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.

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.

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.

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.