Hallmarks of the channelopathies associated with L-type calcium channels : A focus on the Timothy mutations in Cav1.2 channels

Biochimie 93 (2011) 2080e2086

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Hallmarks of the channelopathies associated with L-type calcium channels : A focus on the Timothy mutations in Cav1.2 channels Isabelle Bidaud a, b, c, Philippe Lory a, b, c, * a

CNRS, UMR-5203, Institut de Génomique Fonctionnelle, F-34000 Montpellier, France INSERM, U661, F-34000 Montpellier, France c Universités de Montpellier 1 & 2, UMR-5203, F-34000 Montpellier, France b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 April 2011 Accepted 19 May 2011 Available online 31 May 2011

Within the voltage-gated calcium channels (Cav channels) family, there are four genes coding for the L-type Cav channels (Cav1). The Cav1 channels underly many important physiological functions like excitatione contraction coupling, hormone secretion, neuronal excitability and gene transcription. Mutations found in the genes encoding the Cav channels define a wide variety of diseases called calcium channelopathies and all four genes coding the Cav1 channels are carrying such mutations. L-type calcium channelopathies include muscular, neurological, cardiac and vision syndromes. Among them, the Timothy syndrome (TS) is linked to missense mutations in CACNA1C, the gene that encodes the Cav1.2 subunit. Here we review the important features of the Cav1 channelopathies. We also report on the specific properties of TSeCav1.2 channels, which display non-inactivating calcium current as well as higher plasma membrane expression. Overall, we conclude that both electrophysiological and surface expression properties must be investigated to better account for the functional consequences of mutations linked to calcium channelopathies. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Calcium channelopathies Timothy syndrome L-type calcium channels Calcium current

1. From calcium channels to calcium channelopathies Voltage-gated calcium channels (Cav channels) mediate calcium entry into a wide variety of electrically excitable cells, including cardiac and skeletal muscle cells, neurons, endocrine and sensory cells, thereby controlling numerous physiological processes. Cav channels are classified according to their biophysical and pharmacological properties as well as considering the sequence homology of the various pore subunits (Cav) identified to date (Fig. 1). Ten genes named CACNA1A-S [1] encode the pore-channel Cav subunits that generate the L-types (Cav1: four members), the neuronal P/Q-, N- and R-types (Cav2: three members) and the T-type (Cav3: three members). Cav1 and Cav2 subunits are high voltage-activated (HVA) channels, which also include auxiliary subunits, b (four genes: b1, b2, b3 and b4), a2d (four genes) and g (eight genes) that contribute as regulatory components to these HVA channels [2]. In contrast to HVA channels, it is still unclear whether the low voltage-activated (LVA) channels T-type Cav3 channels associate to any auxiliary subunit [3]. In the early 90’s calcium channel genes were mapped on human chromosomes [1] and mutations in these genes were subsequently * Corresponding author. CNRS, UMR-5203, Institut de Génomique Fonctionnelle, 141 rue de la Cardonille, F-34000 Montpellier, France. Tel.: þ33 434 359 251; fax: þ33 467 542 432. E-mail address: [email protected] (P. Lory). 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.05.015

identified [4]. Identification of mutation(s) in a calcium channel gene is instrumental to qualify the related disease(s) as a “channelopathy”. However, the channelopathy nature of these diseases can be variable, ranging from autosomal dominant monogenic diseases to disease-susceptibility genes with incomplete penetrance. Studies of calcium channelopathies offer two main opportunities : (i) the study of the mutant channels can support the diagnosis of the disease (genetic testing), as well as its clinical phenotyping (episodic manifestations) and ultimately the design of innovative therapeutic strategies; and (ii) the study of channel mutations also illuminate relationships between channel structure and function, and can reveal diverse and unexpected physiological roles for these channels. 2. L-type calcium channels - Cav1 channels L-type Cav1 channels share high sensitivity to the ‘calcium channel blockers’ dihydropyridines (nifedipine, amlodipine), phenylalkylamines (verapamil) and benzothiazepines (diltiazem). Four genes generate the pore subunits Cav1.1 : CACNA1S; Cav1.2 CACNA1C; Cav1.3 : CACNA1D and Cav1.4: CACNA1F (see Fig. 1). The detailed electrophysiological and pharmacological properties of these various Cav1 channels and their splice variants can be found in previous reviews [2]. Here we discuss the expression profile of the Cav1 subunits, as it is highly relevant for a better interpretation of the related channelopathies (summarized in Fig. 1). The Cav1.1 subunit is

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Fig. 1. General properties of voltage-gated calcium channels (Cav channels) including the gene names and the corresponding Cav subunits. HVA stands for high-voltage activated channels (L- P/Q-, N- and R-types) and LVA stands for low-voltage activated (T-types). The expression profile and the diseases related to each channel type, the ‘channelopathies’ column, are indicated. Only the channel properties and channelopathies related to the L-type Cav1 channels are discussed in the text.

expressed specifically in skeletal muscle transverse tubules where it serves as a voltage sensor to induce calcium release from the sarcoplasmic reticulum/ryanodine receptor (Ryr1) compartment and to elicit excitationecontraction coupling. The onset of Cav1.1 expression is associated with skeletal muscle differentiation [5]. The Cav1.2 subunit is widely expressed. It plays a crucial physiological role in the contractile heart where it represents the primary calcium entry mechanism for excitationecontraction coupling. Expectedly, the knockout of the CACNA1C gene in mouse leads to major cardiac defects and premature death during embryonic life. Cav1.2 is also expressed in many other cell types, such as smooth muscle cells, neurons, pancreatic b-cells [6] and T-lymphocytes [7,8] and thereby contributing to many diverse physiological functions. The Cav1.3 channel is expressed together with Cav1.2 in several tissues, especially in the sinoatrial node, in neurons, in chromaffin cells and in pancreatic b-cells. It is particularly involved in cardiac pacemaker activity, as evidenced in homozygous Cav1.3 knockout mice that display significant bradycardia and arrhythmias [9], as well as hearing deficit due to the lack of L-type currents in cochlear inner hair cells and degeneration of outer and inner hair cells [10]. The Cav1.4 subunit exhibits an expression profile restricted to the retina, especially at ribbon synapses of retinal photoreceptors and in bipolar cells where it contributes to neurotransmitter release. Expression of Cav1.4 may also possibly occur in lymphoid tissue and mast cells. 3. L-type calcium channelopathies 3.1. Hypokalemic periodic paralysis (HypoPP1) Mutations in the gene CACNA1S coding the skeletal muscle Cav1.1 subunit are linked to the muscle disease, hypokalemic periodic paralysis (HypoPP1) and account for approximately 55%e 70% of all HypoPP forms. Most CACNA1S mutations are arginine substitutions within the voltage-sensor (S4) segments of domains II

and IV [4] (Fig. 2). Although suggestive of biophysical alterations, these mutations of arginine residues in S4 segment moderately affect calcium current density with mild changes in the gating properties [11,12]. Recently, it was suggested that arginine (gating charge) mutations in S4 segment could result in a gating pore current capable to enhance membrane depolarization and consequently affect action potential [13]. Unfortunately, the low level of Cav1.1 channel expression in heterologous expression systems has not allowed, to date, detection of a gating pore current for Cav1.1eHypoPP1 mutants. Moreover, it remains unclear why the concentration of circulating potassium drops in HypoPP patients. Future studies should examine whether HypoPP1 mutations in Cav1.1 could effectively cause gating pore current and investigate how gating pore mutations could trigger reduction in serum potassium. 3.2. Malignant Hyperthermia Susceptibility type 5 (MHS5) Mutations in the CACNA1S gene account for only 1% of all forms MHS. A R1086H/C mutation within the intracellular IIIeIV linker of the CaV1.1 subunit is linked to Malignant Hyperthermia Susceptibility type 5 (see Fig. 2) [14,15]. This suggests that the IIIeIV linker of CaV1.1 interacts with the ryanodine receptor (RyR1) and heterologous expression experiments have revealed that the R1086H mutation of CaV1.1 enhances calcium release in skeletal muscle cells [15]. Recently, two novel MHS5 mutations were identified. One is located in the S4 segment of domain I, R174W [16]. The other one was found in the extracellular S5eS6 linker in the domain IV (T1354S) of the Cav1.1 channel [17]. It is unexpected that mutations in a transmembrane segment or in an extracellular region could directly influence RyR1 calcium release. However, a change in the activation properties of the Cav1.1 channel, as identified for T1354S [17], could lead to increased RyR1 calcium release under pharmacological trigger and induce the MHS phenotype.

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Fig. 2. Schematic illustration of the calcium channelopathies identified to date for CACNA1S (Cav1.1) and CACNA1C (Cav1.2). The four domains (I to IV) containing the six transmembrane segments (1 to 6) are indicated on the upper part of the figure (Cav1.1 channel). The intracellular linker between domains I and II contains the b-binding domain (see [34] for further details). The HypoPP1 mutations (R528H/G, V876E R897S, R900S, 1239H/G) and the MHS5 mutations (R174W, R1086H/C, T1354S) are presented in the upper part (Cav1.1 channel). The BrS3 mutations (A39V, G490R) and TS mutations (G402S, G406R) are presented in the lower part (Cav1.2 channel).

3.3. Timothy syndrome (TS) CACNA1C (Cav1.2) is the only gene known to be associated with Timothy syndrome (TS). It is a rare childhood multiorgan disorder with less than twenty TS patients identified to date worldwide. The average survival is 2e3 years and few TS patients have survived to puberty thus far. In 2004, Splawski and colleagues [18,19] have described two mutations, G406R and G402S (exon 8 in Cav1.2, see Fig. 2), that are linked to TS. It is a complex physiological and developmental disorder that affects multiple organs systems, including the heart (QT interval prolongation), dysmorphic facial features and syndactyly digits, brain (developmental delays and autism) and the immune and secretory systems. TS is characterized by a severe form of the Long QT syndrome, designated LQT8 [20]. The cardiac defects in TS also include congenital cardiac abnormalities and hypertrophic or dilated cardiomyopathy. This is in direct link with the expression of Cav1.2 channels in cardiac myocytes: Cav1.2 channels being the so-called “cardiac L-type” calcium channels [2]. The onset of cardiac ventricular fibrillation is the major cause of death. Reduced immunity, intractable hypoglycemia, as well as autism and autism spectrum disorder (ASD) are also identified in TS patients. Indeed, non-arrhythmic death in TS patients likely arises from secondary complications, especially severe bronchial and sinus infections due to weakened immune response. It is important to note here that Cav1.2 is expressed in lymphocytes [8,21], as well as in pancreatic b-cells [22]. Cav1.2 is also expressed in the brain and the TS mutation of Cav1.2 could likely impact neuron development and/or excitability. Interestingly, there is increasing information for an association of CACNA1C with psychiatric disorders [23]. However, besides TS there is no evidence to date for a direct link between CACNA1C and autism.

The Cav1.2 channel inactivation is impaired with TS mutations leading to a sustained entry of calcium into the cardiac myocytes during an action potential (AP). Consequently the AP duration is longer, which is in direct link with a longer QT interval. The calcium blocker verapamil has been shown to control cardiac arrhythmias in a TS patient [24]. The TS mutation G406R affects a glycine residue at the border between segment 6 of domain I and the loop between domains I and II, and its study in heterologous expression has revealed a significant loss of voltage-dependent inactivation [18,19]. Because the properties of TSeCav1.2 channels are fascinating, we present and discuss some additional features of TSeCav1.2 channels below in this article. 3.4. Brugada syndrome (BrS3) While TS mutations can be presented as gain of function mutations of Cav1.2, Bragada mutations (A39V and G490R, see Fig. 2) are loss of function [25]. BrS is an inherited cardiac arrhythmia syndrome, which is associated with high risk of ventricular fibrillation and sudden death. Electrocardiograms of these patients exhibit shorter QT intervals with no or very mild other phenotypes. 3.5. Sinoatrial node dysfunction and deafness (SANDD) Recently a loss of function mutation was identified in the CACNA1D gene (Cav1.3) of a large Pakistani family with deafness. The mutation occurs in a Cav1.3 splice variant that is preferentially expressed in inner hair cells and in the sinoatrial node [26]. The mutation results in the insertion of a glycine residue at position 403 in the transmembrane helix IS6 (exon 8B) and is named p403_404insGly. The mutant channel appears to be correctly expressed at the cell

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Fig. 3. A. Typical calcium current traces (recorded in 2 mM external Ca2þ) obtained in a HEK-293 cell transfected with plasmids coding the Cav1.2 (wt), the b3 and the a2d1 subunits, for test pulses at 40 mV, 30 mV, 10 mV, 0 mV and þ30 mV from a holding potential (HP) at 80 mV. B. Typical calcium current traces obtained in a cell transfected with plasmids coding the TSeCav1.2 (G406R), the b3 and the a2d1 subunits. C. Average current-voltage curves for wt (filled squares, n ¼ 6) and G406R (open squares, n ¼ 7). Statistical difference was tested by using Student’s unpaired t-test : *, p < 0.05 and **, p < 0.01. D. Typical current recordings obtained in a Cav1.2- (wt), b3- and a2d1- transfected cell. E. Similar current recordings obtained in a cell expressing the Cav1.2 (G406R), b3 and a2d1 subunits. F. Normalized current amplitudeinterpulse plot for wt (filled squares, n ¼ 5) and G406R (open squares, n ¼ 5). Note that scaling of the two traces (inset) reveals no difference in the recovery timecourse between wt and G406R.

surface but is a non-conducting variant of Cav1.3. Only patients homozygous for the mutation exhibit SANDD. Most SANDD patients showed pronounced bradycardia as well as increased heart rate variability, which is in good agreement with the critical role of Cav1.3 in regulating the heartbeat in mouse (discussed above). 3.6. Congenital stationary night blindness type 2 (CSNB2) CSNB2 is a recessive non-progressive retinal disorder linked to mutations within CACNA1F (Cav1.4). Numerous CSNB2 mutations have been identified to date in CACNA1F, including mutations in splice acceptor and donor sites. Half of the CACNA1F mutations in the coding region are nonsense and frameshift mutations predicted to cause protein truncation and loss of Cav1.4 channel function (reviewed in [27]). The other Cav1.4 mutations are missense mutations, leading to functional channels that show various electrophysiological alterations [27,28]. These CSNB2 mutations may also affect Cav1.4 folding and trafficking. Another recessive retinal disease, the X-linked cone-rod dystrophy type 3 (CORDX3) that share some clinical features with CSNB2, has also be linked to a mutation in CACNA1F leading to altered splicing [29]. It is predicted that this mutation results in non functional CaV1.4 channels.

4. Specific properties of Cav1.2 channels carrying a Timothy mutation We report here on the electrophysiological and surface expression properties of TSeCav1.2 channels (G406R), as well as on the ability of the various b subunits to modulate these properties. Four genes named CACNB1-4 encode the b1, b2, b3 and b4 subunits [2]. In addition to modulation of the biophysical properties of Cav1.2 channels, the b subunits play an important role in controlling expression of the Cav1.2 subunit at the plasma membrane. For the experiments presented below, detailed procedures regarding cell culture and transfection, electrophysiological recordings and analysis, as well surface and total expression measurements can be found elsewhere [30,31]. Briefly, HEK-293 cells were transfected with 0.25 mg of Cav1.2 (wt or G406R) 0.125 mg of auxiliary subunits a2d1 and b1a, b1b, b2, b3 or b4 subunits, as well as 0.01 mg of GFP plasmids. The missense mutation G406R was introduced into the rat Cav1.2 cDNA (M67515) using PCR mutagenesis. Insertion of a hemagglutinin (HA) epitope into the extracellular S5-H5 loop of domain II of a rat Cav1.2 cDNA has been previously described [32].

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4.1. Electrophysiological properties of G406ReCav1.2 subunit Patch-clamp experiments were performed to characterize the electrophysiological properties of the mutant G406R of the Cav1.2 subunit coexpressed with the regulatory subunits, b3 and a2/d1, in HEK-293 cells (Fig. 3). In our recording conditions (2 mM Ca2þ) we observed a marked slowing of the calcium current kinetics for the G406ReCav1.2 subunit at all the depolarizing pulses tested, as compared to that generated by the wt Cav1.2 subunit (Fig. 3A and B). These data are in agreement with those reported originally by Splawski et al. (2004) who described similar inactivation defects for the G406R mutant expressed together with the b2b subunit in Xenopus oocytes or in CHO cells [18,19]. Comparison of the currentevoltage relationship for G406R and wt shows a negative shift in the activation threshold of G406ReCav1.2 channels but no change in the peak current membrane potential (Vm), as well as in the reversal potential values (Fig. 3C). In addition, Fig. 3 (panels DeF) shows that recovery from inactivation is also significantly affected for G406ReCav1.2 channels, compared to wt Cav1.2 channels. Recovery from inactivation is an important mechanism that controls cellular excitability, as it sets up the ability of channels to be activated upon membrane repolarization. Fig. 3D shows that for wt Cav1.2 channels, a w50% (I/ICtrl) recovery in calcium current is obtained 150 ms after a nearly complete inactivation (Fig. 3D and F). Interestingly, G406ReCav1.2 current that inactivates by only w30% using a similar test-pulse (800 ms), presents an instantaneous recovery (I/ICtrl w90%; Fig. 3E and F) that is similar in time course to wt Cav1.2 current upon normalization (Fig. 3F, inset). The G406R mutation takes place at the very beginning of the intracellular loop between domains I and II (IeII loop) of the Cav1.2 subunit. The IeII loop of each high-voltage activated (HVA) Cav subunit includes an interaction domain (AID) where the b subunit binds [33]. The AID site is found 25 amino-acids downstream to the G406 residue and binding of the b subunit to this site is a molecular correlate for the marked effects of this subunit on HVA channels [34]. Whether the G406R missense mutation interferes with the b-subunit modulation of Cav1.2 channels is unravelled. Fig. 4A shows representative current traces recorded at 0 mV (HP-80 mV) on HEK-293 cells expressing the wt Cav1.2 subunit with various b-subunits. Inactivation kinetics was significantly slower when the b2a subunit was present. These data are in good agreement with the bulk of previous studies [34]. When the various b subunit isotypes were coexpressed with G406ReCav1.2 channels, no difference in inactivation kinetics could be observed (Fig. 4B) in our recording conditions (2 mM external Ca2þ). The inactivation time-course of current traces obtained for G406R channels with the b2a subunit were similar to that obtained in the absence of coexpressed b subunit or with other b subunit isotypes (Fig. 4B). These data are supportive of the report by Barrett and Tsien [35]. The percentage of remaining current after 800 ms was 68.1  3.4% for the b2a subunit (n ¼ 5), 69.1  4.3% for the b1b subunit (n ¼ 6), 66.2  3.3% for the b1a subunit (n ¼ 4), 61.3  3.2% for the b3 subunit (n ¼ 8) and 66.1  4.5% (n ¼ 6) in the absence of coexpressed b subunit (Fig. 4C). It has been shown Glycine 406 plays an important role in bringing the channel into the inactivated state and represents a common molecular determinant for both the voltage- and calciumdependent inactivation processes [36]. Here we report that the extremely slow inactivation kinetics of G406R currents cannot be reverted by any of the b subunit isoforms, e.g. the b1b subunit that induced the fastest inactivation kinetics in control condition (Fig. 4A). Similarly to that described for the fast inactivating mutants, E462R [36] and I1624A [35], the lack of effects of any of the b subunits on the inactivation process indicates that the G406R mutation blunts the b subunit modulation of the gating properties of Cav1.2 channels. The data support further that the inactivation

Fig. 4. Inactivation kinetics of wt and G406ReCav1.2 currents in the presence of various b subunit isoforms. A. Typical trace recordings obtained in HEK 293 cells cotransfected with wt Cav1.2 subunit and different b subunits. B. Typical trace recordings obtained in HEK 293 cells co-transfected with G406ReCav1.2 subunit and different b subunits. The a2d1 subunit is present in all conditions. C. Percentage of remaining calcium current after 800 ms pulse to 0 mV in HEK 293 cells co-transfected with G406ReCav1.2 subunit and different b subunits or no b subunit. No statistical difference using Student’s unpaired t-test among these conditions. (n) Number of cells.

kinetics properties of G406ReCav1.2 channels are significantly impaired and that this lack of inactivation is the main substrate for abnormal electrical activity in cells expressing this mutant Cav1.2 subunit. 4.2. Surface expression measurement of G406R/wt Cav1.2 subunit Another important feature of the b-subunit modulation of Cav1.2 channels is the ability of this intracellular subunit to act as a chaperone and to favour plasma membrane expression of the Cav1.2 subunit. In order to characterize the ability of the various b-subunits to the plasma membrane expression of G406ReCav1.2 channels, we took advantage of the Cav1.2 subunits (wt and G406R) being extracellularly tagged with HA to quantify membrane expression using luminometry [32,37]. HA-tagged Cav1.2 subunits were detected at

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cardiac myocytes. Recently, it was shown that spontaneously contracting TS myocytes display irregular calcium transients compared to control myocytes [40]. Here we show that the TS mutation G406R also favours Cav1.2 expression at the plasma membrane. We found similar results when cells were cultivated in the presence of 10 mM nitrendipine, a dihydropyridine inhibitor of Cav1.2 channels (not shown), indicating that the increase in surface expression with the G406R mutant is not related to the channel activity. This property appears to be intrinsic to the G406R mutant of Cav1.2 subunit as it also occurs without coexpression of auxiliary subunits and is conserved upon coexpression of any of the b-subunits. Increased surface expression of TSeCav1.2 channels is a direct consequence of the G406R mutation of Cav1.2 channels and occurs independently of the modulatory effect of the b subunit on Cav1.2 surface expression.

Fig. 5. Plasma membrane expression of wt and G406ReCav1.2 channels in the presence of various b subunit isoforms. Plasma membrane expression (non-permeabilized cells) was normalized over total expression (permeabilized cells). Histograms (mean  SEM) represent an average of >12 data samples obtained in 4 independent experiments. Paired student’s t-test was used to test statistical significance: * p < 0.05, ** p < 0.01.

the cell surface in non-permeabilized cells, while total expression of Cav1.2 subunits was measured in Triton-permeabilized cells. The results are presented as the ratio of plasma membrane expression over total expression (% of membrane/total expression) that reflects the ability of the Cav1.2 subunit to traffic to the plasma membrane in the various conditions (Fig. 5). First, it is important to note that all the b-subunits tested in our coexpression experiments significantly increase the amount of Cav1.2 subunits, wt and G406R, detected at the cell surface. The highest percentage of membrane expression was obtained with the b1b subunit: b1b (5.6 fold) > b4 (5.1 fold) > b3 (4.1 fold) w b1a (3.8 fold) > b2a (2.9 fold) for wt Cav1.2 subunits (Fig. 5). Similar results were obtained with G406ReCav1.2 subunits: b1b (5.3 fold) > b4 (4.8 fold) w b1a (4.7 fold) > b3 (3.9 fold) > b2a (3.0 fold). Second, a striking observation in these experiments is that the G406ReCav1.2 subunit is significantly more expressed at the plasma membrane (G406R/wt w þ30%) than the wt Cav1.2 subunit (Fig. 5). A similar percentage of increase in membrane expression is found with all the b-subunits tested : b1b (þ24%), b2a (þ35%), b3 (þ25%) and b4 (þ23%) and even higher with the b1a subunit (þ62%). Altogether, these data indicate that the mutation G406R intrinsically favours expression of the Cav1.2 subunit at the plasma membrane and that this effect is modulated further by the coexpression of b subunit. 4.3. The Timothy mutation G406R markedly affects the Cav1.2 subunit properties An important finding reported here is that the striking effects of the G406R mutation on the slowing of inactivation is retrieved with all the b-subunits tested. Our data further extend previous observations [18,35]. It indicates that this mutation that significantly impairs voltage- and calcium-dependent inactivation cannot be modulated or compensated by the expression of any of the b subunit isotypes. Interestingly this glycine residue is conserved in all Cav1 and Cav2 channels and its G-to-R mutation either in Cav2.3 channels or Cav2.1 channels similarly affects the gating properties of these channels [36,38]. Single channel analysis of the corresponding mutation in the rabbit Cav1.2 cDNA sequence (G436R) has revealed significant increase in long opening times corresponding to the mode 2 gating, possibly favoured by aberrant phosphorylation [39]. Besides the electrophysiological consequences of the TS mutations of Cav1.2 channels, it is still unknown to date whether the TS mutations impact Cav1.2 expression and calcium homeostasis in

4.4. Altered electrophysiological and plasma membrane expression properties: a mix of effects to account for calcium channelopathies Altogether we provide data that further extend our understanding of the functional consequence of the G406R natural mutation identified in patients with Timothy syndrome [18,20] and the biophysical alterations should not be considered anymore as the solely signature for TS mutations of Cav1.2 channels. Pathogenic mutations are often exhibiting complex phenotypic features that are not limited to electrophysiological alterations [4,41,42]. Folding and trafficking of ion channel proteins, subunit assembly and stability represent critical events in the ion channel’s life that are often underestimated, especially in the case of calcium channelopathies [4,31,43]. As there is increasing evidence for altered trafficking of mutated channels, it is tempting to suggest that future studies of ion channel diseases should also consider how ion channel proteins are trafficked or maintained at the plasma membrane. Acknowledgements This work was supported by Association Française contre les Myopathies, Agence Nationale pour la Recherche Grant ANR-07BLAN-0102-03 and Fédération pour la Recherche sur le Cerveau. We are grateful to Emmanuel Bourinet, Joël Nargeot, Edward PerezReyes, Jean Chemin, Arnaud Monteil and Alexandre Mezghrani for valuable comments on the manuscript. References [1] P. Lory, R.A. Ophoff, J. Nahmias, Towards a unified nomenclature describing voltage-gated calcium channel genes, Hum. Genet. 100 (1997) 149e150. [2] W.A. Catterall, E. Perez-Reyes, T.P. Snutch, J. Striessnig, International Union of Pharmacology. XLVIII. Nomenclature and structure-function relationships of voltage-gated calcium channels, Pharmacol. Rev. 57 (2005) 411e425. [3] S. Huc, A. Monteil, I. Bidaud, G. Barbara, J. Chemin, P. Lory, Regulation of T-type calcium channels: signalling pathways and functional implications, Biochim. Biophys. Acta 1793 (2009) 947e952. [4] I. Bidaud, A. Mezghrani, L.A. Swayne, A. Monteil, P. Lory, Voltage-gated calcium channels in genetic diseases, Biochim. Biophys. Acta 1763 (2006) 1169e1174. [5] I. Bidaud, A. Monteil, J. Nargeot, P. Lory, Properties and role of voltagedependent calcium channels during mouse skeletal muscle differentiation, J. Muscle Res. Cell Motil. 27 (2006) 75e81. [6] S. Moosmang, P. Lenhardt, N. Haider, F. Hofmann, J.W. Wegener, Mouse models to study L-type calcium channel function, Pharmacol. Ther. 106 (2005) 347e355. [7] M.D. Cabral, P.E. Paulet, V. Robert, B. Gomes, M.L. Renoud, M. Savignac, C. Leclerc, M. Moreau, D. Lair, M. Langelot, A. Magnan, H. Yssel, B. Mariame, J.C. Guery, L. Pelletier, Knocking down Cav1 calcium channels implicated in Th2 cell activation prevents experimental asthma, Am. J. Respir. Crit. Care Med. 181 (2010) 1310e1317. [8] B. Gomes, M. Savignac, M. Moreau, C. Leclerc, P. Lory, J.C. Guery, L. Pelletier, Lymphocyte calcium signaling involves dihydropyridine-sensitive L-type calcium channels: facts and controversies, Crit. Rev. Immunol. 24 (2004) 425e447.

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