Asymmetric contribution of a selectivity filter gate in triggering inactivation of CaV1.3 channels.

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultrahighly conserved tryptophan residues in the interdomain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudotetrameric CaV pore domain for feedback inhibition.

Asymmetric Contribution of a Selectivity Filter Gate in Triggering Inactivation of CaV1.3 Channels.

Voltage-dependent and Ca2+-dependent inactivation (VDI and CDI, respectively) of CaV channels are two biologically consequential feedback mechanisms that fine-tune Ca2+ entry into neurons and cardiomyocytes. Although known to be initiated by distinct molecular events, how these processes obstruct conduction through the channel pore remains poorly defined. Here, focusing on ultra-highly conserved tryptophan residues in the inter-domain interfaces near the selectivity filter of CaV1.3, we demonstrate a critical role for asymmetric conformational changes in mediating VDI and CDI. Specifically, mutagenesis of the domain III-IV interface, but not others, enhanced VDI. Molecular dynamics simulations demonstrate that mutations in distinct selectivity filter interfaces differentially impact conformational flexibility. Furthermore, mutations in distinct domains preferentially disrupt CDI mediated by the N- versus C-lobes of CaM, thus uncovering a scheme of structural bifurcation of CaM signaling. These findings highlight the fundamental importance of the asymmetric arrangement of the pseudo-tetrameric CaV pore domain for feedback inhibition.

CaV3.3 Channelopathies.

CaV3.3 is the third member of the low-voltage-activated calcium channel family and the last to be recognized as disease gene. Previously, CACNA1I, the gene encoding CaV3.3, had been described as schizophrenia risk gene. More recently, de novo missense mutations in CACNA1I were identified in patients with variable degrees of neurodevelopmental disease with and without epilepsy. Their functional characterization indicated gain-of-function effects resulting in increased calcium load and hyperexcitability of neurons expressing CaV3.3. The amino acids mutated in the CaV3.3 disease variants are located in the vicinity of the channel's activation gate and thus are classified as gate-modifying channelopathy mutations. A persistent calcium leak during rest and prolonged calcium spikes due to increased voltage sensitivity of activation and slowed kinetics of channel inactivation, respectively, may be causal for the neurodevelopmental defects. The prominent expression of CaV3.3 in thalamic reticular nucleus neurons and its essential role in generating the rhythmic thalamocortical network activity are consistent with a role of the mutated channels in the etiology of epileptic seizures and thus suggest T-type channel blockers as a viable treatment option.

STAC3 determines the slow activation kinetics of CaV 1.1 currents and inhibits its voltage-dependent inactivation.

The skeletal muscle CaV 1.1 channel functions as the voltage-sensor of excitation-contraction (EC) coupling. Recently, the adaptor protein STAC3 was found to be essential for both CaV 1.1 functional expression and EC coupling. Interestingly, STAC proteins were also reported to inhibit calcium-dependent inactivation (CDI) of L-type calcium channels (LTCC), an important negative feedback mechanism in calcium signaling. The same could not be demonstrated for CaV 1.1, as STAC3 is required for its functional expression. However, upon strong membrane depolarization, CaV 1.1 conducts calcium currents characterized by very slow kinetics of activation and inactivation. Therefore, we hypothesized that the negligible inactivation observed in CaV 1.1 currents reflects the inhibitory effect of STAC3. Here, we inserted a triple mutation in the linker region of STAC3 (ETLAAA), as the analogous mutation abolished the inhibitory effect of STAC2 on CDI of CaV 1.3 currents. When coexpressed in CaV 1.1/STAC3 double knockout myotubes, the mutant STAC3-ETLAAA failed to colocalize with CaV 1.1 in the sarcoplasmic reticulum/membrane junctions. However, combined patch-clamp and calcium recording experiments revealed that STAC3-ETLAAA supports CaV 1.1 functional expression and EC coupling, although at a reduced extent compared to wild-type STAC3. Importantly, STAC3-ETLAAA coexpression dramatically accelerated the kinetics of activation and inactivation of CaV 1.1 currents, suggesting that STAC3 determines the slow CaV 1.1 currents kinetics. To examine if STAC3 specifically inhibits the CDI of CaV 1.1 currents, we performed patch-clamp recordings using calcium and barium as charge carriers in HEK cells. While CaV 1.1 displayed negligible CDI with STAC3, this did not increase in the presence of STAC3-ETLAAA. On the contrary, our data demonstrate that STAC3 specifically inhibits the voltage-dependent inactivation (VDI) of CaV 1.1 currents. Altogether, these results designate STAC3 as a crucial determinant for the slow activation kinetics of CaV 1.1 currents and implicate STAC proteins as modulators of both components of inactivation of LTCC.

Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1.

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as a voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming α1S subunit and the auxiliary α2δ-1 and γ1 subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage dependence of activation to positive potentials outside the physiological range. We generated new HEK293 cell lines stably expressing α2δ-1, ß3, and STAC3. When the adult (CaV1.1a) and embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further coexpressed the γ1 subunit was the current density of CaV1.1a, but not that of CaV1.1e, reduced by >50%. In addition, γ1 caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of γ1, unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the γ1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the γ1-dependent reduction of current density, suggesting that structural rearrangements in CaV1.1a induced by inclusion of exon 29 may allosterically empower the γ1 subunit to exert its inhibitory action on CaV1.1 calcium currents.

Counteractive and cooperative actions of muscle ß-catenin and CaV1.1 during early neuromuscular synapse formation.

Activity-dependent calcium signals in developing muscle play a crucial role in neuromuscular junction (NMJ) formation. However, its downstream effectors and interactions with other regulators of pre- and postsynaptic differentiation are poorly understood. Here, we demonstrate that the skeletal muscle calcium channel CaV1.1 and ß-catenin interact in various ways to control NMJ development. They differentially regulate nerve branching and presynaptic innervation patterns during the initial phase of NMJ formation. Conversely, they cooperate in regulating postsynaptic AChR clustering, synapse formation, and the proper organization of muscle fibers in mouse diaphragm. CaV1.1 does not directly regulate ß-catenin expression but differentially controls the activity of its transcriptional co-regulators TCF/Lef and YAP. These findings suggest a crosstalk between CaV1.1 and ß-catenin in the activity-dependent transcriptional regulation of genes involved in specific pre- and postsynaptic aspects of NMJ formation.

Ion-pair interactions between voltage-sensing domain IV and pore domain I regulate CaV1.1 gating.

The voltage-gated calcium channel CaV1.1 belongs to the family of pseudo-heterotetrameric cation channels, which are built of four structurally and functionally distinct voltage-sensing domains (VSDs) arranged around a common channel pore. Upon depolarization, positive gating charges in the S4 helices of each VSD are moved across the membrane electric field, thus generating the conformational change that prompts channel opening. This sliding helix mechanism is aided by the transient formation of ion-pair interactions with countercharges located in the S2 and S3 helices within the VSDs. Recently, we identified a domain-specific ion-pair partner of R1 and R2 in VSD IV of CaV1.1 that stabilizes the activated state of this VSD and regulates the voltage dependence of current activation in a splicing-dependent manner. Structure modeling of the entire CaV1.1 in a membrane environment now revealed the participation in this process of an additional putative ion-pair partner (E216) located outside VSD IV, in the pore domain of the first repeat (IS5). This interdomain interaction is specific for CaV1.1 and CaV1.2 L-type calcium channels. Moreover, in CaV1.1 it is sensitive to insertion of the 19 amino acid peptide encoded by exon 29. Whole-cell patch-clamp recordings in dysgenic myotubes reconstituted with wild-type or E216 mutants of GFP-CaV1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-CaV1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V1/2, whereas no further shift of V1/2 was observed on substitution of E216 with A or Q. Together with our previous findings, these results demonstrate that inter- and intradomain ion-pair interactions cooperate in the molecular mechanism regulating VSD function and channel gating in CaV1.1.

Structural determinants of voltage-gating properties in calcium channels.

Voltage-gated calcium channels control key functions of excitable cells, like synaptic transmission in neurons and the contraction of heart and skeletal muscles. To accomplish such diverse functions, different calcium channels activate at different voltages and with distinct kinetics. To identify the molecular mechanisms governing specific voltage sensing properties, we combined structure modeling, mutagenesis, and electrophysiology to analyze the structures, free energy, and transition kinetics of the activated and resting states of two functionally distinct voltage sensing domains (VSDs) of the eukaryotic calcium channel CaV1.1. Both VSDs displayed the typical features of the sliding helix model; however, they greatly differed in ion-pair formation of the outer gating charges. Specifically, stabilization of the activated state enhanced the voltage dependence of activation, while stabilization of resting states slowed the kinetics. This mechanism provides a mechanistic model explaining how specific ion-pair formation in separate VSDs can realize the characteristic gating properties of voltage-gated cation channels.

Communication in our body runs on electricity. Between the exterior and interior of every living cell, there is a difference in electrical charge, or voltage. Rapid changes in this so-called membrane potential activate vital biological processes, ranging from muscle contraction to communication between nerve cells. Ion channels are cellular structures that maintain membrane potential and help 'excitable' cells like nerve and muscle cells produce electrical impulses. They are specialized proteins that form highly specific conduction pores in the cell surface. When open, these channels let charged particles (such as calcium ions) through, rapidly altering the electrical potential between the inside and outside the cell. To ensure proper control over this process, most ion channels open in response to specific stimuli, which is known as 'gating'. For example, voltage-gated calcium channels contain charge-sensing domains that change shape and allow the channel to open once the membrane potential reaches a certain threshold. These channels play important roles in many tissues and, when mutated, can cause severe brain or muscle disease. Although the basic principle of voltage gating is well-known, the properties of individual voltage-gated calcium channels still vary. Different family members open at different voltage levels and at different speeds. Such fine-tuning is thought to be key to their diverse roles in various parts of the body, but the underlying mechanisms are still poorly understood. Here, Fernández-Quintero, El Ghaleb et al. set out to determine how this variation is achieved. The first step was to create a dynamic computer simulation showing the detailed structure of a mammalian voltage-gated calcium channel, called Ca_V1.1. The simulation was then used to predict the movements of the voltage sensing regions while the channel opened. The computer modelling experiments showed that although the voltage sensors looked superficially similar, they acted differently. The first of the four voltage sensors of the studied calcium channel controlled opening speed. This was driven by shifts in its configuration that caused oppositely charged parts of the protein to sequentially form and break molecular bonds; a process that takes time. In contrast, the fourth sensor, which set the voltage threshold at which the channel opened, did not form these sequential bonds and accordingly reacted fast. Experimental tests in muscle cells that had been engineered to produce channels with mutations in the sensors, confirmed these results. These findings shed new light on the molecular mechanisms that shape the activity of voltage-gated calcium channels. This knowledge will help us understand better how ion channels work, both in healthy tissue and in human disease.

CACNA1I gain-of-function mutations differentially affect channel gating and cause neurodevelopmental disorders.

T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.

Multiple Sequence Variants in STAC3 Affect Interactions with CaV1.1 and Excitation-Contraction Coupling.

STAC3 is a soluble protein essential for skeletal muscle excitation-contraction (EC) coupling. Through its tandem SH3 domains, it interacts with the cytosolic II-III loop of the skeletal muscle voltage-gated calcium channel. STAC3 is the target for a mutation (W284S) that causes Native American myopathy, but multiple other sequence variants have been reported. Here, we report a crystal structure of the human STAC3 tandem SH3 domains. We analyzed the effect of five disease-associated variants, spread over both SH3 domains, on their ability to bind to the CaV1.1 II-III loop and on muscle EC coupling. In addition to W284S, we find the F295L and K329N variants to affect both binding and EC coupling. The ability of the K329N variant, located in the second SH3 domain, to affect the interaction highlights the importance of both SH3 domains in association with CaV1.1. Our results suggest that multiple STAC3 variants may cause myopathy.

A homozygous missense variant in CACNB4 encoding the auxiliary calcium channel beta4 subunit causes a severe neurodevelopmental disorder and impairs channel and non-channel functions.

P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and ß4 subunits. ß4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat ß4b cDNA. Heterologously expressed wild-type ß4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the ß4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, ß4b and ß4b-L125P augmented the calcium current amplitudes, however, ß4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of ß4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype ß4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of ß4b in cultured myotubes and hippocampal neurons. While binding of ß4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between ß4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of ß4b.

Skeletal muscle CaV1.1 channelopathies.

CaV1.1 is specifically expressed in skeletal muscle where it functions as voltage sensor of skeletal muscle excitation-contraction (EC) coupling independently of its functions as L-type calcium channel. Consequently, all known CaV1.1-related diseases are muscle diseases and the molecular and cellular disease mechanisms relate to the dual functions of CaV1.1 in this tissue. To date, four types of muscle diseases are known that can be linked to mutations in the CACNA1S gene or to splicing defects. These are hypo- and normokalemic periodic paralysis, malignant hyperthermia susceptibility, CaV1.1-related myopathies, and myotonic dystrophy type 1. In addition, the CaV1.1 function in EC coupling is perturbed in Native American myopathy, arising from mutations in the CaV1.1-associated protein STAC3. Here, we first address general considerations concerning the possible roles of CaV1.1 in disease and then discuss the state of the art regarding the pathophysiology of the CaV1.1-related skeletal muscle diseases with an emphasis on molecular disease mechanisms.

Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder.

Background: There is increasing evidence that de novo CACNA1D missense mutations inducing increased Cav1.3 L-type Ca2+-channel-function confer a high risk for neurodevelopmental disorders (autism spectrum disorder with and without neurological and endocrine symptoms). Electrophysiological studies demonstrating the presence or absence of typical gain-of-function gating changes could therefore serve as a tool to distinguish likely disease-causing from non-pathogenic de novo CACNA1D variants in affected individuals. We tested this hypothesis for mutation S652L, which has previously been reported in twins with a severe neurodevelopmental disorder in the Deciphering Developmental Disorder Study, but has not been classified as a novel disease mutation. Methods: For functional characterization, wild-type and mutant Cav1.3 channel complexes were expressed in tsA-201 cells and tested for typical gain-of-function gating changes using the whole-cell patch-clamp technique. Results: Mutation S652L significantly shifted the voltage-dependence of activation and steady-state inactivation to more negative potentials (~ 13-17 mV) and increased window currents at subthreshold voltages. Moreover, it slowed tail currents and increased Ca2+-levels during action potential-like stimulations, characteristic for gain-of-function changes. To provide evidence that only gain-of-function variants confer high disease risk, we also studied missense variant S652W reported in apparently healthy individuals. S652W shifted activation and inactivation to more positive voltages, compatible with a loss-of-function phenotype. Mutation S652L increased the sensitivity of Cav1.3 for inhibition by the dihydropyridine L-type Ca2+-channel blocker isradipine by 3-4-fold.Conclusions and limitationsOur data provide evidence that gain-of-function CACNA1D mutations, such as S652L, but not loss-of-function mutations, such as S652W, cause high risk for neurodevelopmental disorders including autism. This adds CACNA1D to the list of novel disease genes identified in the Deciphering Developmental Disorder Study. Although our study does not provide insight into the cellular mechanisms of pathological Cav1.3 signaling in neurons, we provide a unifying mechanism of gain-of-function CACNA1D mutations as a predictor for disease risk, which may allow the establishment of a more reliable diagnosis of affected individuals. Moreover, the increased sensitivity of S652L to isradipine encourages a therapeutic trial in the two affected individuals. This can address the important question to which extent symptoms are responsive to therapy with Ca2+-channel blockers.

Postsynaptic CaV1.1-driven calcium signaling coordinates presynaptic differentiation at the developing neuromuscular junction.

Proper formation of neuromuscular synapses requires the reciprocal communication between motor neurons and muscle cells. Several anterograde and retrograde signals involved in neuromuscular junction formation are known. However the postsynaptic mechanisms regulating presynaptic differentiation are still incompletely understood. Here we report that the skeletal muscle calcium channel (CaV1.1) is required for motor nerve differentiation and that the mechanism by which CaV1.1 controls presynaptic differentiation utilizes activity-dependent calcium signaling in muscle. In mice lacking CaV1.1 or CaV1.1-driven calcium signaling motor nerves are ectopically located and aberrantly defasciculated. Axons fail to recognize their postsynaptic target structures and synaptic vesicles and active zones fail to correctly accumulate at the nerve terminals opposite AChR clusters. These presynaptic defects are independent of aberrant AChR patterning and more sensitive to deficient calcium signals. Thus, our results identify CaV1.1-driven calcium signaling in muscle as a major regulator coordinating multiple aspects of presynaptic differentiation at the neuromuscular synapse.

Correcting the R165K substitution in the first voltage-sensor of CaV1.1 right-shifts the voltage-dependence of skeletal muscle calcium channel activation.

The voltage-gated calcium channel CaV1.1a primarily functions as voltage-sensor in skeletal muscle excitation-contraction (EC) coupling. In embryonic muscle the splice variant CaV1.1e, which lacks exon 29, additionally function as a genuine L-type calcium channel. Because previous work in most laboratories used a CaV1.1 expression plasmid containing a single amino acid substitution (R165K) of a critical gating charge in the first voltage-sensing domain (VSD), we corrected this substitution and analyzed its effects on the gating properties of the L-type calcium currents in dysgenic myotubes. Reverting K165 to R right-shifted the voltage-dependence of activation by ~12 mV in both CaV1.1 splice variants without changing their current amplitudes or kinetics. This demonstrates the exquisite sensitivity of the voltage-sensor function to changes in the specific amino acid side chains independent of their charge. Our results further indicate the cooperativity of VSDs I and IV in determining the voltage-sensitivity of CaV1.1 channel gating.

STAC proteins: The missing link in skeletal muscle EC coupling and new regulators of calcium channel function.

Excitation-contraction coupling is the signaling process by which action potentials control calcium release and consequently the force of muscle contraction. Until recently, three triad proteins were known to be essential for skeletal muscle EC coupling: the voltage-gated calcium channel CaV1.1 acting as voltage sensor, the SR calcium release channel RyR1 representing the only relevant calcium source, and the auxiliary CaV ß1a subunit. Whether CaV1.1 and RyR1 are directly coupled or whether their interaction is mediated by another triad protein is still unknown. The recent identification of the adaptor protein STAC3 as fourth essential component of skeletal muscle EC coupling prompted vigorous research to reveal its role in this signaling process. Accumulating evidence supports its possible involvement in linking CaV1.1 and RyR1 in skeletal muscle EC coupling, but also indicates a second, much broader role of STAC proteins in the regulation of calcium/calmodulin-dependent feedback regulation of L-type calcium channels.

STAC3 incorporation into skeletal muscle triads occurs independent of the dihydropyridine receptor.

Excitation-contraction (EC) coupling in skeletal muscles operates through a physical interaction between the dihydropyridine receptor (DHPR), acting as a voltage sensor, and the ryanodine receptor (RyR1), acting as a calcium release channel. Recently, the adaptor protein SH3 and cysteine-rich containing protein 3 (STAC3) has been identified as a myopathy disease gene and as an additional essential EC coupling component. STAC3 interacts with DHPR sequences including the critical EC coupling domain and has been proposed to function in linking the DHPR and RyR1. However, we and others demonstrated that incorporation of recombinant STAC3 into skeletal muscle triads critically depends only on the DHPR but not the RyR1. On the contrary, here, we provide evidence that endogenous STAC3 incorporates into triads in the absence of the DHPR in myotubes and muscle fibers of dysgenic mice. This finding demonstrates that STAC3 interacts with additional triad proteins and is consistent with its proposed role in directly or indirectly linking the DHPR with the RyR1.

Role of putative voltage-sensor countercharge D4 in regulating gating properties of CaV1.2 and CaV1.3 calcium channels.

Voltage-dependent calcium channels (CaV) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of CaV1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of CaV1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of CaV1.3 and in VSDs II and IV of CaV1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of CaV1.3 and in two splice variants of CaV1.2. In both channels the D4N (VSD IV) mutation resulted in a  Ì´5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in CaV1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined CaV1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle CaV1.1 channel.

Calcium Influx and Release Cooperatively Regulate AChR Patterning and Motor Axon Outgrowth during Neuromuscular Junction Formation.

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (CaV1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth.

STAC proteins associate to the IQ domain of CaV1.2 and inhibit calcium-dependent inactivation.

The adaptor proteins STAC1, STAC2, and STAC3 represent a newly identified family of regulators of voltage-gated calcium channel (CaV) trafficking and function. The skeletal muscle isoform STAC3 is essential for excitation-contraction coupling and its mutation causes severe muscle disease. Recently, two distinct molecular domains in STAC3 were identified, necessary for its functional interaction with CaV1.1: the C1 domain, which recruits STAC proteins to the calcium channel complex in skeletal muscle triads, and the SH3-1 domain, involved in excitation-contraction coupling. These interaction sites are conserved in the three STAC proteins. However, the molecular domain in CaV1 channels interacting with the STAC C1 domain and the possible role of this interaction in neuronal CaV1 channels remained unknown. Using CaV1.2/2.1 chimeras expressed in dysgenic (CaV1.1-/-) myotubes, we identified the amino acids 1,641-1,668 in the C terminus of CaV1.2 as necessary for association of STAC proteins. This sequence contains the IQ domain and alanine mutagenesis revealed that the amino acids important for STAC association overlap with those making contacts with the C-lobe of calcium-calmodulin (Ca/CaM) and mediating calcium-dependent inactivation of CaV1.2. Indeed, patch-clamp analysis demonstrated that coexpression of either one of the three STAC proteins with CaV1.2 opposed calcium-dependent inactivation, although to different degrees, and that substitution of the CaV1.2 IQ domain with that of CaV2.1, which does not interact with STAC, abolished this effect. These results suggest that STAC proteins associate with the CaV1.2 C terminus at the IQ domain and thus inhibit calcium-dependent feedback regulation of CaV1.2 currents.