CaV1.2 and the CaV1.3
(129). A comprehensive CaV channel expression profile of
mouse brain and cultured hippocampal neurons has recently
been presented (253). The CaV1.3 and CaV1.2 channels are
necessary to mediate cocaine-induced GluA1 trafficking in the
nucleus accumbens (252). Neuronal CaV1.2 activity affects
not only intracellular trafficking but inhibits axon growth in
DRG neurons (77).
Conditioned fear responses are a popular paradigm to study
associative learning and anxiety disorders. Amygdalae express
both L-type calcium channels (129). Early studies used L-type
Ca2⫹ channel blockers to show that consolidation and extinction depended on the activity of L-type Ca2⫹ channels (19, 44,
45, 296). These studies were limited, because the compounds
inhibited several distinct CaV1 channels and produced a protracted stress response (44, 296). Two groups used neuron
specific deletion of the cacna1 gene to delineate the potential
importance of CaV1.2 for fear consolidation and extinction. It
was reported that deletion of CaV1.2 did not affect these parameters (196). However, this negative result was most likely
caused by an upregulation of the GluR1 receptor that allows
postsynaptic Ca2⫹ influx as shown in an elegant study by
Langwieser et al. (171). McKinney et al. (196) did not investigate if the deletion of CaV1.2 induced the upregulation of
other proteins involved in synaptic Ca2⫹ homeostasis. Langwieser et al. (171) showed that impairment of the CaV1.2
channel decreased fear acquisition. Direct injection of verapamil or nifedipine in the basolateral nucleus of the amygdale
indicated that the L-type calcium channel blockers interfered
with extinction memory (61). These latter experiments prevented the stress response, but did not distinguish between
CaV1.3 and CaV1.2 channels. On the other hand, forebrain-specific deletion of the CaV1.2␣1 subunit gene increased fear behavior as measured by the time spent in an
open arm (173).
X. CaV1.2 AND SMOOTH MUSCLE
Smooth muscle (SM) is widely distributed in the organism and
is the major constituent of blood vessels, intestines, urinary
bladder, penis, and uterus. They show a great diversity with
respect to embryological, anatomic, or physiological belongings (90). SM can be separated into phasic versus tonic contracting SM. Phasic SM are predominantly present in the gastrointestinal systems and characterized by rhythmic contractile activity. Tonic SMs mainly exist in large arteries and build
up the myogenic tone which represents a long-lasting contraction. Blood vessels exhibit both phasic and tonic contractions.
For example, portal venous smooth muscle is regarded as a
prototypical phasic SM (9), whereas small arteries exhibit a
mixture of tonic contractions and phasic contractile activity
(reviewed in Ref. 117). The most important task of SM is to
contract in response to physiological needs. Contractile force
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A polymorphism rs4765913 in the cacna1c (CaV1.2 ␣1) gene
is strongly associated with familial bipolar disorder including
an increased risk for schizophrenia (86, 121, 265). Interestingly, mice with a reduced neuronal expression of the CaV1.2
␣1 protein show a reduced depressive behavior (150), supporting the human findings. Furthermore, observational fear
learning is reduced after deletion of the CaV1.2 gene in the
murine nucleus anterior cingulate cortex (145). The CaV␤4
protein is highly expressed in the cerebellar Purkinje cells and
acts as a repressor recruiting platform to control neuronal gene
expression (280). CaV␤4 appears to be associated most likely
with the CaV2.1 channel. The lethargic mouse (lh/lh) has a
four-nucleotide insertion into a splice donor site of the CaV␤4
gene leading to frameshift and a truncated protein with loss of
the CaV␣1-binding site (43). Neither full-length nor truncated
CaV␤4 protein is expressed in lh/lh mice (194). This lethargic
mutation induces ataxia, focal motor seizures, and cortical
spike wave epilepsy and defective cell-mediated immune responses. However, unaltered P-type currents in dissociated
lethargic Purkinje neurons indicate that these CaV2.1␣1Acontaining channels retain regulation by other CaV␤ subunits
(41). In the cochlear hair cell, the CaV␤4 subunit associates
most likely with the CaV1.3 channel (165). Deletion decreases
peak ICa,L. The CaV␤3 prevails in the cochlear outer hair cells
and associates there with the CaV1.3 channel. Again, its deletion decreases peak ICa,L (165).
Hippocampus and neocortex-specific deletion of the cacna1C
gene (CaV1.2 ␣1) induced a defect in long LTP, spatial learning, and induction of nuclear gene transcription (203),
whereas deletion of the CaV1.3 gene was without effect on
these parameters (55). In very elegant work, it was shown that
Ca2⫹ flowing through activated postsynaptic CaV1.2 channels
regulated the phosphorylation of CREB (306). Surprisingly,
CaV2.1 channels were ⬃10-fold less effective in signaling to
the nucleus than were CaV1.2 channels for the same bulk
[Ca2⫹]i increase.
HOFMANN ET AL.
The major form of CaV1.2␣1 subunit in smooth muscle is the
CaV1.2b isoform (28) (see also sect. IIIA). The importance of
the CaV1.2 channel for smooth muscle function has been revealed in a mouse model, in which the CaV1.2␣1 subunit was
selectively deleted in smooth muscle (206) using a Cre/lox site
specific recombination system (164). These mice died within
21 days after Cre activation due to a paralytic ileus (298).
Mean arterial blood pressure was reduced by ⬃30 mmHg in
these mice (206). The development of myogenic tone in response to intravascular pressure according to Bayliss (21) was
abolished (206). About 50% of the phenylephrine-induced
resistance was lost in hindlimb perfusion experiments using
this mouse line, indicating that CaV1.2␣1 is essential for the
hormonal regulation of blood pressure and development of
myogenic tone (206). Rhythmic contractions of the small and
large intestine of these mice were absent, and hormoneinduced contractions were diminished resulting in the observed reduced feces excretion (298). These mice exhibited
also a severely reduced micturition and bladder hypertrophy
reminiscent to the symptoms of bladder outlet obstruction
(299). In addition, carbachol-induced contractions were diminished by ⬃90%, probably related to the loss of the PKCmediated regulation of the CaV1.2 channel (141, 299). Together, these studies strongly support the notion that the
CaV1.2 channel is critical for the contractile function of various types of smooth muscle.
Ca2⫹ released from intracellular stores (Ca2⫹ sparks) has been
implicated in vascular SM relaxation (305). Ca2⫹ sparks stimulate nearby Ca2⫹-activated K⫹ channels (BK) that hyperpolarize the membrane and close L-type Ca2⫹ channels and,
thus, play an important role in the regulation of arterial diameter and appear to be involved in the action of a variety of
vasodilators like NO (305). CaV1.2␣1 gene inactivation reduced Ca2⫹ spark frequency and amplitude by ⬃50% and
80%, respectively, in SM cells from tibial and basilar arteries
(81). In addition, these cells show lower global cytosolic Ca2⫹
levels and reduced sarcoplasmic reticulum Ca2⫹ load. Fixing
intracellular Ca2⫹ by EGTA-AM restored global Ca2⫹ levels
316
in the CaV1.2␣1-deficient SM cells and restored spontaneous
transient outward currents (STOC) frequencies. Elevating cytosolic Ca2⫹ levels reversed the effects completely. This study
provided evidence that the local and tight coupling between
the CaV1.2 channels and ryanodine receptors as observed in
cardiac CMs is not required to initiate Ca2⫹ sparks in arterial
smooth muscle. Instead, CaV1.2 channels contribute to global
cytosolic [Ca2⫹]i, which in turn influences luminal SR calcium
and thus Ca2⫹ sparks. Together, this study supports the notion that the CaV1.2 channel is not only critical for the contractile function but also mediates partially dilatory responses
in smooth muscle.
The CaV1.2 channel has been implicated to function as
metabotropic receptor in arterial smooth muscle (293).
In recent years, a new SR Ca2⫹ release mechanism, depolarization-induced Ca 2⫹ release, has been postulated
(66). This pathway involves the CaV1.2 channel as membrane potential sensor that induces sarcoplasmic reticulum Ca2⫹ release through G protein and phospholipase C
activation followed by stimulation of the RhoA/RhoAassociated kinase pathway (84). The ion-conducting
property of CaV1.2 is not used in this pathway. Deletion
of CaV1.2␣1 gene in smooth muscle abolished this pathway (83) in line with its potentially functional relevance
for hormone-induced SM contraction.
SM was shown to express mRNA for all CaV␤ subunits, but
only two of the four CaV␤ subunits, namely, CaV␤2 and
CaV␤3, were clearly detected at the protein level (123, 137,
210). The role of CaV␤2 in smooth muscle could so far not be
clarified in a mouse model, since global deletion of CaV␤2 was
embryonic lethal because of cardiovascular dysfunction (302).
In contrast, CaV␤3-deficient mice show no major cardiovascular phenotype in the absence of a high-salt diet (210). In ileum
SM cells, deletion of CaV␤3 has only subtle effects on L-type
Ca2⫹ current and was not required for spontaneous and potassium-induced contraction (123). However, it has been suggested that CaV␤3 might have a function beyond being a Ca2⫹
channel subunit (23, 123) (see also sect. X).
XI. CaV␤2 AND CaV␤3 KNOCKOUT MICE
The cardiac CaV1.2␣1 subunit is associated with the CaV␤2
subunit (137, 181, 239), whereas the non-cardiac CaV1.2 ␣1
channel may be associated with the CaV␤2 and/or the CaV␤3
subunit (23, 123, 208, 209). The CaV␤3 subunit like CaV␤1,
CaV␤2, and CaV␤4 regulates also the function of the CaV1.3
channel and the neuronal CaV2.1 and CaV2.2 channel (207,
208, 216, 278). Inactivation of the CaV␤2 gene leads to death
at embryonic day 9.5 (13, 302). The same phenotype was
observed after cardiomyocyte-specific CaV␤2 gene deletion in the embryo (302). Cardiac-specific inactivation of
the CaV␤2 gene in adult mice results in viable animals and
a slightly reduced CaV1.2 current (⫺25%) (198). Extracardiac inactivation of CaV␤2 affected the b-wave of the
electroretinogram (13).
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is mainly determined by the amount of phosphorylated myosin light chain (MLC), which is regulated by the balance between the activities of the MLC kinase (MLCK) and the myosin phosphatase (MLCP) (211, 250, 271). Contraction of SM
can be initiated in two distinct ways: 1) increasing [Ca2⫹]i
through increased Ca2⫹ influx or Ca2⫹ release from the endoplasmatic reticulum thereby activating MLCK, or 2) activation of second messenger systems that inhibit MLCP activity
thereby sensitizing the myofilaments to calcium activation.
Ca2⫹ influx through L-type Ca2⫹ channels has been accepted
to be the major source for the increase in [Ca2⫹]i leading to
contraction. CaV1.2 channels play a particularly important
role in smooth muscle reactivity and tone at least in the microcirculation (60). The important role of the SM CaV1.2 channel
is generally accepted. High blood pressure is successfully
treated with DHPs that inhibit SM CaV1.2 channels.
IN VIVO FUNCTION OF L-TYPE CaV1.2 CALCIUM CHANNELS
XII. CaV1.2 AND SECRETION
Early studies identified Ca2⫹ as intracellular coupling agent in
the process of stimulus-secretion coupling (72). Ca2⫹ influx
mediated by CaV channels triggered the secretion of hormones
from excitable endocrine cells, like the pancreatic ␤-cells (318)
or the chromaffin cells from the adrenal gland (102). The
contribution of single CaV channel types to this Ca2⫹ influx
varies among species, with the CaV1.2 channel being the main
mediator of Ca2⫹ influx in ␤-cells and chromaffin cells from
the mouse but not from humans (36, 99).
The predominant CaV channel form expressed in murine pancreatic ␤-cells are the CaV1.2 and CaV1.3 channel (249).
␤-Cell specific deletion of the CaV1.2 gene decreased fastphase insulin release (256), a phenotype reproduced by the
mice expressing the DHP-insensitive CaV1.2 (264). In contrast, ␤-cells from CaV1.3 knockout mice showed almost unaltered insulin secretion, probably due to a compensatory
overexpression of CaV1.2 channels (215). The CaV1.3 channel
has been associated with insulin release in humans (242). Surprisingly, deletion of the CaV␤3 subunit had a strong effect on
insulin secretion in mice. The lack of the CaV␤3 subunit facilitated insulin release, but only at high glucose, eventually because the CaV␤3 subunit inhibited inositol trisphosphatestimulated Ca2⫹ release, an effect not mediated by the CaV1.2
channel (23). Interestingly, patients with the Timothy syndrome frequently showed hypoglycemia pointing to a possible
increase in ␤-cell-mediated insulin secretion due to the enhanced Ca2⫹ influx through the modified CaV1.2 channel that
displays a reduced voltage-dependent inactivation (273).
Similar to pancreatic ␤-cells, murine chromaffin cells from the
adrenal gland express predominantly CaV1.2 and CaV1.3
channels (188). Unfortunately, the effect of CaV1.2 gene inactivation in chromaffin cells could not be studied due to the
lethality of the CaV1.2 knockout mice (260). Inactivation of
the CaV1.3 gene resulted in a reduced spontaneous firing rate
in chromaffin cells, indicating a reduced catecholamine secretion (188). Recently, mutations of the human CaV1.3 channel,
which shifts the voltage dependence of activation to more hyperpolarized potentials, have been associated in adrenal glomerulosa cells with primary hyperaldosteronism leading to
hypertension (254).
Currents through CaV1.2 channels can be activated by cAMP/
PKA signaling cascade in both ␤-cells and chromaffin cells
(155, 185). Furthermore, cGMP decreased L-type current in
chromaffin cells (185). PKA and PKG modulated CaV1.2 and
CaV1.3 currents in the same direction (185). The molecular
mechanism of this regulation has not been studied and may
differ from the ␤-adrenergic regulation of the cardiac CaV1.2
channel.
XIII. CONCLUSION
The generation of various mouse lines with defects in genes of
the CaV1.2 channel subunits has fundamentally extended the
knowledge about the wide-ranged biological functions of
CaV1.2 channel-dependent signaling processes and fruitfully
complemented previous theories obtained solely by pharmacological tools. However, many potential functions have
probably not yet been revealed. Limitations of the genemodifying approaches using mutant mice may involve the
functional redundancy of CaV1.2 channel isoforms, induction
of compensatory processes, or early embryonic lethality. To
reduce these problems, researchers have started to generate
mice with amino acid specific mutations and/or mutations related to specific CaV1.2 subunit isoforms. With the introduction of these probably more valuable mouse lines, it may be
possible to get new interesting insights into the specific role of
CaV1.2 channel subunits in vivo.
ACKNOWLEDGMENTS
Address for reprint requests and other correspondence: F.
Hofmann, FOR 923, Institut für Pharmakologie und
Toxikologie, TU München, Biedersteiner Str. 29, 80802
München, Germany (e-mail: [email protected]).
GRANTS
The work of the authors was supported by grants from
Deutsche Forschungsgemeinschaft and Fond der Chemischen
Industrie.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by
the authors.
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