Distinct roles for CaV1.1’s voltage-sensing domains

Study reveals how a slowly activating calcium channel is able to control rapid excitation–contraction coupling in skeletal muscle.

Skeletal muscle contraction is initiated by action potentials that depolarize the muscle fiber and trigger the rapid release of Ca²⁺ from the SR via RYR1 channels. This process of excitation–contraction coupling depends on voltage-gated Ca_V1.1 channels in the plasma membrane, or sarcolemma, of muscle fibers. But Ca_V1.1 channels are only slowly activated by changes in the sarcolemma membrane potential, and it is therefore unclear how they are able to trigger the much faster activation of RYR1 channels. In this issue of JGP, Savalli et al. reveal that this paradox can be explained by the fact that each of Ca_V1.1’s four voltage-sensing domains (VSDs) have distinct biophysical properties (1).Nicoletta Savalli (left), Riccardo Olcese (center), and colleagues reveal the distinct physical properties of the Ca_V1.1 channel’s four voltage-sensing domains (VSD I–IV, right). VSD-I shows slow activation kinetics and is the main contributor to the opening of Ca_V1.1. The other VSDs activate much faster and may therefore be coupled to RYR1 to mediate the rapid release of Ca²⁺ from the SR during skeletal muscle contraction.RYR1 channels have no voltage-sensing machinery of their own and therefore rely on a physical connection to Ca_V1.1 channels to release Ca²⁺ and initiate muscle contraction in response to muscle fiber depolarization. But RYR1 channels open ∼25 times faster than Ca_V1.1 channels. “So, how can these slowly activating Ca_V1.1 channels trigger the rapid release of Ca²⁺ from the SR?” asks Riccardo Olcese, a professor at the David Geffen School of Medicine, UCLA.Olcese and colleagues, including Assistant Project Scientist Nicoletta Savalli, suspected that the answer might lie in the fact that, like many other voltage-gated ion channels, Ca_V1.1 has four VSDs that alter their conformation in response to voltage changes. These domains are similar, but not identical, to each other, potentially enabling them to have distinct biophysical properties and perform distinct functions. Indeed, Olcese and colleagues previously demonstrated that, in the closely related channel Ca_V1.2, only VSDs II and III are involved in pore opening (2, 3).Savalli et al. used voltage-clamp fluorometry to compare the properties of Ca_V1.1’s VSDs, expressing the channel in Xenopus oocytes and labeling each of its VSDs in turn with an environmentally sensitive fluorophore to report voltage-dependent changes in their conformation (1). “We found that the four VSDs were very heterogenous in both their kinetics and voltage dependencies,” says Olcese. “VSD-I had very slow kinetics, compatible with the slow activation of the Ca_V1.1 pore. The other three VSDs had much faster kinetics and could, therefore, be good candidates to be the voltage sensors for RYR1 activation.”Olcese and colleagues confirmed the importance of VSD-I for Ca_V1.1 activation by analyzing a naturally occurring, charge-neutralizing mutation in this domain, R174W, that is linked to malignant hyperthermia (4). The team found that this mutation reduced the voltage-sensitivity of VSD-I and abolished the ability of Ca_V1.1 to conduct Ca²⁺ at physiological membrane potentials, but had no effect on the behavior of the other three VSDs.Finally, Savalli et al. applied their data on both the wild-type and mutant VSDs to an allosteric model of Ca_V activation (2, 3), which predicted that VSD-I contributes most of the energy required to stabilize the open state of Ca_V1.1, while the other VSDs contribute little to nothing.Thus, Ca_V1.1 activation is mainly driven by a single VSD—a mechanism that hasn’t been seen in any other voltage-gated ion channel—leaving the other VSDs free to perform other functions, such as the rapid activation of RYR1. Olcese and colleagues now want to pinpoint exactly which VSD(s) are coupled to RYR1 and determine how they trigger rapid Ca²⁺ release from the SR.     

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

The voltage-gated calcium channel Ca_V1.1 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 Ca_V1.1 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 Ca_V1.1 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 Ca_V1.1 and Ca_V1.2 L-type calcium channels. Moreover, in Ca_V1.1 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-Ca_V1.1e (lacking exon 29) showed that charge neutralization (E216Q) or removal of the side chain (E216A) significantly shifted the voltage dependence of activation (V_1/2) to more positive potentials, suggesting that E216 stabilizes the activated state. Insertion of exon 29 in the GFP-Ca_V1.1a splice variant strongly reduced the ionic interactions with R1 and R2 and caused a substantial right shift of V_1/2, whereas no further shift of V_1/2 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 Ca_V1.1.     

Linker flexibility of IVS3-S4 loops modulates voltage-dependent activation of L-type Ca2+ channels

Extracellular S3-S4 linkers of domain IV (IVS3-S4) of L-type Ca²⁺ channels (Ca_V1) are subject to alternative splicing, resulting into distinct gating profiles serving for diverse physiological roles. However, it has remained elusive what would be the determining factor of IVS3-S4 effects on Ca_V1 channels. In this study, we systematically compared IVS3-S4 variants from Ca_V1.1-1.4, and discover that the flexibility of the linker plays a prominent role in gating characteristics. Chimeric analysis and mutagenesis demonstrated that changes in half activation voltage (V_1/2) or activation time constant (τ) are positively correlated with the numbers of flexible glycine residues within the linker. Moreover, antibodies that reduce IVS3-S4 flexibility negatively shifted V_1/2, emerging as a new category of Ca_V1 enhancers. In summary, our results suggest that the flexibility or rigidity of IVS3-S4 linker underlies its modulations on Ca_V1 activation (V_1/2 and τ), paving the way to dissect the core mechanisms and to develop innovative perturbations pertaining to voltage-sensing S4 and its vicinities.     

Excitation-Contraction Coupling: The distinct role of the four voltage sensors of the skeletal CaV1.1 channel in voltage-dependent activation

Initiation of skeletal muscle contraction is triggered by rapid activation of RYR1 channels in response to sarcolemmal depolarization. RYR1 is intracellular and has no voltage-sensing structures, but it is coupled with the voltage-sensing apparatus of Ca_V1.1 channels to inherit voltage sensitivity. Using an opto-electrophysiological approach, we resolved the excitation-driven molecular events controlling both Ca_V1.1 and RYR1 activations, reported as fluorescence changes. We discovered that each of the four human Ca_V1.1 voltage-sensing domains (VSDs) exhibits unique biophysical properties: VSD-I time-dependent properties were similar to ionic current activation kinetics, suggesting a critical role of this voltage sensor in Ca_V1.1 activation; VSD-II, VSD-III, and VSD-IV displayed faster activation, compatible with kinetics of sarcoplasmic reticulum Ca²⁺ release. The prominent role of VSD-I in governing Ca_V1.1 activation was also confirmed using a naturally occurring, charge-neutralizing mutation in VSD-I (R174W). This mutation abolished Ca_V1.1 current at physiological membrane potentials by impairing VSD-I activation without affecting the other VSDs. Using a structurally relevant allosteric model of Ca_V activation, which accounted for both time- and voltage-dependent properties of Ca_V1.1, to predict VSD-pore coupling energies, we found that VSD-I contributed the most energy (~75 meV or ∼3 kT) toward the stabilization of the open states of the channel, with smaller (VSD-IV) or negligible (VSDs II and III) energetic contribution from the other voltage sensors (<25 meV or ∼1 kT). This study settles the longstanding question of how Ca_V1.1, a slowly activating channel, can trigger RYR1 rapid activation, and reveals a new mechanism for voltage-dependent activation in ion channels, whereby pore opening of human Ca_V1.1 channels is primarily driven by the activation of one voltage sensor, a mechanism distinct from that of all other voltage-gated channels.     

Excitation–Contraction Coupling: Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1

The skeletal muscle voltage-gated calcium channel (Ca_V1.1) primarily functions as a voltage sensor for excitation–contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming α_1S 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 Ca_V1.1a, 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, β₃, and STAC3. When the adult (Ca_V1.1a) and embryonic (Ca_V1.1e) 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 Ca_V1.1a. Only when we further coexpressed the γ₁ subunit was the current density of Ca_V1.1a, but not that of Ca_V1.1e, 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 Ca_V1.1a. 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 Ca_V1.1a induced by inclusion of exon 29 may allosterically empower the γ₁ subunit to exert its inhibitory action on Ca_V1.1 calcium currents.     

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

Voltage-dependent calcium channels (Ca_V) 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 Ca_V1.1 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 Ca_V1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of Ca_V1.3 and in VSDs II and IV of Ca_V1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of Ca_V1.3 and in two splice variants of Ca_V1.2. In both channels the D4N (VSD IV) mutation resulted in a ?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 Ca_V1.2 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 Ca_V1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle Ca_V1.1 channel.     

Gating pore currents occur in CaV1.1 domain III mutants associated with HypoPP

Mutations in the voltage sensor domain (VSD) of Ca_V1.1, the α_1S subunit of the L-type calcium channel in skeletal muscle, are an established cause of hypokalemic periodic paralysis (HypoPP). Of the 10 reported mutations, 9 are missense substitutions of outer arginine residues (R1 or R2) in the S4 transmembrane segments of the homologous domain II, III (DIII), or IV. The prevailing view is that R/X mutations create an anomalous ion conduction pathway in the VSD, and this so-called gating pore current is the basis for paradoxical depolarization of the resting potential and weakness in low potassium for HypoPP fibers. Gating pore currents have been observed for four of the five Ca_V1.1 HypoPP mutant channels studied to date, the one exception being the charge-conserving R897K in R1 of DIII. We tested whether gating pore currents are detectable for the other three HypoPP Ca_V1.1 mutations in DIII. For the less conserved R1 mutation, R897S, gating pore currents with exceptionally large amplitude were observed, correlating with the severe clinical phenotype of these patients. At the R2 residue, gating pore currents were detected for R900G but not R900S. These findings show that gating pore currents may occur with missense mutations at R1 or R2 in S4 of DIII and that the magnitude of this anomalous inward current is mutation specific.     

Physiological and Pharmacological Modulation of the Embryonic Skeletal Muscle Calcium Channel Splice Variant CaV1.1e

Ca_V1.1e is the voltage-gated calcium channel splice variant of embryonic skeletal muscle. It differs from the adult Ca_V1.1a splice variant by the exclusion of exon 29 coding for 19 amino acids in the extracellular loop connecting transmembrane domains IVS3 and IVS4. Like the adult splice variant Ca_V1.1a, the embryonic Ca_V1.1e variant functions as voltage sensor in excitation-contraction coupling, but unlike Ca_V1.1a it also conducts sizable calcium currents. Consequently, physiological or pharmacological modulation of calcium currents may have a greater impact in Ca_V1.1e expressing muscle cells. Here, we analyzed the effects of L-type current modulators on whole-cell current properties in dysgenic (Ca_V1.1-null) myotubes reconstituted with either Ca_V1.1a or Ca_V1.1e. Furthermore, we examined the physiological current modulation by interactions with the ryanodine receptor using a chimeric Ca_V1.1e construct in which the cytoplasmic II-III loop, essential for skeletal muscle excitation-contraction coupling, has been replaced with the corresponding but nonfunctional loop from the Musca channel. Whereas the equivalent substitution in Ca_V1.1a had abolished the calcium currents, substitution of the II-III loop in Ca_V1.1e did not significantly reduce current amplitudes. This indicates that Ca_V1.1e is not subject to retrograde coupling with the ryanodine receptor and that the retrograde coupling mechanism in Ca_V1.1a operates by counteracting the limiting effects of exon 29 inclusion on the current amplitude. Pharmacologically, Ca_V1.1e behaves like other L-type calcium channels. Its currents are substantially increased by the calcium channel agonist Bay K 8644 and inhibited by the calcium channel blocker nifedipine in a dose-dependent manner. With an IC50 of 0.37 μM for current inhibition by nifedipine, Ca_V1.1e is a potential drug target for the treatment of myotonic dystrophy. It might block the excessive calcium influx resulting from the aberrant expression of the embryonic splice variant Ca_V1.1e in the skeletal muscles of myotonic dystrophy patients.     

Conformations of voltage-sensing domain III differentially define NaV channel closed- and open-state inactivation

Voltage-gated Na⁺ (Na_V) channels underlie the initiation and propagation of action potentials (APs). Rapid inactivation after Na_V channel opening, known as open-state inactivation, plays a critical role in limiting the AP duration. However, Na_V channel inactivation can also occur before opening, namely closed-state inactivation, to tune the cellular excitability. The voltage-sensing domain (VSD) within repeat IV (VSD-IV) of the pseudotetrameric Na_V channel α-subunit is known to be a critical regulator of Na_V channel inactivation. Yet, the two processes of open- and closed-state inactivation predominate at different voltage ranges and feature distinct kinetics. How inactivation occurs over these different ranges to give rise to the complexity of Na_V channel dynamics is unclear. Past functional studies and recent cryo-electron microscopy structures, however, reveal significant inactivation regulation from other Na_V channel components. In this Hypothesis paper, we propose that the VSD of Na_V repeat III (VSD-III), together with VSD-IV, orchestrates the inactivation-state occupancy of Na_V channels by modulating the affinity of the intracellular binding site of the IFMT motif on the III-IV linker. We review and outline substantial evidence that VSD-III activates in two distinct steps, with the intermediate and fully activated conformation regulating closed- and open-state inactivation state occupancy by altering the formation and affinity of the IFMT crevice. A role of VSD-III in determining inactivation-state occupancy and recovery from inactivation suggests a regulatory mechanism for the state-dependent block by small-molecule anti-arrhythmic and anesthetic therapies.

IntroductionVoltage-gated Na⁺ (Na_V) channels initiate excitation in neurons and myocytes, enabling rapid conduction over large distances that is decoupled from intracellular Ca²⁺ signaling (Hille, 2001). During excitation, Na_V channel opening releases a large inward Na⁺ current (I_Na) that is followed by rapid inactivation, which occurs within milliseconds and makes most channels nonconductive. This inactivation from the open state is required to allow outward repolarizing currents to bring the cell membrane back to the resting potential. However, inactivation recovery is not instantaneous at hyperpolarized membrane potentials, nor is it limited to depolarized potentials. For example, in cardiac myocytes, Na_V channels remain inactivated for 10s to 100s of milliseconds following action potential repolarization, rendering the myocyte refractory to excitation for a brief period and preventing reentrant arrhythmia (Zipes et al., 2017). In addition, neuronal memory of previous excitation can be conferred by inactivation of Na_V channels that reduces the subsequent firing rate (Marom, 1998; Toib et al., 1998). At modest depolarized membrane potentials (less than −30 mV), Na_V channels can also become inactivated before the activation gate opening but require longer depolarizations (Aldrich et al., 1983; Bean, 1981). This closed-state inactivation results in fewer available channels to initiate excitation. Thus, the regulation of Na_V channel inactivation by time and membrane potential is essential for the functioning of excitable cells, allowing neurons and myocytes to appropriately respond to changes in membrane potential that span multiple time domains (Silva, 2014).Mammalian Na_V channels are formed by a single protein with four homologous repeats (I–IV; Fig. 1 A), each constituting six membrane-spanning segments (S1–S6). Within each repeat contains a voltage-sensing domain (VSD) that consists of segments S1–S4. The Na⁺-selective pore is formed jointly by the S5 and S6 segments (Yu and Catterall, 2003), with the region between S5 and S6 constituting the P-loop responsible for Na⁺ ion selectivity. Mammalian Na_V channels additionally feature intracellular linkers and a unique C-terminal domain (CTD), which is known to bind various accessory subunits, including calmodulin and fibroblast growth factor homologous factors (Abriel, 2010). Given the complex time and voltage dependence of Na_V channel inactivation, it must be connected to multiple different conformational states, whose occupancy is determined by the positions of the VSDs, the state of the channel pore, and the position of the CTD (Ulbricht, 2005).Open in a separate windowFigure 1.The Na_V channel inactivation mechanism.(A) A schematic representation of mammalian Na_V channel shows four homologous repeats (I–IV) and the IFMT motif on the III–IV linker (yellow). (B) The structure of rNa_V1.5 (PDB accession no. 6UZ3) shows the IFMT motif enclosed by repeat III S5 and repeat IV S4–S5 linker and S5 and S6 segments, causing an allosteric block during inactivation.Indeed, several reports have connected the activation of the VSD-IV to the onset of fast inactivation after channel opening (Capes et al., 2013; Goldschen-Ohm et al., 2013). Mutations in VSD-IV also suggest a role in closed-state inactivation (Kambouris et al., 2000; Chahine et al., 1994; Groome et al., 2011). Charge neutralization mutations within VSD-IV cause a large hyperpolarizing shift in voltage-dependent channel availability and a large fraction of inactivated channels at voltages where the channels are closed (Capes et al., 2013; Brake et al., 2021 Preprint). Early experiments demonstrated that the addition of intracellular pronase removed Na_V channel inactivation and revealed the participation of intracellular components (Armstrong et al., 1973; Armstrong, 1981; Salgado et al., 1985). The model of Na_V channel inactivation was then suggested to resemble the “ball-and-chain” model of K⁺ channel N-type inactivation, where a ball that is attached to the inner part of the channel causes inactivation by occluding the pore (Armstrong and Bezanilla, 1977; Hoshi et al., 1990). Subsequent experiments that disrupted the intracellular linker between repeats III and IV produced functional channels but lacked inactivation (Stühmer et al., 1989; Vassilev et al., 1988; Vassilev et al., 1989), further confining the sites of the inactivation gate to the III-IV linker. Finally, site-specific mutagenesis identified a hydrophobic cluster of amino acids, Ile-Phe-Met-Thr (IFMT), as an essential component for the inactivation mechanism (Hartmann et al., 1994), and a “hinged-lid” model was proposed (Eaholtz et al., 1994; Kellenberger et al., 1997; Rohl et al., 1999; West et al., 1992). In this model, the loop between two hinged points serves as a rigid lid that folds over the channel pore, with the IFMT motif acting as a hydrophobic latch to stabilize the inactivated conformation. Additional mutagenesis and the discovery of inherited proarrhythmic mutations within cardiac Na_V channels further suggested that the binding site or the “receptor” for the IFMT motif involves repeats III and IV S4–S5 linkers and IV S6 segment (Smith and Goldin, 1997; McPhee et al., 1995; McPhee et al., 1998). Recent structures of eukaryotic Na_V channels, however, reveal that the IFMT motif resembles more of a wedge that squeezes into the crevice formed by repeat III S5 and repeat IV S4–S5 linker and S5 and S6 segments (Yan et al., 2017; Pan et al., 2018, 2019; Shen et al., 2019; Jiang et al., 2020; Li et al., 2021) and allosterically blocks the Na_V channel conduction pore (Fig. 1 B).Apart from the inactivation gate, the VSD-III, the CTD, and the state of the channel pore have also been implicated in Na_V channel inactivation (Cha et al., 1999; Deschênes et al., 2001; Hsu et al., 2017; Mantegazza et al., 2001; Motoike et al., 2004; Pitt and Lee, 2016, Mangold et al., 2017). Based on new structural data in combination with numerous previous functional studies, we hypothesize that the activation of the VSD-III, in addition to the VSD-IV, facilitates different inactivated states by modulating the binding affinity of IFMT crevice. In this Hypothesis paper, we will first outline the models of each inactivated state. Then, we will present the structural and electrophysiological evidence that supports our hypothesis and leads to the proposed model. Finally, we will discuss the model implications on the modulation of Na_V channel function by accessory subunits and therapeutic drugs.The models of open- and closed-state Na_V channel inactivationWe propose a VSD-III and VSD-IV-centric inactivation model (created at https://biorender.com; Fig. 2), which incorporates structural motifs throughout the channel. In this model, the VSD-III can activate in two distinct conformations over different voltage ranges as detected by fluorescent tracking of voltage sensor conformational change upon membrane depolarization (Chanda and Bezanilla, 2002; Zhu et al., 2017) and various supportive electrophysiological evidence (Varga et al., 2015; Hsu et al., 2017). The intermediate and fully activated conformations of VSD-III then unmask distinct crevices with low and high affinity for the IFMT motif, as suggested by eukaryotic Na_V channel structures (Yan et al., 2017; Jiang et al., 2020), that leads to unique kinetics of closed- versus open-state inactivation. Different inactivated states are therefore connected to distinct activated conformations of VSD-III.Open in a separate windowFigure 2.The models of open- and closed-state inactivation based on two VSD-III depolarized conformations.(A) During open-state inactivation, strong membrane depolarization leads to the activation of repeat I and II VSDs and the intermediate activation of VSD-III, resulting in the opening of the activation gate and the conduction of Na⁺ ions (I). Activation of the VSD-IV exposes the low-affinity binding site for the IFMT motif (II). Further translation of the VSD-III into its fully activated conformation establishes the stable inactivated configuration with high-affinity IFMT motif binding (III). (B) For closed-state inactivation at hyperpolarized membrane potential, VSD-III activates while repeats I and II VSDs are at rest, resulting in closed activation gate and no I_Na (I). Subsequent VSD-IV activation forms the low-affinity binding site for the IFMT motif (II). Since VSD-III occupies primarily in its intermediate conformation, the stable inactivated configuration is not established.During the process of open-state inactivation (Fig. 2 A), the VSDs of repeats I and II rapidly move outward into their activated position upon membrane depolarization. At the same time, VSD-III adopts its intermediate conformation, and the activation gate opens (step I), allowing the channel to pass current. Shortly thereafter, VSD-IV activates and a partial crevice for the IFMT motif is formed. The III–IV linker may also be released from CTD interaction upon VSD-IV activation, as suggested by the structure of the α-scorpion toxin AaH2-bound hNa_V1.7 VSD-IV–Na_VPaS hybrid, where the intracellular end of resting IV S4 (K7 and R8) interacts with the CTD, causing the sequestration of the III–IV linker (Clairfeuille et al., 2019). The IFMT motif can move into the hydrophobic cleft formed by repeat IV S4–S5 linker and S6 segment and establish low-affinity inactivation binding (step II). Concurrently, further translation of VSD-III into its fully activated conformation repositions the III S4–S5 linker to increasingly interact with the helix along III–IV linker, providing additional support for the stable inactivated conformation (step III). The binding of the IFMT motif leads to a rearrangement around S6 helices and an allosteric block of the conduction pathway. During fast inactivation, the VSDs of repeats I and II are free to deactivate upon membrane hyperpolarization. VSD-III and VSD-IV, however, are immobilized by the IFMT motif. Importantly, the deactivation of VSD-III and VSD-IV determines inactivation recovery time course, with the slowness of VSD-III deactivation playing a rate-limiting role (Hsu et al., 2017).For closed-state inactivation, at membrane potentials less than −30 mV (Fig. 2 B), VSD-III activates to its intermediate conformation while VSD-I and VSD-II are at rest (step I). When VSD-IV moves up to its activated conformation, the crevice for IFMT motif is partially exposed and low-affinity IFMT binding is established (step II). Because of the hyperpolarized potential, VSD-III primarily occupies only its intermediate conformation, incapable of providing supportive interactions around the inactivation gate, and thus the channel is unable to adopt a stable inactivated configuration. The inactivated state with low-affinity IFMT motif binding results in the slow kinetics of closed-state inactivation. As the VSD-IV activates at more positive potentials than the VSD-III (Varga et al., 2015), the rate limiting of closed-state inactivation is reflected by the overlap in voltage range at which VSD-IV activates and the steady-state inactivation occurs (Capes et al., 2013).In the following section, we will review supporting evidence that underlies the key aspects and implications of our proposed hypothesis.Differential Na_V channel–inactivated states are determined by the IFMT binding affinity that is modulated by the conformations of repeats III and IV VSDsThe closed and open Na_V channel inactivation states differ not only in the voltage range over which they are occupied but also prominently in their kinetics, where inactivation proceeds at a much faster rate from the open-channel conformation (Aldrich et al., 1983; Goldman, 1995). As shown by structural and functional studies, inactivation is due to the binding of the IFMT motif to a crevice (Fig. 1 B). If the final step in both closed- and open-state inactivation is identical, then how can these two inactivation processes feature distinct kinetics? One possible explanation is that the conformation of IFMT binding site is dynamic and its binding affinity can be tuned. The crevice is formed by the S4–S5 linker of repeat IV, S5 of repeat III, and the S5 and S6 segments of repeat IV with additional interactions from repeat III S4–S5 linker aiding in the stabilization of the IFMT motif binding, as indicated by many recent structures (Yan et al., 2017; Pan et al., 2018; Shen et al., 2019; Pan et al., 2019; Jiang et al., 2020) and past mutagenesis studies (Kellenberger et al., 1997; McPhee et al., 1995, 1998; Smith and Goldin, 1997).In the resting state model of rat Na_V1.5 (rNa_V1.5), fitted after the resting bacterial Na_VAb structure (Wisedchaisri et al., 2019), the IFMT crevice is blocked by the intracellular end of repeat III S4 and the S4–S5 linkers of repeats III and IV (Jiang et al., 2020). The outward translation of repeat III and IV VSDs is therefore a prerequisite for rendering the inactivation crevice available. The movement of III– or IV–S4 segments will pull on their corresponding S4–S5 linkers and position them accordingly. In other words, the extent of VSD activation (i.e., the number of gating charges that moves from an internal to an external side) directly affects the placement of the S4–S5 linker, which in repeats III and IV form the integral component of the IFMT crevice. Thus, different combinations of the VSD-III and VSD-IV depolarized conformations may give rise to different binding affinities of the IFMT crevice, resulting in the distinct inactivation kinetics observed over different membrane potentials. We expect that there are also differences in the contribution from VSD-III and VSD-IV in regulating the IFMT crevice between different Na_V channel homologues (Chanda and Bezanilla, 2002; Varga et al., 2015; Brake et al., 2021 Preprint).Facilitation of inactivation by the depolarized VSD-IV conformation has been convincingly shown via multiple charge neutralization and toxin studies (Capes et al., 2013; Chen et al., 1996; Kontis et al., 1997; Kühn and Greeff, 1999; Clairfeuille et al., 2019). Further studies have revealed that VSD-III may also play a key role in modulating IFMT binding. During fast inactivation, a fraction of the gating charge was found to be immobilized by the inactivation gate (Armstrong and Bezanilla, 1975; Bezanilla and Armstrong, 1974; Bezanilla et al., 1982). These gating charges were identified as components of the repeat III and IV VSDs, suggesting their interaction with the III–IV linker (Armstrong and Bezanilla, 1977; Cha et al., 1999). Specific disruption of the inactivation particle through an IFM/ICM mutation did not eliminate the immobilization of VSD-IV, yet entirely abolished VSD-III immobilization (Sheets and Hanck, 2005), emphasizing a functional linkage between the IFMT motif and the VSD-III depolarized conformation.The role of VSD-III in regulating Na_V channel closed- and open-state inactivation was additionally highlighted through various protocols. Particularly, biasing VSD-III to the depolarized conformation by tethering extracellular MTSEA-biotin on repeat III S4 through an R3C mutation dramatically reduced Na_V channel availability via enhanced closed-state inactivation and its slope factor but showed minimal effects on the peak I–V relationship (Sheets and Hanck, 2007). The R1128C (R4C on repeat III S4 segment) mutation in rat skeletal muscle sodium channel (rNa_V1.4) promoted closed-state inactivation, leading to reduced steady-state availability (Groome et al., 2011, 2014a). Similar mutations (R4H on repeat III S4 segment) in human skeletal muscle Na_V channel (hNa_V1.4 R1135H) and human cardiac sodium channel (hNa_V1.5 R1309H) showed enhanced entry into inactivated states with prolonged recovery from inactivation (Groome et al., 2014b; Wang et al., 2016). When the three outermost gating charges of VSD-III were neutralized, the fraction of inactivated channels at subthreshold potentials (−60 and −50 mV), increased and the delay to the onset of open-state inactivation after a conditioning pulse decreased, but to a lesser extent than the neutralization of VSD-IV (Capes et al., 2013). The charge neutralization in VSD-III of Na_V1.5 caused the similar significant shift in steady-state inactivation curve toward hyperpolarizing potentials, which is an indicative of increased closed-state inactivation, as observed when the VSD-IV charges were neutralized (Brake et al., 2021 Preprint). All these results illustrate that the modulation of the repeat III S4 either through a chemical manipulation or a reduction in the number of VSD-III gating charges that facilitate VSD-III activation enhance closed-state inactivation and reduce channel availability, emphasizing the significant role of VSD-III activated conformations in regulating the states of Na_V channel inactivation.Interestingly, open-state and closed-state inactivation feature distinct kinetics: open-state inactivation kinetics show little change in rate over the voltages at which it occurs (Aldrich et al., 1983), whereas the time constant of closed-state inactivation is highly voltage dependent (Goldman, 1995; Sheets and Hanck, 1995). An inactivation gate peptide (KIFMK) that mimics the IFMT motif and restores the fast inactivation in defective mutant Na_V channels illustrates weak voltage dependence in terms of inactivation time constants at depolarized potential of more than −30 mV (Eaholtz et al., 1994; Peter et al., 1999). This observation, together with the absence of gating current component with a time course that tracks inactivation, suggests that inactivation after channel opening has no intrinsic voltage dependence (Armstrong, 2006). We hypothesize that the voltage dependence of closed-state inactivation arises from movements of the VSDs of repeats III and IV exposing the crevice for IFMT binding, while during open-state inactivation, rapid and complete VSD-III and VSD-IV activation fully reveals the crevice and inactivation kinetics are determined by voltage-independent III-IV linker diffusion. Previously, closed-state inactivation was postulated to occur when the repeats III and IV VSDs move to depolarized conformations, while the VSDs of repeats I and II remain at rest (Armstrong, 2006). We further propose that the position of the VSD-III is crucial in determining the conformation of the IFMT binding site and that inactivation state occupancy is derived from the multiple steps traversed by the VSD-III as it activates. In the next section, we will examine studies that imply different VSD-III activated conformations.Unique VSD-III activated conformation determines the differential binding of the IFMT motifRecent structures of eukaryotic Na_V channels featured components of Na_V channel inactivation machinery that were missing from prior structures of bacterial Na_V channels. The first eukaryotic Na_V channel structure from the American cockroach, Na_VPaS, captured closed pore domain with all four VSDs in an “activated” conformation (Shen et al., 2017), defined by two positively charges within the S4 segment having crossed the hydrophobic constriction site (HCS), a cluster of hydrophobic residues that prevents ion leakage in voltage sensor (Yang et al., 1996; Starace and Bezanilla, 2004; Tao et al., 2010; Catterall, 2014). The III–IV linker, although similar in length to other eukaryotic Na_V channels, lacks the key hydrophobic motif required for inactivation and is shown to interact with the CTD. Subsequently, the electric eel Na_V1.4 (eeNa_V1.4), human Na_V1.2 (hNa_V1.2), hNa_V1.4, hNa_V1.7, and rat Na_V1.5 (rNa_V1.5) structures were resolved. These structures are all highly similar, with high resolution of key elements that are critical for inactivation mechanism. In these structures, the III–IV linker contains the inactivation motif (LFM for eeNa_V1.4) binding to the crevice formed by repeat III S5, repeat IV S4–S5 linker, and S5 and S6 segments (Fig. 1 B), likely representing the inactivated state structure (Yan et al., 2017; Pan et al., 2018; Shen et al., 2019; Pan et al., 2019; Jiang et al., 2020). All four VSDs are also captured in the activated positions, albeit some with different conformations in the Na_VPaS structure. The CTD is not well resolved and is thus omitted.Comparison between Na_VPaS and other eukaryotic Na_V channels reveals VSD-III can activate in two steps. First, the positions of the VSD-III differ in the extent of their depolarized conformation or activation. Even though Na_VPaS channel is nonfunctional and may not represent a relevant physiological state, Na_VPaS contains highly conserved transmembrane segments that are similar to and superimpose well with other eukaryotic Na_V channels. The number of arginines on repeat III S4 segment is also comparable among two groups. Differences observed may provide an opportunity for dissecting Na_V channel mechanism. In Na_VPaS, two gating charges on repeat III S4 are transferred across the HCS, while in other structures, a total of four charges on III S4 segment are displaced from the intracellular to the extracellular side (Fig. 3 A). These structural variations reveal that the VSD-III might adopt two distinct activated conformations correlating to different numbers of charges across the HCS. The trajectory of the VSD-III activation resembles a sigmoidal curve, with an intracellular tip moving laterally away from the pore and the helix rotating upward and bending downward at the extracellular end (Yan et al., 2017).Open in a separate windowFigure 3.A comparison between Na_VPaS and rNa_V1.5 structures. (A) Structures of eukaryotic Na_V channel reveal two distinct conformations of the depolarized VSD-III, varying in the number of gating charges (K and R) across the HCS. In Na_VPaS (left, light blue; PDB accession no. 5X0M), the activated VSD-III transfers two positively charged residues from an internal to an external side. The other VSD-III activated conformation from rNa_V1.5 (right, cyan; PDB accession no. 6UZ3) captures the total of four gating charges transfer. (B) An overlay of two Na_V channel structures shows that the VSD-III fully activated conformation (cyan) is needed to facilitate the binding of the IFMT motif.Two-step VSD-III activation is further supported by functional experiments that tracked Na_V channel VSD movement optically via the voltage-clamp fluorometry (VCF) technique (Zhu et al., 2016; Rudokas et al., 2014; Chanda and Bezanilla, 2002; Mannuzzu et al., 1996). The VCF protocol was developed to observe the conformational dynamics of individual S4 segments and can reveal structurally correlated kinetic information (Cowgill and Chanda, 2019). By attaching a fluorophore to the extracellular S3–S4 linker site through a cysteine mutation, the movement of the proximal S4 segment can be detected upon the changes in fluorescence emission caused by differences in the surrounding environment (Cha et al., 1999b; Glauner et al., 1999; Bezanilla, 2000; Chanda and Bezanilla, 2002). Simultaneous recordings of fluorescence emission and ionic current identified the concurrent activation of repeat I–III VSDs during Na_V channel activation, with a slight delay of VSD-IV activation lagging the rise of the I_Na (Fig. 4 A). The VSD-III is most sensitive to hyperpolarized potentials, having the half-maximal voltage of the fluorescence–voltage curve at the most negative potential among all VSDs (Fig. 4 B; Varga et al., 2015). The VSD-III is already activated at highly negative potentials where closed-state inactivation occurs.Open in a separate windowFigure 4.Experimental data supporting a two-step VSD-III activation model. (A) VCF recordings of rNa_V1.4 show simultaneous initiation of repeats I–III VSD activation (blue, red, and green) but a delay in VSD-IV activation (black), which is reduced with increasing voltages. Adapted from Chanda and Bezanilla (2002). (B) Fluorescence–voltage (FV) curves of hNa_V1.5 VCF constructs show that VSD-III activates at hyperpolarized potential, earlier than other repeat VSDs. Adapted from Varga et al. (2015). LFS, large fluorescence signal. (C) VCF recording of S1113C in rNa_V1.4 elicits two stages of VSD-III movement, with an initial increase followed by a decrease in fluorescence emission. Adapted from Chanda and Bezanilla (2002). (D) Coexpression of hNa_V1.5 and β3-subunit at high ratio (1:4 and 1:6) yields two distinct components of VSD-III activation kinetics, as detected by fluorescence emission (inset) and fluorescence–voltage curves. Adapted from Zhu et al. (2017). (E) A correlation between VSD-III deactivation time constants and the depolarization duration in WT hNa_V1.5 suggests the multiple steps of VSD-III activation, which is dependent on the IFMT motif as illustrated by the loss of correlation in IQM mutation. Adapted from Hsu et al. (2017). Error bars represent the standard error of the mean from the sample size of 3–6 measurements.Two stages of VSD-III activation were demonstrated in the VCF recordings of rNa_V1.4 VSD-III with a fluorophore attached to S1113C, showing an initial increase in the fluorescence emission followed by a reduction (Fig. 4 C; Chanda and Bezanilla, 2002). Similarly, in another independent recording of hNa_V1.5 in the presence of high β3-subunit expressions, the fluorescence emission tracking VSD-III activation at M1296C showed two components with distinct kinetics (Fig. 4 D; Zhu et al., 2017). The curves also displayed two elements of VSD-III activation, implying that VSD-III activates with different kinetics over different voltage ranges. Recently, the VCF recordings of neonatal form Na_V1.5 (Na_V1.5e) reported the biphasic relationship of VSD-III fluorescence–voltage curve reaching its maximum at −50 mV and declining with more depolarized potentials (Brake et al., 2021 Preprint). Finally, the time constant of VSD-III deactivation, unlike other VSDs, varied with depolarization duration, hinting at the multiple-step VSD-III activation mechanism (Fig. 4 E; Hsu et al., 2017). If the VSD activation progressed to only a single conformation, then prolonged depolarization would not affect the time it takes to return to its resting position. Instead, when the S4 segment travels across two distinct states, longer depolarizing pulses bias the transition to the second step and result in longer deactivation times. Altogether, extensive structural and functional evidence strongly supports two-step VSD-III activation.The two conformations of VSD-III activation lead to an interesting consequence that each activation step may dictate a different conformation of the IFMT crevice. Because of the highly similar structures in the transmembrane core and the same length of III-IV linker across eukaryotic Na_V channels, the relative positions of repeat III S4 and its ensuing S4–S5 linker can provide relevant, useful insights. In the Na_VPaS structure, the position of the III S4 segment orients the S4–S5 linker so that it interferes with IFMT binding and likely obstructs the formation of the IFMT crevice (Fig. 3 B). The short section on the III–IV linker before the IFMT motif is also restricted from moving near the membrane, further constraining the range over which the IFMT motif can travel. In contrast, further translation of the III S4 in other eukaryotic structures pulls the S4–S5 linker up and away from the pore such that a crevice is exposed and direct interactions with the IFMT motif can be established (Fig. 3 B; Jiang et al., 2020). Consecutive helices on the III–IV linker provide additional interactions with the S4–S5 linkers of repeats III and IV that further stabilize inactivated state. Specifically, charged residues near the IFMT motif allosterically interact with repeats III and IV VSDs (Groome et al., 2003; Groome et al., 2007).Electrostatic interactions from the III–IV linker cause the immobilization of repeats III and IV VSDs during prolonged depolarization (Armstrong and Bezanilla, 1977; Kuo and Bean, 1994; Cha et al., 1999). The recovery from inactivation is thus affected by the kinetics of VSD-III and VSD-IV deactivation. When the IFMT motif was mutated to IQMT, fast inactivation was greatly impaired, and the deactivation of repeats III and IV VSDs was significantly accelerated (Hsu et al., 2017). The time constant of VSD-III deactivation that normally increases with depolarizing pulse duration no longer shows such correlation in the IQMT mutant channel (Fig. 4 E), suggesting that IFMT binding specifically stabilizes the second VSD-III activated state (Hsu et al., 2017). The same study also showed that inactivation recovery after >200 ms depolarization is defined exclusively by the deactivation of VSD-III (Hsu et al., 2017). This result coincides with the second step VSD-III activation, transferring the highest number of gating charges across the HCS and hence likely returning last to the resting position. Other arrhythmogenic mutations along the IFMT binding sites on S4–S5 linkers of III and IV (N1325S, A1330, and N1659A) also showed enhanced recovery from inactivation, accompanying the decreased time constant of VSD-III deactivation (Hsu et al., 2017). Together, the differential states of VSD-III activation are likely to provide the basis for distinct Na_V channel inactivation kinetics at hyperpolarized and depolarized potentials.Physiological and pharmacological modulation of repeat III VSD activation affects Na_V channel inactivationMultiple factors involved in the regulation of Na_V channel inactivation, such as the β-subunits, drugs and toxins, were shown to modulate VSD-III activation. Resolved structures of hNa_V1.2, hNa_V1.4, and hNa_V1.7 with coexpressed β1-subunit identified its docking site near the VSD-III with interactions between the Ig domain of β1-subunit and repeat IV L6 (extracellular linker between P-loop and S6) and repeat I L5 (extracellular linker between S5 and P-loop) loops (Pan et al., 2018; Shen et al., 2019; Pan et al., 2019). Such interaction sites imply β-subunit modulation of VSDs III and IV kinetics. Careful study of the mechanism of noncovalent β-subunits (β1 and β3) demonstrated a unique impact on the initiation of repeat III and IV VSDs activation, which results in distinct modulatory effects on the hNa_V1.5 (Zhu et al., 2017). Consistent with the resolved structures, coexpression of β3-subunit shifts VSD-III and VSD-IV activation, leading to altered steady-state inactivation, activation, and fast inactivation kinetics. Switching the charge of a β3-subunit transmembrane glutamic acid (E176K) altered VSD-III activation of hNa_V1.5 and affected the channel recovery from inactivation (Salvage et al., 2019). To the contrary, the β1-subunit regulates Na_V channel inactivation solely through the modulation of the VSD-IV, resulting in a shift in steady-state inactivation, while having no impact on I_Na activation or fast inactivation kinetics (Zhu et al., 2017). This unique interaction between cardiac Na_V channel and β1-subunit is further supported by rNa_V1.5 and hNa_V1.5 structures that failed to resolve the coexpressed β1-subunit (Jiang et al., 2020; Li et al., 2021). The different regulating mechanisms between β1 and β3 subunits on hNa_V1.5, therefore, emphasize the significance of the VSD-III conformation in determining the state and kinetics of Na_V channel inactivation.The action of the local anesthetic (LA) lidocaine has also been intimately linked to VSD-III function. LAs are normally used for local and regional anesthesia and the treatment of excitatory pathologies, including epilepsy and cardiac arrhythmia. Their significant therapeutic effect is achieved through use-dependent block, an increase in channel blocking over repetitive activation. This effect is due to the higher-affinity binding when the channel is in the open and inactivated states (Bean, 1981), resulting in a cumulative increase of inactivated channels over successive pulses of depolarization. The modulated receptor hypothesis postulates that the accessibility to the high-affinity binding site depends on the state of the channel, which in turn involves voltage-dependent VSD activation (Hille, 2001). Lidocaine stabilizes VSD-III in its activated state in rNa_V1.4 channel coexpressed with β1-subunit (Muroi and Chanda, 2009). The prepositioning of both repeats III and IV VSDs in the outward position (through MTSEA-biotin modified R3C III and R2C IV) enhances lidocaine block significantly in hNa_V1.5 α-subunit alone, and when the inactivation gate is disrupted via IFM/ICM mutation, the stabilization of individual III or IV S4 increases lidocaine affinity similar to when both VSDs are stabilized, suggesting an equally significant contribution of VSD-IV as VSD-III to lidocaine block (Sheets and Hanck, 2003; Sheets and Hanck, 2007). The discrepancy that is observed between two studies by Muroi et al. and Sheets et al. could be attributed to the intrinsic properties of different Na_V channel isoforms as well as the presence of β1-subunit. We found that lidocaine induces hyperpolarizing shifts in VSD-III and VSD-IV activations, but β1-subunit coexpression subdues the VSD-IV modulation (Zhu et al., 2021). The inactivation gate, though not necessary for drug binding, drastically reduces drug binding affinity when it is removed (Bennett et al., 1995; Sheets and Hanck, 2007). How the inactivation gate contributes to the kinetics of lidocaine block is still not fully understood (Fozzard et al., 2011).It is, nevertheless, interesting to note the parallel molecular determinants between lidocaine and the IFMT motif binding affinity. Both are dependent upon the positions of VSD-III and VSD-IV. Even though lidocaine is found to bind in the inner pore of Na_V channel through interaction with phenylalanine and tyrosine on the repeat IV S6 segment (Ragsdale et al., 1994; Yarov-Yarovoy et al., 2002; F1759 and Y1766 in Na_V1.5) and directly blocks Na⁺ conductance, binding of the IFMT motif on the rim of the activation gate might also shape the local interaction around the lidocaine-binding site. For one, the IFMT binding stabilizes the outward positions of VSD-III and VSD-IV, which are integral to the drug receptor formation. Thus, the VSD translocation of repeats III and IV could have twofold implications, directly through the VSD-pore coupling and indirectly through the binding of the IFMT motif. According to our hypothesis, the preactivation of VSD-III by lidocaine would favor the high-affinity binding of the IFMT motif and facilitate the inactivation, which in turn enables the high drug affinity–binding mode. A chemical accessibility study of the inactivation gate position during lidocaine block in rNa_V1.4 F1304C (IFMT motif) by MTSET modification showed that lidocaine favors the inactivation gate closure, hypothetically by promoting the transition along the activation pathway (Vedantham and Cannon, 1999).Finally, a study of a lidocaine derivative, mexiletine, also shows that the level of VSD-III activation strongly affects drug efficacy (Zhu et al., 2019; Moreno et al., 2019). Mexiletine preferentially blocks late I_Na, which is enhanced in many cardiac pathologies, including long-QT type 3 syndrome. Investigation into varied mexiletine sensitivity of different long-QT type 3 mutations identified a correlation to the fraction of activated VSD-III. These results resonate with the findings from the lidocaine studies on the significance of VSD-III position on a determination of LA like drug affinity. Taken together, these studies of Na_V channel accessory subunits and its blockers emphasize the essential role of VSD-III activation in the regulation of Na_V channel inactivation kinetics.Future perspectiveThere is often a singular focus on individual channel domains in the regulation of the Na_V channel inactivation, consistent with the m³h gating model of Hodgkin and Huxley, where a single “h” gate mediates inactivation. However, a myriad of studies has proved the process to be more elaborate and complicated than the simplified representation of this model, and intricate inactivation is not unique to Na_V channels. Homotetrameric potassium channels and prokaryotic Na_V channels display complexity in the coupling of inactivation to channel activation (Hoshi et al., 1990; Yang et al., 2018). Toxin and pharmacological studies suggest the possibility of a drug-induced alteration in Na_V channel conducting conformation (Baumgarten et al., 1991) or a unique drug-bound inactivated state (Finol-Urdaneta et al., 2019a; Finol-Urdaneta et al., 2019b; Ong et al., 2000). Distinct channel isoforms also tend to respond to drug and toxin differently, such as the differential sensitivity to tetrodotoxin or a unique external lidocaine-binding site in the cardiac Na_V channel (Baumgarten et al., 1991). More importantly, variabilities in the voltage-dependent activation and inactivation curves among different Na_V isoforms suggest their distinctive propensity to closed-state inactivation that likely stems from the unique sequence of VSDs activation (Brake et al., 2021 Preprint) determined by their rates of activation and the midpoint of activation curve (Capes et al., 2013). Because of the heterogeneity and complexity of the channel kinetics, combined results from multiple methods should be interpreted with careful consideration.Through multiple Na_V channel structures and electrophysiological studies combined with the fluorescent labeling technique, we argue here that VSD-III, together with VSD-IV, governs the binding affinity for the inactivation gate and thus affects the different inactivated states. Still missing, however, is the discrete structure of state-dependent conformations such as those of the resting-state and closed-state inactivation, where some VSDs are not activated. One possible means to overcome this obstacle is the use of toxin, small-molecule modulators, or biochemical methods to force certain states of the channel (Clairfeuille et al., 2019; Wisedchaisri et al., 2019; Moreno et al., 2019). Linking channel structure to channel functional states is also challenging but is achievable via dynamic tools such as FRET. FRET is a distance-dependent energy transfer between two fluorophores, namely the excited donor and the emitting acceptor (Sekar and Periasamy, 2003), that can be attached to any protein or peptide. This method allows for the measurement of molecular proximity between two different parts within angstrom distances and thus is often used to test molecular interactions. When combined with patch clamp, FRET measurement could be used to determine the interactions between the channel’s different domains at various functional states. Integrating such knowledge to the available structures would provide the means to derive a state-dependent structure and a dynamic change in its conformation. Recent work by Kubota et al. (2017) used a lanthanide-based resonance energy transfer, which allows distance measurement, to map the voltage sensor positions of rNa_V1.4 during the resting and inactivated states. To gain better insight into Na_V channel inactivation, the ability to label the intracellular linkers or region is critical. Conventional fluorescent proteins and genetically encoded tags produce large stable fluorescent signals but are disruptive to the protein structure due to their bulkiness (Toseland, 2013). To overcome such limitations, genetic code expansion through site-specific mutation of fluorescent unnatural amino acids such as ANAP (Shandell et al., 2019; Kalstrup and Blunck, 2017) could serve as potential alternatives. Using these techniques to track activation gating and intracellular inactivation particle binding over different membrane potentials would allow differentiation of the states of inactivation, namely closed and open, and ultimately dissection of the regulatory components required for the physiological function of Na_V channels.     

Rem uncouples excitation–contraction coupling in adult skeletal muscle fibers

In skeletal muscle, excitation–contraction (EC) coupling requires depolarization-induced conformational rearrangements in L-type Ca²⁺ channel (Ca_V1.1) to be communicated to the type 1 ryanodine-sensitive Ca²⁺ release channel (RYR1) of the sarcoplasmic reticulum (SR) via transient protein–protein interactions. Although the molecular mechanism that underlies conformational coupling between Ca_V1.1 and RYR1 has been investigated intensely for more than 25 years, the question of whether such signaling occurs via a direct interaction between the principal, voltage-sensing α_1S subunit of Ca_V1.1 and RYR1 or through an intermediary protein persists. A substantial body of evidence supports the idea that the auxiliary β_1a subunit of Ca_V1.1 is a conduit for this intermolecular communication. However, a direct role for β_1a has been difficult to test because β_1a serves two other functions that are prerequisite for conformational coupling between Ca_V1.1 and RYR1. Specifically, β_1a promotes efficient membrane expression of Ca_V1.1 and facilitates the tetradic ultrastructural arrangement of Ca_V1.1 channels within plasma membrane–SR junctions. In this paper, we demonstrate that overexpression of the RGK protein Rem, an established β subunit–interacting protein, in adult mouse flexor digitorum brevis fibers markedly reduces voltage-induced myoplasmic Ca²⁺ transients without greatly affecting Ca_V1.1 targeting, intramembrane gating charge movement, or releasable SR Ca²⁺ store content. In contrast, a β_1a-binding–deficient Rem triple mutant (R200A/L227A/H229A) has little effect on myoplasmic Ca²⁺ release in response to membrane depolarization. Thus, Rem effectively uncouples the voltage sensors of Ca_V1.1 from RYR1-mediated SR Ca²⁺ release via its ability to interact with β_1a. Our findings reveal Rem-expressing adult muscle as an experimental system that may prove useful in the definition of the precise role of the β_1a subunit in skeletal-type EC coupling.     

Molecular regions underlying the activation of low- and high-voltage activating calcium channels

We have studied two aspects of calcium channel activation. First, we investigated the molecular regions that are important in determining differences in activation between low- and high-voltage activated channels. For this, we made chimeras between the low-voltage activating Ca_V3.1 channel and the high-voltage activating Ca_V1.2 channel. Chimeras were expressed in oocytes, and calcium channel currents recorded by voltage clamp. For domain I, we found that the molecular region that is important in determining the voltage dependence of activation comprises the pore regions S5-P as well as P-S6, but surprisingly not the voltage sensor S1–S4 region, which might have been expected to play a major part. By contrast, the smaller, but still significant, modulating effects of domain II on activation properties were due to effects involving both S1–S4 and S5–S6 but not the I/II linker. Second, during channel activation we studied movement of the S4 segment in domain I of one of the chimeras, using cysteine-scanning mutagenesis. The reagent parachloromercuribenzensulfonate inhibited currents for mutants V263, A265, L266 and A268, but not for F269 and V271, and voltage dependence of inhibition for residue V263 indicated S4 movement, which occurred before channel opening. The data indicate movement outwards upon depolarisation so as to expose amino acids up to residue 268 in S4.Junying Li and Louisa Stevens contributed equally to this work.     

S3-S4 Linker Length Modulates the Relaxed State of a Voltage-Gated Potassium Channel

Voltage-sensing domains (VSDs) are membrane protein modules found in ion channels and enzymes that are responsible for a large number of fundamental biological tasks, such as neuronal electrical activity. The VSDs switch from a resting to an active conformation upon membrane depolarization, altering the activity of the protein in response to voltage changes. Interestingly, numerous studies describe the existence of a third distinct state, called the relaxed state, also populated at positive potentials. Although some physiological roles for the relaxed state have been suggested, little is known about the molecular determinants responsible for the development and modulation of VSD relaxation. Several lines of evidence have suggested that the linker (S3-S4 linker) between the third (S3) and fourth (S4) transmembrane segments of the VSD alters the equilibrium between resting and active conformations. By measuring gating currents from the Shaker potassium channel, we demonstrate here that shortening the S3-S4 linker stabilizes the relaxed state, whereas lengthening the linker or splitting it and coinjecting two fragments of the channel have little effect. We propose that natural variations of the length of the S3-S4 linker in various VSD-containing proteins may produce differential VSD relaxation in vivo.     

Different pathways for activation and deactivation in CaV1.2: a minimal gating model

Point mutations in pore-lining S6 segments of Ca_V1.2 shift the voltage dependence of activation into the hyperpolarizing direction and significantly decelerate current activation and deactivation. Here, we analyze theses changes in channel gating in terms of a circular four-state model accounting for an activation R–A–O and a deactivation O–D–R pathway. Transitions between resting-closed (R) and activated-closed (A) states (rate constants x(V) and y(V)) and open (O) and deactivated-open (D) states (u(V) and w(V)) describe voltage-dependent sensor movements. Voltage-independent pore openings and closures during activation (A–O) and deactivation (D–R) are described by rate constants α and β, and γ and δ, respectively. Rate constants were determined for 16-channel constructs assuming that pore mutations in IIS6 do not affect the activating transition of the voltage-sensing machinery (x(V) and y(V)). Estimated model parameters of 15 Ca_V1.2 constructs well describe the activation and deactivation processes. Voltage dependence of the “pore-releasing” sensor movement ((x(V)) was much weaker than the voltage dependence of “pore-locking” sensor movement (y(V)). Our data suggest that changes in membrane voltage are more efficient in closing than in opening Ca_V1.2. The model failed to reproduce current kinetics of mutation A780P that was, however, accurately fitted with individually adjusted x(V) and y(V). We speculate that structural changes induced by a proline substitution in this position may disturb the voltage-sensing domain.     

Probing α-310 Transitions in a Voltage-Sensing S4 Helix

The S4 helix of voltage sensor domains (VSDs) transfers its gating charges across the membrane electrical field in response to changes of the membrane potential. Recent studies suggest that this process may occur via the helical conversion of the entire S4 between α and 3₁₀ conformations. Here, using LRET and FRET, we tested this hypothesis by measuring dynamic changes in the transmembrane length of S4 from engineered VSDs expressed in Xenopus oocytes. Our results suggest that the native S4 from the Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) does not exhibit extended and long-lived 3₁₀ conformations and remains mostly α-helical. Although the S4 of NavAb displays a fully extended 3₁₀ conformation in x-ray structures, its transplantation in the Ci-VSP VSD scaffold yielded similar results as the native Ci-VSP S4. Taken together, our study does not support the presence of long-lived extended α-to-3₁₀ helical conversions of the S4 in Ci-VSP associated with voltage activation.     

Cooperative Activation of the T-type CaV3.2 Channel: INTERACTION BETWEEN DOMAINS II AND III*

T-type Ca_V3 channels are important mediators of Ca²⁺ entry near the resting membrane potential. Little is known about the molecular mechanisms responsible for channel activation. Homology models based upon the high-resolution structure of bacterial Na_V channels predict interaction between the S4-S5 helix of Domain II (IIS4-S5) and the distal S6 pore region of Domain II (IIS6) and Domain III (IIIS6). Functional intra- and inter-domain interactions were investigated with a double mutant cycle analysis. Activation gating and channel kinetics were measured for 47 single mutants and 20 pairs of mutants. Significant coupling energies (ΔΔG_interact ≥ 1.5 kcal mol⁻¹) were measured for 4 specific pairs of mutants introduced between IIS4-S5 and IIS6 and between IIS4-S5 and IIIS6. In agreement with the computer based models, Thr-911 in IIS4-S5 was functionally coupled with Ile-1013 in IIS6 during channel activation. The interaction energy was, however, found to be stronger between Val-907 in IIS4-S5 and Ile-1013 in IIS6. In addition Val-907 was significantly coupled with Asn-1548 in IIIS6 but not with Asn-1853 in IVS6. Altogether, our results demonstrate that the S4-S5 and S6 helices from adjacent domains are energetically coupled during the activation of a low voltage-gated T-type Ca_V3 channel.     

S3-S4 Linker Length Modulates the Relaxed State of a Voltage-Gated Potassium Channel

Voltage-sensing domains (VSDs) are membrane protein modules found in ion channels and enzymes that are responsible for a large number of fundamental biological tasks, such as neuronal electrical activity. The VSDs switch from a resting to an active conformation upon membrane depolarization, altering the activity of the protein in response to voltage changes. Interestingly, numerous studies describe the existence of a third distinct state, called the relaxed state, also populated at positive potentials. Although some physiological roles for the relaxed state have been suggested, little is known about the molecular determinants responsible for the development and modulation of VSD relaxation. Several lines of evidence have suggested that the linker (S3-S4 linker) between the third (S3) and fourth (S4) transmembrane segments of the VSD alters the equilibrium between resting and active conformations. By measuring gating currents from the Shaker potassium channel, we demonstrate here that shortening the S3-S4 linker stabilizes the relaxed state, whereas lengthening the linker or splitting it and coinjecting two fragments of the channel have little effect. We propose that natural variations of the length of the S3-S4 linker in various VSD-containing proteins may produce differential VSD relaxation in vivo.     

CaV1.2 I-II linker structure and Timothy syndrome

Ca_V channels are multi-subunit protein complexes that enable inward cellular Ca²⁺ currents in response to membrane depolarization. We recently described structure-function studies of the intracellular α1 subunit domain I-II linker, directly downstream of domain IS6. The results show the extent of the linker’s helical structure to be subfamily dependent, as dictated by highly conserved primary sequence differences. Moreover, the difference in structure confers different biophysical properties, particularly the extent and kinetics of voltage and calcium-dependent inactivation. Timothy syndrome is a human genetic disorder due to mutations in the Ca_V1.2 gene. Here, we explored whether perturbation of the I-II linker helical structure might provide a mechanistic explanation for a Timothy syndrome mutant’s (human Ca_V1.2 G406R equivalent) biophysical effects on inactivation and activation. The results are equivocal, suggesting that a full mechanistic explanation for this Timothy syndrome mutation requires further investigation.     

Molecular Determinants of the CaVβ-induced Plasma Membrane Targeting of the CaV1.2 Channel

Ca_Vβ subunits modulate cell surface expression and voltage-dependent gating of high voltage-activated (HVA) Ca_V1 and Ca_V2 α1 subunits. High affinity Ca_Vβ binding onto the so-called α interaction domain of the I-II linker of the Ca_Vα1 subunit is required for Ca_Vβ modulation of HVA channel gating. It has been suggested, however, that Ca_Vβ-mediated plasma membrane targeting could be uncoupled from Ca_Vβ-mediated modulation of channel gating. In addition to Ca_Vβ, Ca_Vα2δ and calmodulin have been proposed to play important roles in HVA channel targeting. Indeed we show that co-expression of Ca_Vα2δ caused a 5-fold stimulation of the whole cell currents measured with Ca_V1.2 and Ca_Vβ3. To gauge the synergetic role of auxiliary subunits in the steady-state plasma membrane expression of Ca_V1.2, extracellularly tagged Ca_V1.2 proteins were quantified using fluorescence-activated cell sorting analysis. Co-expression of Ca_V1.2 with either Ca_Vα2δ, calmodulin wild type, or apocalmodulin (alone or in combination) failed to promote the detection of fluorescently labeled Ca_V1.2 subunits. In contrast, co-expression with Ca_Vβ3 stimulated plasma membrane expression of Ca_V1.2 by a 10-fold factor. Mutations within the α interaction domain of Ca_V1.2 or within the nucleotide kinase domain of Ca_Vβ3 disrupted the Ca_Vβ3-induced plasma membrane targeting of Ca_V1.2. Altogether, these data support a model where high affinity binding of Ca_Vβ to the I-II linker of Ca_Vα1 largely accounts for Ca_Vβ-induced plasma membrane targeting of Ca_V1.2.     

Ca2+ Binding/Permeation via Calcium Channel,CaV1.1, Regulates the Intracellular Distribution of the Fatty Acid Transport Protein,CD36, and Fatty Acid Metabolism

Ca²⁺ permeation and/or binding to the skeletal muscle L-type Ca²⁺ channel (Ca_V1.1) facilitates activation of Ca²⁺/calmodulin kinase type II (CaMKII) and Ca²⁺ store refilling to reduce muscle fatigue and atrophy (Lee, C. S., Dagnino-Acosta, A., Yarotskyy, V., Hanna, A., Lyfenko, A., Knoblauch, M., Georgiou, D. K., Poché, R. A., Swank, M. W., Long, C., Ismailov, I. I., Lanner, J., Tran, T., Dong, K., Rodney, G. G., Dickinson, M. E., Beeton, C., Zhang, P., Dirksen, R. T., and Hamilton, S. L. (2015) Skelet. Muscle 5, 4). Mice with a mutation (E1014K) in the Cacna1s (α₁ subunit of Ca_V1.1) gene that abolishes Ca²⁺ binding within the Ca_V1.1 pore gain more body weight and fat on a chow diet than control mice, without changes in food intake or activity, suggesting that Ca_V1.1-mediated CaMKII activation impacts muscle energy expenditure. We delineate a pathway (Ca_v1.1→ CaMKII→ NOS) in normal skeletal muscle that regulates the intracellular distribution of the fatty acid transport protein, CD36, altering fatty acid metabolism. The consequences of blocking this pathway are decreased mitochondrial β-oxidation and decreased energy expenditure. This study delineates a previously uncharacterized Ca_V1.1-mediated pathway that regulates energy utilization in skeletal muscle.     

Evolutionary imprint of activation: The design principles of VSDs

Voltage-sensor domains (VSDs) are modular biomolecular machines that transduce electrical signals in cells through a highly conserved activation mechanism. Here, we investigate sequence–function relationships in VSDs with approaches from information theory and probabilistic modeling. Specifically, we collect over 6,600 unique VSD sequences from diverse, long-diverged phylogenetic lineages and relate the statistical properties of this ensemble to functional constraints imposed by evolution. The VSD is a helical bundle with helices labeled S1–S4. Surrounding conserved VSD residues such as the countercharges and the S2 phenylalanine, we discover sparse networks of coevolving residues. Additional networks are found lining the VSD lumen, tuning the local hydrophilicity. Notably, state-dependent contacts and the absence of coevolution between S4 and the rest of the bundle are imprints of the activation mechanism on the VSD sequence ensemble. These design principles rationalize existing experimental results and generate testable hypotheses.