Voltage-sensing domains (VSDs) are highly conserved protein modules that regulate the activation of voltage-gated ion channels. In response to membrane depolarization, positive gating charges in the S4 helix of VSDs move across the membrane electric field, which is focused at the hydrophobic constriction site (HCS) in the center of the VSD. This conformational change is translated into opening of the channel gate. Transient interactions of the gating charges with negatively charged countercharges in the adjacent helices are critical for catalyzing this state transition and for determining its voltage dependence and kinetics. However, the mechanism by which the sequential interactions between the multiple gating- and countercharges regulate these properties remains poorly understood. Here, we analyze the state transitions of the first VSD of CaV1.1 using MD simulation of the channel exposed to an electric field and site-directed mutagenesis of gating and countercharges to investigate the role of their interactions in determining the gating properties of CaV1.1. Alanine substitutions of gating charges differentially altered the kinetics or voltage dependence of activation, depending on whether they pass the HCS (R2 and R3) or not (K0, R1, and R4). Alanine substitutions of countercharges differentially altered kinetics and voltage dependence, depending on whether they facilitate the transfer of gating charges across the HCS (E100 and D126), and whether they stabilize the activated (E87, E90, and E140) or the resting state (E100, D126). Thus, our results reveal basic mechanistic principles by which variable interactions between gating charges and countercharges regulate the gating properties of voltage-gated calcium channels.

Voltage-gated calcium channels communicate electrical signals in membranes of excitable cells into cellular responses like secretion of hormones and neurotransmitters, or the contraction of heart and skeletal muscle cells. Their activation properties are tuned to match their specific functions. Consequently, the different members of the calcium channel family activate over a wide range of voltages and with greatly differing speeds. The skeletal muscle Ca V 1.1 and the cardiac/neuronal Ca V 1.2 represent two structurally closely related channels with particularly slow and fast activation kinetics, respectively. Both channel paralogs associate with the auxiliary calcium channel subunit α 2 δ-1, which is a known regulator of activation properties. By expressing Ca V 1.1 and Ca V 1.2 with and without α 2 δ-1 in a new double-knockout muscle cell line, we demonstrate that α 2 δ-1 regulates activation kinetics of the two channels in opposite directions. Molecular dynamics simulation revealed a string of charged amino acids connecting α 2 δ-1 to the intrinsic speed-control mechanism of voltage-sensing domain I (VSD I) in Ca V 1.1. Charge-neutralizing mutations of any of these charged amino acids abolished the α 2 δ-1 modulation and accelerated current kinetics. Together, these results reveal the molecular mechanism by which the α 2 δ-1 subunit regulates the intrinsic speed-control mechanism in the VSD I of Ca V 1.1 calcium channels.

Voltage-gated calcium channels communicate electrical signals in membranes of excitable cells into cellular responses like secretion of hormones and neurotransmitters, or the contraction of heart and skeletal muscle cells. Their activation properties are tuned to match their specific functions. Consequently, the different members of the calcium channel family activate over a wide range of voltages and with greatly differing speeds. The skeletal muscle CaV1.1 and the cardiac/neuronal CaV1.2 represent two structurally closely related channels with particularly slow and fast activation kinetics, respectively. Both channel paralogs associate with the auxiliary calcium channel subunit α2δ-1, which is a known regulator of activation properties. By expressing CaV1.1 and CaV1.2 with and without α2δ-1 in a new double-knockout muscle cell line, we demonstrate that α2δ-1 regulates activation kinetics of the two channels in opposite directions. Molecular dynamics simulation revealed a string of charged amino acids connecting α2δ-1 to the intrinsic speed-control mechanism of voltage-sensing domain I (VSD I) in CaV1.1. Charge-neutralizing mutations of any of these charged amino acids abolished the α2δ-1 modulation and accelerated current kinetics. Together, these results reveal the molecular mechanism by which the α2δ-1 subunit regulates the intrinsic speed-control mechanism in the VSD I of CaV1.1 calcium channels. ### Competing Interest Statement The authors have declared no competing interest. FWF Austrian Science Fund, https://ror.org/013tf3c58, P35618 FWF Austrian Science Fund, https://ror.org/013tf3c58, P33776 FWF Austrian Science Fund, DOC30

The T-type voltage-gated calcium channel CaV3.3 is expressed in GABAergic neurons of the thalamic reticular nucleus (TRN), where its pacemaking activity controls sleep spindle rhythmogenesis during the non-rapid eye movement (NREM) phase of natural sleep. Previously, we established CACNA1I, the gene coding for CaV3.3, as a disease gene for neurodevelopmental disease with or without epilepsy. Here we report three newly identified activation-gate-modifying heterozygous missense variants of CACNA1I, found in four unrelated patients with neurodevelopmental disease with or without seizures. One of these variants, p.(Met1425Val), is an amino-acid substitution at the same position as previously published variant p.(Met1425Ile). Notably, the other two variants studied here are also a pair of two different substitutions of the same amino acid: p.(Ala398Val) and p.(Ala398Glu). By using site-directed mutagenesis, voltage-clamp electrophysiology, computational modelling of neuronal excitability, and structure modelling, we found that the two substitutions of M1425 both result in a gain of channel function including left-shifted voltage-dependence of activation and inactivation, slowed inactivation and deactivation kinetics, and increased neuronal excitability. Remarkably, the two substitutions of A398 show opposite effects on channel function. While substitution A398E leads to a gain of channel function, A398V results in decreased current density, accelerated gating kinetics, and a decreased neuronal excitability. The lack of seizures in the two independent p.(Ala398Val) patients correlates with the absence of increased neuronal excitability in this variant. This is the first report of a gate-modifying CaV3.3 channel variant with partial loss-of-function effects associated with developmental delay and intellectual disability without seizures. Our study corroborates the role of CaV3.3 dysfunction in the etiology of neurodevelopmental disorders. Moreover, our data suggest that substantial gain-of-function of CaV3.3 leads to the development of seizures, whereas both gain- and loss-of-function variants of CACNA1I can cause neurodevelopmental disease.

IntroductionThe CACNA1A gene encodes the pore-forming subunit of the Cav2.1 (P/Q type) neuronal calcium channel and pathogenic variants cause a variety of neurological disorders including episodic and congenital ataxia, familial hemiplegic migraine, developmental delay and epilepsy. Multiple types of seizures have been described in affected patients, including status epilepticus as the first manifestation. In mice harboring the homozygous gain-of-function variant p.Ser218Leu, seizures leading to SUDEP triggered by brainstem spreading depolarization with subsequent apnea and cardiac arrest have been reported.MethodsClinical, genetic and functional data are presented.Results and discussionThe 9-year-old boy with global developmental delay and congenital ataxia developed recurrent seizures and status epilepticus with prolonged, life-threatening apnea implying a high risk for SUDEP. Genetic testing showed a novel de novo missense variant in CACNA1A (c.5398T>A, p.Phe1800Ile). Functional analysis revealed a gain of channel function as the molecular pathomechanism. Therefore, an increased risk of SUDEP in patients with CACNA1-associated epilepsy seems reasonable and preventive strategies should be discussed with caregivers.

The voltage-gated calcium channel CaV1.1 is the voltage sensor for skeletal muscle excitation-contraction (EC) coupling. Upon depolarization of the membrane, it rapidly triggers calcium release from the sarcoplasmic reticulum by conformational coupling to the type 1 ryanodine receptor. Strong depolarization further gives rise to slowly activating L-type calcium currents. Activation of these two processes at distinct voltages and with distinct kinetics is accomplished by the specific actions and properties of CaV1.1's four voltage-sensing domains (VSDs). Although they jointly regulate the gating of the channel pore, only a single VSD (VSD III) controls EC coupling. How CaV1.1 VSD III operates these two functions and to what degree it contributes to channel gating, if at all, are still incompletely known. Here, we analyze the molecular mechanism by which VSD III S3-S4 loop chimeras shift the voltage dependence of EC coupling to negative potentials without affecting the current properties. Furthermore, we report on point mutations in VSD III that shift the voltage dependence of both processes to more positive potentials and on one combined mutant construct that abolishes the currents while retaining the EC coupling function. Together, these findings demonstrate that, in addition to its primary function in EC coupling, CaV1.1 VSD III contributes to channel gating. Moreover, the data indicate that the mechanisms in VSD III operating the two processes can be experimentally separated from one another and probably represent two divergent state transitions initiated by voltage changes.

Voltage-sensing domains (VSDs) are structural modules of voltage-gated ion channels, which sense changes in the membrane potential and, in response, open and close the channel’s ion conduction pore. VSDs comprise a bundle of four antiparallel transmembrane helices (S1–S4). Their basic function is well described by the sliding helix model. Upon membrane depolarization, the positively charged S4 helix slides upward and several of its positive gating charges cross the focused membrane electric field. This state transition is conformationally coupled to the opening of the channel gate. While this essential mechanism is common to all VSDs, different VSDs display a considerable structural and functional diversity, including the number of the gating charges, the nature of their countercharges, and the range, speed, and voltage dependence of the S4 movement upon activation. Here, we review these differences and discuss how they might function to determine the distinct gating properties of voltage-gated ion channels.

The response of voltage-gated calcium channels to membrane depolarization is mediated by four distinct voltage-sensing domains (VSD I-IV) coupled to a common pore. Each of these VSDs consist of four transmembrane helices (S1-S4) with S4 containing 4-5 gating charges. Upon membrane depolarization, these gating charges sense the change in the membrane potential and initiate an upward movement of the S4 segment, resulting in the opening of the channel pore. This sliding S4 helix movement is enabled by the formation of transient interactions between the gating charges and negative countercharges in the surrounding helices.

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.

Skeletal muscle CaV1.1 is a voltage gated calcium channel, which primarily functions as voltage sensor for excitation-contraction coupling (ECC). Upon membrane depolarization, CaV1.1 triggers the opening of RyR1, which releases calcium into the myoplasm, initiating contraction. Under strong stimulation, CaV1.1 also elicits calcium currents (ICa) with kinetics and voltage-dependence different from those of ECC. Four independently functioning voltage-sensing domains (VSDs) gate a common channel pore.

Skeletal muscle contractions are initiated by action potentials, which are sensed by the voltage-gated calcium channel (CaV1.1) and are conformationally coupled to calcium release from intracellular stores. Notably, CaV1.1 contains four separate voltage-sensing domains (VSDs), which activate channel gating and excitation-contraction (EC-) coupling at different voltages and with distinct kinetics. Here we show that a single VSD of CaV1.1 controls skeletal muscle EC-coupling. Whereas mutations in VSDs I, II and IV affect the current properties but not EC-coupling, only mutations in VSD III alter the voltage-dependence of depolarization-induced calcium release. Molecular dynamics simulations reveal comprehensive, non-canonical state transitions of VSD III in response to membrane depolarization. Identifying the voltage sensor that activates EC-coupling and detecting its unique conformational changes opens the door to unraveling the downstream events linking VSD III motion to the opening of the calcium release channel, and thus resolving the signal transduction mechanism of skeletal muscle EC-coupling.

The murine embryonic diaphragm is a primary model for studying myogenesis and neuro-muscular synaptogenesis, both representing processes regulated by spatially organized genetic programs of myonuclei located in distinct myodomains. However, a spatial gene expression pattern of embryonic mouse diaphragm has not been reported. Here, we provide spatially resolved gene expression data for horizontally sectioned embryonic mouse diaphragms at embryonic days E14.5 and E18.5. These data reveal gene signatures for specific muscle regions with distinct maturity and fiber type composition, as well as for a central neuromuscular junction (NMJ) and a peripheral myotendinous junction (MTJ) compartment. Comparing spatial expression patterns of wild -type mice with those of transgenic mice lacking either the skeletal muscle calcium channel Ca V 1.1 or b- catenin, reveals curtailed muscle development and dysregulated expression of genes potentially involved in NMJ formation. Altogether, these datasets provide a powerful resource for further studies of muscle development and NMJ formation in the mouse.

Voltage-gated calcium channels are highly functional proteins important for signal transduction and muscle contraction. They consist of four homologous voltage-sensing domains (VSDs), which regulate the opening and closing of a common pore upon depolarization and repolarization. According to this mechanism, the sliding helix hypothesis illustrates the movement of the S4 helices in the VSDs across the membrane electrical field leading to the ion channel opening/closing. However, the VSDs have varying numbers and positions of countercharges and hence, affect the opening and closing thermodynamics and kinetics differently.

Skeletal muscle CaV1.1 is a voltage gated calcium channel, which primarily functions as voltage sensor of excitation-contraction coupling (ECC). Upon depolarization, CaV1.1 triggers the opening of the RyR1 in the sarcoplasmic reticulum, causing the calcium release event essential for skeletal muscle contraction. Upon strong stimulation, CaV1.1 elicits a calcium current with kinetics and voltage-dependence different from those of ECC. Probably, these distinct activation properties arise from the differential roles of the four CaV1.1 voltage sensing domains (VSDs). Each VSD comprises of four transmembrane helices (S1-S4); S4 segments contain 4-5 positive charges, which interact with negative countercharges in the surrounding helices. These transient ion-pair interactions facilitate and tune the S4 motion in response to changes of the membrane potential. Using structure-guided mutagenesis with patch-clamp analysis and fluorescence calcium recordings, our group identified crucial countercharges in VSD I and IV able to regulate the channel's voltage sensitivity (I and IV) and activation kinetics (IV), without interfering with ECC. However, analogous mutations in VSDs II and III showed little or no effects on either channel gaiting or ECC, corroborating the notion that the four VSDs use different molecular mechanism for regulating their activation properties. Recently, two independent voltage-clamp fluorometry studies suggested that either VSD II or III is the main activator of ECC, based on their specific kinetics and voltage sensitivity matching those of ECC. While both studies agree that perhaps only a single VSD may activate ECC, they disagree on which one that might be. Here we address this problem using site-directed mutagenesis and chimeric CaV1.1 channels expressed in dysgenic myotubes. Constructs shifting the voltage-dependence of either ECC or channel activation reveal the specific contributions of the mutated VSDs in one or the other function of CaV1.1.

Previously we identified CACNA1I, encoding CaV3.3, as a disease gene for neurodevelopmental disorders and epilepsy. Here we examine two further de novo missense variants in the CACNA1I gene, affecting the same residue located in the activation gate at the cytoplasmic end of the IS6 helix. A398E was identified in a patient with seizures, severe intellectual disability, hypotonia and developmental delay, while the patient with the A398V variant presented with microcephaly and mild to moderate developmental delay but no seizures. We analyzed current properties using whole-cell patch-clamp recording in tsA201 cells and examined the effects of the amino acid substitutions in our CaV3.3 structure model. A398E showed no effect on current density, but significantly left-shifted voltage-dependence of activation and inactivation, and substantial slowed activation, deactivation, and inactivation. In contrast, A398V caused a reduction of current density, a mild shift of activation and little to no changes in the gating kinetics. Structure modeling indicated that the two substitutions differentially affect stabilization of the different gating states of CaV3.3. Thus, gain-of-function effects of A398E are in line with the severe patient phenotype. The increased calcium influx at rest and after action potentials, due to the increased window currents and slowed kinetics, likely leads to calcium toxicity in developing neurons. Thus, altered differentiation and cell death might explain the neurodevelopmental defects. Additionally, hyperpolarized activation could cause increased excitability, explaining the seizures. The effects of A398V are much smaller and correspond to the mild patient phenotype. The absence of severely altered gating seemingly protected the patient from developing seizures. This corroborates the link between CaV3.3 and neurodevelopmental disorders, and underlines that not solely the position, but also the type of substitution determines the severity and nature of the disease.

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.

Voltage-gated calcium channels control a variety of processes in excitable cells. However, the exact mechanisms regulating kinetics and voltage-dependence of channel activation are not fully understood. Voltage-gated activation is determined by four distinct voltage-sensing domains (VSD I-IV) coupled to a common pore. Each VSD consists of four transmembrane helices (S1-S4) with S4 containing four to five gating charges. Upon membrane depolarization, consecutive interactions of these gating charges with negatively charged countercharges are believed to facilitate an upward movement of the S4 helices, leading to the opening of the pore and activation of the channel. Previous studies linked naturally occurring mutations of the innermost gating charge R4 (R174W) and its negative countercharge (E100) in CaV1.1 to muscle disease. To investigate the contribution of VSD I and the roles of its gating- and countercharges in channel gating, we combined structure-guided site-directed mutagenesis with patch-clamp analysis in dysgenic myotubes (CaV1.1-null). As E100 in helix S2 together with D126 in helix S3 form the highly conserved charge-transfer center of VSDs, we included this second negative countercharge (D126) in our analysis. While mutation of R174A resulted in a strong right-shift of voltage dependence, charge neutralizing mutations of the countercharges E100 and D126 both lead to a left-shift of voltage-dependence and to a slowing of channel activation. The double mutant of both countercharges resulted in an additive effect on activation kinetics, but left the voltage dependence of activation unaffected compared to the single mutants. Our findings indicate that VSD I substantially contributes to the regulation of both kinetics and voltage-dependence of activation; that the same ionic interactions are critical for both properties; but that the molecular mechanisms governing the two gating properties are functionally separate.