Coupling between the voltage-sensing and pore domains in a voltage-gated potassium channel

Voltage-dependent potassium (Kv), sodium (Nav), and calcium channels open and close in response to changes in transmembrane (TM) potential, thus regulating cell excitability by controlling ion flow across the membrane. An outstanding question concerning voltage gating is how voltage-induced conformational changes of the channel voltage-sensing domains (VSDs) are coupled through the S4-S5 interfacial linking helices to the opening and closing of the pore domain (PD). To investigate the coupling between the VSDs and the PD, we generated a closed Kv channel configuration from Aeropyrum pernix (KvAP) using atomistic simulations with experiment-based restraints on the VSDs. Full closure of the channel required, in addition to the experimentally determined TM displacement, that the VSDs be displaced both inwardly and laterally around the PD. This twisting motion generates a tight hydrophobic interface between the S4-S5 linkers and the C-terminal ends of the pore domain S6 helices in agreement with available experimental evidence.     

Mapping the membrane-aqueous border for the voltage-sensing domain of a potassium channel

Voltage-sensing domains (VSDs) play diverse roles in biology. As integral components, they can detect changes in the membrane potential of a cell and couple these changes to activity of ion channels and enzymes. As independent proteins, homologues of the VSD can function as voltage-dependent proton channels. To sense voltage changes, the positively charged fourth transmembrane segment, S4, must move across the energetically unfavorable hydrophobic core of the bilayer, which presents a barrier to movement of both charged species and protons. To reduce the barrier to S4 movement, it has been suggested that aqueous crevices may penetrate the protein, reducing the extent of total movement. To investigate this hypothesis in a system containing fully functional channels in a native environment with an intact membrane potential, we have determined the contour of the membrane-aqueous border of the VSD of KvAP in Escherichia coli by examining the chemical accessibility of introduced cysteines. The results revealed the contour of the membrane-aqueous border of the VSD in its activated conformation. The water-inaccessible regions of S1 and S2 correspond to the standard width of the membrane bilayer (~28 A), but those of S3 and S4 are considerably shorter (> or = 40%), consistent with aqueous crevices pervading both the extracellular and intracellular ends. One face of S3b and the entire S3a were water-accessible, reducing the water-inaccessible region of S3 to just 10 residues, significantly shorter than for S4. The results suggest a key role for S3 in reducing the distance S4 needs to move to elicit gating.     

A localized interaction surface for voltage-sensing domains on the pore domain of a K+ channel

Voltage-gated K+ channels contain a central pore domain and four surrounding voltage-sensing domains. How and where changes in the structure of the voltage-sensing domains couple to the pore domain so as to gate ion conduction is not understood. The crystal structure of KcsA, a bacterial K+ channel homologous to the pore domain of voltage-gated K+ channels, provides a starting point for addressing this question. Guided by this structure, we used tryptophan-scanning mutagenesis on the transmembrane shell of the pore domain in the Shaker voltage-gated K+ channel to localize potential protein-protein and protein-lipid interfaces. Some mutants cause only minor changes in gating and when mapped onto the KcsA structure cluster away from the interface between pore domain subunits. In contrast, mutants producing large changes in gating tend to cluster near this interface. These results imply that voltage-sensing domains interact with localized regions near the interface between adjacent pore domain subunits.     

Functional diversity of potassium channel voltage-sensing domains

Voltage-gated potassium channels or Kv's are membrane proteins with fundamental physiological roles. They are composed of 2 main functional protein domains, the pore domain, which regulates ion permeation, and the voltage-sensing domain, which is in charge of sensing voltage and undergoing a conformational change that is later transduced into pore opening. The voltage-sensing domain or VSD is a highly conserved structural motif found in all voltage-gated ion channels and can also exist as an independent feature, giving rise to voltage sensitive enzymes and also sustaining proton fluxes in proton-permeable channels. In spite of the structural conservation of VSDs in potassium channels, there are several differences in the details of VSD function found across variants of Kvs. These differences are mainly reflected in variations in the electrostatic energy needed to open different potassium channels. In turn, the differences in detailed VSD functioning among voltage-gated potassium channels might have physiological consequences that have not been explored and which might reflect evolutionary adaptations to the different roles played by Kv channels in cell physiology.     

The orientation and molecular movement of a k(+) channel voltage-sensing domain

Voltage-gated channels operate through the action of a voltage-sensing domain (membrane segments S1-S4) that controls the conformation of gates located in the pore domain (membrane segments S5-S6). Recent structural studies on the bacterial K(v)AP potassium channel have led to a new model of voltage sensing in which S4 lies in the lipid at the channel periphery and moves through the membrane as a unit with a portion of S3. Here we describe accessibility probing and disulfide scanning experiments aimed at determining how well the K(v)AP model describes the Drosophila Shaker potassium channel. We find that the S1-S3 helices have one end that is externally exposed, S3 does not undergo a transmembrane motion, and S4 lies in close apposition to the pore domain in the resting and activated state.     

The cooperative voltage sensor motion that gates a potassium channel

The four arginine-rich S4 helices of a voltage-gated channel move outward through the membrane in response to depolarization, opening and closing gates to generate a transient ionic current. Coupling of voltage sensing to gating was originally thought to operate with the S4s moving independently from an inward/resting to an outward/activated conformation, so that when all four S4s are activated, the gates are driven to open or closed. However, S4 has also been found to influence the cooperative opening step (Smith-Maxwell et al., 1998a), suggesting a more complex mechanism of coupling. Using fluorescence to monitor structural rearrangements in a Shaker channel mutant, the ILT channel (Ledwell and Aldrich, 1999), that energetically isolates the steps of activation from the cooperative opening step, we find that opening is accompanied by a previously unknown and cooperative movement of S4. This gating motion of S4 appears to be coupled to the internal S6 gate and to two forms of slow inactivation. Our results suggest that S4 plays a direct role in gating. While large transmembrane rearrangements of S4 may be required to unlock the gating machinery, as proposed before, it appears to be the gating motion of S4 that drives the gates to open and close.     

The voltage-sensing domain of a hERG1 mutant is a cation-selective channel

A cationic leak current known as an “omega current” may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li⁺, K⁺, Cs⁺, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.     

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.     

The S4 voltage sensor packs against the pore domain in the KAT1 voltage-gated potassium channel

In voltage-gated ion channels, the S4 transmembrane segment responds to changes in membrane potential and controls channel opening. The local environment of S4 is still unknown, even regarding the basic question as to whether S4 is close to the pore domain. Relying on the ability of functional KAT1 channels to rescue potassium (K+) transport-deficient yeast, we have performed an unbiased mutagenesis screen aimed at determining whether S4 packs against S5 of the pore domain. Starting with semilethal mutations of surface-exposed S5 residues of the KAT1 pore domain, we have screened randomly mutagenized libraries of S4 or S1-S3 for second-site suppressors. Our study identifies two S4 residues that interact in a highly specific manner with two S5 residues in the middle of the membrane-spanning regions, supporting a model in which the S4 voltage sensor packs against the pore domain in the hyperpolarized, or "down," state of S4.     

TWIK-2, a new weak inward rectifying member of the tandem pore domain potassium channel family

Potassium channels are found in all mammalian cell types, and they perform many distinct functions in both excitable and non-excitable cells. These functions are subserved by several different families of potassium channels distinguishable by primary sequence features as well as by physiological characteristics. Of these families, the tandem pore domain potassium channels are a new and distinct class, primarily distinguished by the presence of two pore-forming domains within a single polypeptide chain. We have cloned a new member of this family, TWIK-2, from a human brain cDNA library. Primary sequence analysis of TWIK-2 shows that it is most closely related to TWIK-1, especially in the pore-forming domains. Northern blot analysis reveals the expression of TWIK-2 in all human tissues assayed except skeletal muscle. Human TWIK-2 expressed heterologously in Xenopus oocytes is a non-inactivating weak inward rectifier with channel properties similar to TWIK-1. Pharmacologically, TWIK-2 channels are distinct from TWIK-1 channels in their response to quinidine, quinine, and barium. TWIK-2 is inhibited by intracellular, but not extracellular, acidification. This new clone reveals the existence of a subfamily in the tandem pore domain potassium channel family with weak inward rectification properties.     

Expression of the two pore domain potassium channel TREK-1 in human intervertebral disc cells

Potassium channels play a major role in intracellular homeostasis and regulation of cell volume. Intervertebral disc cells respond to mechanical loading in a complex manner. Mechanical loading may play a role in disc degeneration. Lumbar intervertebral disc samples from 5 patients (average age: 47 years, range: 25-64 years) were used for this study, investigating cells from the nucleus pulposus and the annulus fibrosus duplicate samples to determine RNA expression and protein expression. Analysis of mRNA expression by RT-PCR demonstrated that TREK 1 was expressed by nucleus pulposus (n=5) and annulus fibrosus (n=5) cells. Currently, TREK-1 is the only potassium channel known to be activated by intracellular acidosis, and responds to mechanical and chemical stimuli. Whilst the precise role of potassium channels in cellular homeostasis remains to be determined, TREK-1 may be important to protect disc cells against ischaemic damage, and subsequent disc degeneration, and may also play a role in effecting mechanotransduction. Further research is required to fully elucidate the role of the TREK-1 ion channel in intervertebral disc cells.     

A specific two-pore domain potassium channel blocker defines the structure of the TASK-1 open pore

Two-pore domain potassium (K(2P)) channels play a key role in setting the membrane potential of excitable cells. Despite their role as putative targets for drugs and general anesthetics, little is known about the structure and the drug binding site of K(2P) channels. We describe A1899 as a potent and highly selective blocker of the K(2P) channel TASK-1. As A1899 acts as an open-channel blocker and binds to residues forming the wall of the central cavity, the drug was used to further our understanding of the channel pore. Using alanine mutagenesis screens, we have identified residues in both pore loops, the M2 and M4 segments, and the halothane response element to form the drug binding site of TASK-1. Our experimental data were used to validate a K(2P) open-pore homology model of TASK-1, providing structural insights for future rational design of drugs targeting K(2P) channels.     

Sensing charges of the Ciona intestinalis voltage-sensing phosphatase

Voltage control over enzymatic activity in voltage-sensitive phosphatases (VSPs) is conferred by a voltage-sensing domain (VSD) located in the N terminus. These VSDs are constituted by four putative transmembrane segments (S1 to S4) resembling those found in voltage-gated ion channels. The putative fourth segment (S4) of the VSD contains positive residues that likely function as voltage-sensing elements. To study in detail how these residues sense the plasma membrane potential, we have focused on five arginines in the S4 segment of the Ciona intestinalis VSP (Ci-VSP). After implementing a histidine scan, here we show that four arginine-to-histidine mutants, namely R223H to R232H, mediate voltage-dependent proton translocation across the membrane, indicating that these residues transit through the hydrophobic core of Ci-VSP as a function of the membrane potential. These observations indicate that the charges carried by these residues are sensing charges. Furthermore, our results also show that the electrical field in VSPs is focused in a narrow hydrophobic region that separates the extracellular and intracellular space and constitutes the energy barrier for charge crossing.     

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.     

Contributions of counter-charge in a potassium channel voltage-sensor domain

Voltage-sensor domains couple membrane potential to conformational changes in voltage-gated ion channels and phosphatases. Highly coevolved acidic and aromatic side chains assist the transfer of cationic side chains across the transmembrane electric field during voltage sensing. We investigated the functional contribution of negative electrostatic potentials from these residues to channel gating and voltage sensing with unnatural amino acid mutagenesis, electrophysiology, voltage-clamp fluorometry and ab initio calculations. The data show that neutralization of two conserved acidic side chains in transmembrane segments S2 and S3, namely Glu293 and Asp316 in Shaker potassium channels, has little functional effect on conductance-voltage relationships, although Glu293 appears to catalyze S4 movement. Our results suggest that neither Glu293 nor Asp316 engages in electrostatic state-dependent charge-charge interactions with S4, likely because they occupy, and possibly help create, a water-filled vestibule.     

Coupling between the voltage-sensing and phosphatase domains of Ci-VSP

The Ciona intestinalis voltage sensor–containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.     

Hinge-bending motions in the pore domain of a bacterial voltage-gated sodium channel

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na(+) ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.     

Substitutions in the domain III voltage-sensing module enhance the sensitivity of an insect sodium channel to a scorpion beta-toxin

Scorpion β-toxins bind to the extracellular regions of the voltage-sensing module of domain II and to the pore module of domain III in voltage-gated sodium channels and enhance channel activation by trapping and stabilizing the voltage sensor of domain II in its activated state. We investigated the interaction of a highly potent insect-selective scorpion depressant β-toxin, Lqh-dprIT(3), from Leiurus quinquestriatus hebraeus with insect sodium channels from Blattella germanica (BgNa(v)). Like other scorpion β-toxins, Lqh-dprIT(3) shifts the voltage dependence of activation of BgNa(v) channels expressed in Xenopus oocytes to more negative membrane potentials but only after strong depolarizing prepulses. Notably, among 10 BgNa(v) splice variants tested for their sensitivity to the toxin, only BgNa(v)1-1 was hypersensitive due to an L1285P substitution in IIIS1 resulting from a U-to-C RNA-editing event. Furthermore, charge reversal of a negatively charged residue (E1290K) at the extracellular end of IIIS1 and the two innermost positively charged residues (R4E and R5E) in IIIS4 also increased the channel sensitivity to Lqh-dprIT(3). Besides enhancement of toxin sensitivity, the R4E substitution caused an additional 20-mV negative shift in the voltage dependence of activation of toxin-modified channels, inducing a unique toxin-modified state. Our findings provide the first direct evidence for the involvement of the domain III voltage-sensing module in the action of scorpion β-toxins. This hypersensitivity most likely reflects an increase in IIS4 trapping via allosteric mechanisms, suggesting coupling between the voltage sensors in neighboring domains during channel activation.     

Functional interactions at the interface between voltage-sensing and pore domains in the Shaker K(v) channel

Voltage-activated potassium (K(v)) channels contain a central pore domain that is partially surrounded by four voltage-sensing domains. Recent X-ray structures suggest that the two domains lack extensive protein-protein contacts within presumed transmembrane regions, but whether this is the case for functional channels embedded in lipid membranes remains to be tested. We investigated domain interactions in the Shaker K(v) channel by systematically mutating the pore domain and assessing tolerance by examining channel maturation, S4 gating charge movement, and channel opening. When mapped onto the X-ray structure of the K(v)1.2 channel the large number of permissive mutations support the notion of relatively independent domains, consistent with crystallographic studies. Inspection of the maps also identifies portions of the interface where residues are sensitive to mutation, an external cluster where mutations hinder voltage sensor activation, and an internal cluster where domain interactions between S4 and S5 helices from adjacent subunits appear crucial for the concerted opening transition.     

The distal C-terminal region of the KcsA potassium channel is a pH-dependent tetramerization domain

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition. Here, we investigate the involvement of this domain in pH-mediated channel opening by NMR using a soluble peptide corresponding to residues 128-160 of the CTD (CTD34). At neutral pH, CTD34 exhibits concentration-dependent spectral changes consistent with oligomer formation. We prove this slowly tumbling species to be a tetramer with a dissociation constant of (2.0±0.5)×10(-)(11)?M(3) by NMR and sedimentation equilibrium experiments. Whereas monomeric CTD34 is only mildly helical, secondary chemical shifts prove that the tetrameric species adopts a tight native-like helical bundle conformation. The tetrameric species undergoes pH-dependent dissociation, and CTD34 is fully monomeric below pH?5.0. The structural basis for this phenomenon is the destabilization of the tetrameric CTD34 by protonation of residue H145 in the monomeric form of the peptide. We conclude that (i) the CTD34 peptide is independently capable of forming a tetrameric helical bundle, and (ii) this structurally significant conformational shift is modulated by the effects of solution pH on residue H145. Therefore, the involvement of this domain in the pH gating of the channel is strongly suggested.