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1. Introduction
The voltage-sensitive domain (VSD) is a structure shared by a large number of proteins (Fig. 1). VSDs are composed of four transmembrane (TM) segments. Most VSDs are associated with voltage-gated ion channels (VGICs) and ensure the optimal modulation of VGICs in response to environmental signals. Proteins featuring VSDs are essential for cardiac, motor, cognitive, male fertility, and immune functions. Several mutations in VSDs have been reported to cause hypokalemic periodic paralysis (HypoPP) and normokalemic periodic paralysis (NormoPP), cardiac arrhythmias associated with dilated cardiomyopathy, peripheral nerve hyperexcitability, and epilepsy. Most of the mutations related to these channelopathies are located on the S4 segment of the VSDs of the Nav1.1, Nav1.2, Nav1.4, Nav1.5, Cav1.1, and Kv7.4 channels (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahine2012b, Reference Gosselin-Badaroudine, Moreau and Chahine2013; Matthews et al. Reference Matthews, Labrum, Sweeney, Sud, Haworth, Chinnery, Meola, Schorge, Kullmann, Davis and Hanna2009; Misra et al. Reference Misra, Kahlig and George2008; Volkers et al. Reference Volkers, Kahlig, Verbeek, Das, Van Kempen, Stroink, Augustijn, Van Nieuwenhuizen, Lindhout, George, Koeleman and Rook2011; Wuttke et al. Reference Wuttke, Jurkat-Rott, Paulus, Garncarek, Lehmann-Horn and Lerche2007). The biophysical characterization of several of these mutations has revealed the presence of an alternative permeation pathway through VSDs, which are usually non-conductive. This alternative permeation pathway has been called a gating pore or omega pore to differentiate it from the alpha pore (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahine2012b, Reference Gosselin-Badaroudine, Moreau and Chahine2013; Sokolov et al. Reference Sokolov, Scheuer and Catterall2005, Reference Sokolov, Scheuer and Catterall2007; Struyk & Cannon, Reference Struyk and Cannon2007; Tombola et al. Reference Tombola, Pathak and Isacoff2005). Gating pores are one of the major discoveries in the field of VGICs. They have been used to investigate the structure and function of VSDs as well as differences between VSDs (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a).
The VSD superfamily. The VSD motif is a protein structure shared by a large number of proteins. These proteins form the VSD superfamily, which can be divided into four families: 4 TM, 6 TM, 12 TM, and 24 TM proteins. Four TM proteins (purple) make up 2% of the superfamily. Their TM domains are composed solely of a VSD. VSDs can also be associated with a phosphatase, as is the case with TPTE, or can feature a built-in permeation pathway, as in the case with Hv1. Six TM (red) is the largest family and accounts for 78% of the members of the superfamily. This family includes the Kv, TRP, CNG, KCa, and CatSper channels. Twelve TM (green) is the smallest family (2%) and is composed of TPCs. Twenty-four TM accounts for 18% of the members of the VSD superfamily and is composed of the Nav and Cav channels (blue).
The aim of the present review is to summarize existing knowledge of gating pores. It describes the gating, ionic selectivity, and modulators of this alternative permeation pathway. It also describes the major aspects of the structure and function of VSDs.
2. The VSD
2.1. The VSD super familly
Over 60 years ago, Hodgkin and Huxley described the voltage-gated movement of ions (Hodgkin & Huxley, Reference Hodgkin and Huxley1952). It is now known that these ion movements are mediated by VGICs, which have been cloned and expressed in several expression systems over the past two decades. It is now clear that most VGICs feature two main domains: a pore domain (PD) and a VSD.
The PD defines the voltage-gated-like protein superfamily (Yu & Catterall, Reference Yu and Catterall2004; Yu et al. Reference Yu, Yarov-Yarovoy, Gutman and Catterall2005). These proteins all feature at least two TM segments akin to the S5–S6 segments of Kv channels. The TM segments assemble in a fourfold symmetry to form a selective ion permeation pathway. The re-entrant loop between the TM segments is composed of turret loops on which the selectivity filter is located.
VSDs are created by the assembly of four TM segments. Their most recognizable features are the presence of several highly conserved positive charges on the S4 segments (Fig. 2) and several highly conserved acidic residues on the S2 and S3 segments (Fig. 2). The assembly of the four TM segments also leads to the creation of two water crevices that are responsible for focusing the electric field (Ahern & Horn, Reference Ahern and Horn2005; Starace & Bezanilla, Reference Starace and Bezanilla2004) and amplifying changes in the electric field inside the VSD.
Conserved residues in the TM segments of VSD superfamily members. Amino-acid sequence alignments of the TM segments of several members of the VSD superfamily. The alignment was built using a protein blast with the NavAb sequence as the query for identifying VSD superfamily members in the UniprotKB database. Only human proteins were included. The hits were used to build a multiple sequence alignment with Clustal omega from which the sequences shown here were extracted. The TM segments were delimited using the crystal structure of NavAb (Payandeh et al. Reference Payandeh, Scheuer, Zheng and Catterall2011). The conserved positive, negative, and aromatic residues are highlighted in blue, red, and orange, respectively. The conserved residues of the GCTC and its interacting partner, the S4 segment, are identified on top of the corresponding columns (F1, E1, D1, K0, R1–R4, K5).
The crystal structures of several proteins featuring VSDs have been determined (Jiang et al. Reference Jiang, Lee, Chen, Ruta, Cadene, Chait and Mackinnon2003; Long et al. Reference Long, Tao, Campbell and Mackinnon2007; Payandeh et al. Reference Payandeh, Scheuer, Zheng and Catterall2011, Reference Payandeh, Gamal El-Din, Scheuer, Zheng and Catterall2012; Takeshita et al. Reference Takeshita, Sakata, Yamashita, Fujiwara, Kawanabe, Kurokawa, Okochi, Matsuda, Narita, Okamura and Nakagawa2014; Zhang et al. Reference Zhang, Ren, Decaen, Yan, Tao, Tang, Wang, Hasegawa, Kumasaka, He, Wang, Clapham and Yan2012), which has brought to light a permeation pathway through the VSD that functions in both physiological (Berger & Isacoff, Reference Berger and Isacoff2011; Musset et al. Reference Musset, Smith, Rajan, Morgan, Cherny and Decoursey2011; Ramsey et al. Reference Ramsey, Mokrab, Carvacho, Sands, Sansom and Clapham2010) and pathological conditions (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a, Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahineb, Reference Gosselin-Badaroudine, Moreau and Chahine2013; Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007, Reference Sokolov, Scheuer and Catterall2008; Struyk & Cannon, Reference Struyk and Cannon2007; Struyk et al. Reference Struyk, Markin, Francis and Cannon2008). This gating structure is shared by many proteins (Fig. 1), most of which are VGICs. However, the family of proteins featuring a VSD also includes phosphatases and uncharacterized proteins (Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014).
More than 100 human protein structures containing a VSD motif have been identified to date (Fig. 1). These proteins are broadly expressed in many living organisms and are crucial for many physiological functions. They can be divided into four families (proteins featuring 24 TM, 12 TM, 6 TM, and 4 TM segments), which can be further separated into subfamilies, including Na+ (Nav) and Ca2+ (Cav) channels, voltage-gated K+ (Kv) channels, two-pore channels (TPC), transient receptor potential (TRP) channels, cyclic nucleotide gated (CNG) channels, CatSper channels, Ca2+ activated K+ channels (KCa), and H+ channels (Hv).
Nav and Cav channels are large proteins formed by 24 TM segments organized into four homologous domains (DI to DIV). They are involved in depolarizing inward currents associated with the onset of the action potential (AP) (Catterall, Reference Catterall2011; Koopmann et al. Reference Koopmann, Bezzina and Wilde2006). These two subfamilies support relatively fast activating and inactivating currents although inactivation is an exclusively voltage-dependent process for Nav channels as opposed to a mixture of Ca2+ and voltage-dependent processes for Cav channels.
TPCs are present in rat kidney, liver, and lung cells (Ishibashi et al. Reference Ishibashi, Suzuki and Imai2000) and may mediate nicotinic acid adenine dinucleotide phosphate (NAADP)-dependent Ca2+ release in organelles (Calcraft et al. Reference Calcraft, Ruas, Pan, Cheng, Arredouani, Hao, Tang, Rietdorf, Teboul, Chuang, Lin, Xiao, Wang, Zhu, Lin, Wyatt, Parrington, Ma, Evans, Galione and Zhu2009). This physiological role is the subject of some debate as TPCs may be more selective for Na+ than for Ca2+ ions (Wang et al. Reference Wang, Zhang, Dong, Samie, Li, Cheng, Goschka, Shen, Zhou, Harlow, Zhu, Clapham, Ren and Xu2012). In addition, some studies have shown that NAADP does not directly activate TPCs (Wang et al. Reference Wang, Zhang, Dong, Samie, Li, Cheng, Goschka, Shen, Zhou, Harlow, Zhu, Clapham, Ren and Xu2012).
Kv channels are the hallmark subfamily of the six TM families. Six TM proteins tetramerize to form functional channels that are activated by membrane depolarization and that determine the resting membrane potential. They are also associated with the membrane repolarization phase of the AP. This subfamily has been well characterized given that the proteins are robustly expressed in various expression systems (Gonzalez et al. Reference Gonzalez, Baez-Nieto, Valencia, Oyarzun, Rojas, Naranjo and Latorre2012).
The structure of CNG channels is very similar to that of Kv channels. They are tetramers, and each monomer is composed of a VSD (S1–S4) and a PD (S5–S6). They also feature a cyclic nucleotide-binding domain in their C-terminal region that binds c-AMP and c-GMP and that modulates channel activity. These channels are expressed in the heart and the central nervous system where they play an important role in cell rhythmicity. Hyperpolarization-activated cyclic nucleotide-gated CNG channels (HCN) are distinguished by their voltage sensitivity. The movement of the S4 segment of HCN channels in response to changes in voltage is similar to the movement of the S4 segment of depolarization-activated channels except that the coupling of the VSD to the PD is inverted in HCN channels (Mannikko et al. Reference Mannikko, Elinder and Larsson2002).
TRP channels are perhaps the most diverse subfamily, with over 100 known genes in members of the animal kingdom. They play important physiological roles such as sensory, taste sensation, and ionic homeostasis. Like Kv channels, they are composed of six TM segments, while S1–S4 form the VSD and S5–S6 form the PD that functions as a Ca2+-permeable cation channel. Interestingly, they all feature a VSD although the gating of many of those channels depends exclusively on ions or ligands. For a review see Nilius and Owsianik (Reference Nilius and Owsianik2011).
KCa channels are widely expressed and may play different physiological roles. They are known to be involved in setting the membrane potential in neurons and, although they all feature a VSD, most KCa channels are voltage insensitive or display low-voltage sensitivity (Yuan et al. Reference Yuan, Santi, Wei, Wang, Pollak, Nonet, Kaczmarek, Crowder and Salkoff2003).
Catsper are cationic channels found in sperm and are associated with spermatozoa maturation. They are activated by an increase in intracellular pH and membrane depolarization. Knocking down this channel in mice is associated with male infertility (Quill et al. Reference Quill, Sugden, Rossi, Doolittle, Hammer and Garbers2003; Ren et al. Reference Ren, Navarro, Perez, Jackson, Hsu, Shi, Tilly and Clapham2001).
There are only two known members of the four TM VSD-featuring protein superfamilies. These proteins possess unexpected biological functions that are not shared by VGICs, indicating that VSDs play many more roles than previously suspected. Members of the VSD-featuring superfamily do not all have PDs (Fig. 1). VSDs also form part of a membrane-bound Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP) and its human homolog (transmembrane phosphatase with tensin homology (TPTE)) and have been shown to regulate phosphatase activity (Murata et al. Reference Murata, Iwasaki, Sasaki, Inaba and Okamura2005). In addition, a newly identified H+ channel, Hv1 or VSOP for voltage sensor only protein, is a self-contained voltage sensor. It does not have a PD but functions as a voltage-dependent H+ permeation channel (Sasaki et al. Reference Sasaki, Takagi and Okamura2006). The VSDs of Ci-VSP and Hv1 exhibit gating currents, the hallmark of a functional voltage sensor.
2.2. The S4 segment
2.2.1. Gating currents
Functionally, VGICs pass through several states in a voltage-dependent manner during their course of action. Membrane depolarization activates VGICs, opening the channel pore and allowing ions to cross through the pore. The channels then shift from the open to the inactivated states. Membrane repolarization allows VGICs to recover from inactivation.
In 1952, Hodgkin and Huxley hypothesized that voltage dependence was mediated by a change in the distribution or orientation of a charged particle or molecule with a dipole moment (Hodgkin & Huxley, Reference Hodgkin and Huxley1952). Twenty-one years later, this charge movement was observed in squid giant axons and was called the gating current (Armstrong & Bezanilla, Reference Armstrong and Bezanilla1973). The identification of VSDs as dedicated structures responsible for this sensing process began with the identification of a charge segment in Na+ channels (Noda et al. Reference Noda, Shimizu, Tanabe, Takai, Kayano, Ikeda, Takahashi, Nakayama, Kanaoka, Minamino, Kangawa, Matsuo, Raftery, Hirose, Inayama, Hayashida, Miyat and Numa1984). The first evidence of the S4 segment operating as a voltage sensor received support from mutagenesis studies of the Nav channel (Stuhmer et al. Reference Stuhmer, Conti, Suzuki, Wang, Noda, Yahagi, Kubo and Numa1989). The S4 segment is considered as a voltage sensor because of two fundamental properties: (1) it is located in the lipid bilayer of the membrane because it is hydrophobic; and (2) it contains several positively charged amino acids, mostly arginine residues.
When the outermost S4 arginine is replaced by cysteine in the DIV-S4 of Nav channels, the cysteine residue becomes accessible to extracellular methanethiosulfonate (MTS) reagents in response to membrane depolarization. The charged S4 segment moves outward from the membrane under depolarized conditions (Yang & Horn, Reference Yang and Horn1995). Subsequent studies showed that several S4 segment arginines move from the inside to the outside of the membrane in response to depolarization (Yang et al. Reference Yang, George and Horn1996). The S4 segment is thus in a resting conformation, i.e., in a downward configuration, at hyperpolarized voltages. During the gating process, VSDs rapidly undergo conformational changes by the outward movement of S4 segments. Approximately 12 charges (e 0) buried inside the membrane move across the membrane during the activation of Shaker channels (Sigworth, Reference Sigworth1994). The charge movement can be traced as a function of the voltage relationship (Q–V), which fits a two-state Boltzmann distribution. The Q–V curve describes the probability of finding a defined population of VSDs in various states at a given voltage. The two opposing plateau phases account for the two principal states of the VSD, i.e., activated and resting.
2.2.2. Traditional models of S4 segment movement
Two main traditional models for the S4 motion have been proposed (Fig. 3). The first consists of the outward movement of the α-helical S4 structure and is called the helical screw or sliding helix model (Catterall, Reference Catterall1986; Guy & Seetharamulu, Reference Guy and Seetharamulu1986; Yang et al. Reference Yang, George and Horn1996), which predicts the rotation and translation of the α-helical S4 segment from one side of the plasma membrane to the other (Fig. 3) (Yang et al. Reference Yang, George and Horn1996). The second is called the paddle model (Jiang et al. Reference Jiang, Lee, Chen, Ruta, Cadene, Chait and Mackinnon2003) and predicts an α-helically hairpin-like paddle motif created by the S4 segment helix and the second part of S3 (S3b). The paddle motif extends into the fluid interior of the membrane from the channel core (Jiang et al. Reference Jiang, Lee, Chen, Ruta, Cadene, Chait and Mackinnon2003). In the resting state, the paddle is loosely located inside the membrane near the intracellular surface and extends into the cytoplasm from the channel core (Jiang et al. Reference Jiang, Lee, Chen, Ruta, Cadene, Chait and Mackinnon2003). When the channel opens, the paddle moves to the outside surface (Fig. 3) much like rowing a boat. However, experimental findings, notably from luminescence experiments on a functional Shaker K+ channel, do not support the paddle model (Posson et al. Reference Posson, Ge, Miller, Bezanilla and Selvin2005).
Models of S4 movement. Schematic representation of the different models of S4 movement caused by membrane depolarization. Depolarization induces a conformational rearrangement of the VSD from the resting to the activated state. During this process, the S4 segment moves upward. (a) Representation of the sliding helix or helical screw (top) and paddle (bottom) models of S4 movement. The helical screw model predicts an upward motion associated with a rotation of the S4 segment. The paddle model predicts a major tilt of the S3b (orange cylinder) and S4 segments from the cytoplasmic side of the membrane due to depolarization. (b) The currently accepted model for S4 movement, which is a combination of the helical screw and paddle models, predicts a large upward movement of the S4 segment associated with a slight rotation and tilt.
2.2.3. Current model of S4 segment movement
Recent molecular dynamic simulations modeling the movement of S4 have accounted for the transition from the resting to the activated state (Delemotte et al. Reference Delemotte, Tarek, Klein, Amaral and Treptow2011). Membrane depolarization causes S4 to move toward the extracellular milieu in a way that is most likely a combination of the helical screw and paddle models (Delemotte et al. Reference Delemotte, Tarek, Klein, Amaral and Treptow2011). Based on the Kv1.2 model, Delemotte et al. proposed that membrane depolarization initiates an upward 10–15-Å movement associated with a 60° rotation and 15° tilt of the S4 segment (Fig. 3) (Delemotte et al. Reference Delemotte, Tarek, Klein, Amaral and Treptow2011; Henrion et al. Reference Henrion, Renhorn, Borjesson, Nelson, Schwaiger, Bjelkmar, Wallner, Lindahl and Elinder2012; Jensen et al. Reference Jensen, Jogini, Borhani, Leffler, Dror and Shaw2012; Vargas et al. Reference Vargas, Yarov-Yarovoy, Khalili-Araghi, Catterall, Klein, Tarek, Lindahl, Schulten, Perozo, Bezanilla and Roux2012).
The VSD of Ci-VSP has recently been isolated and crystallized in both the activated and resting states (Li et al. Reference Li, Wanderling, Paduch, Medovoy, Singharoy, Mcgreevy, Villalba-Galea, Hulse, Roux, Schulten, Kossiakoff and Perozo2014). Li et al. reported that, following the depolarization of the membrane, S4 would most likely move toward the extracellular milieu using the helical screw mechanism described 28 years ago (Catterall, Reference Catterall1986; Guy & Seetharamulu, Reference Guy and Seetharamulu1986). Li et al. studied the R217E mutation in the S4 segment, which results in a large Q–V shift. At 0 mV, the WT VSD is in a resting state while the R217E VSD is in an activated state (Li et al. Reference Li, Wanderling, Paduch, Medovoy, Singharoy, Mcgreevy, Villalba-Galea, Hulse, Roux, Schulten, Kossiakoff and Perozo2014). The study of the WT and R217E VSDs revealed that, in depolarized conditions, the S4 segment moves upward by 5 Å with a 60° clockwise rotation to reach the activated conformation. However, Li et al. pointed out that, while this movement could be considered a general mechanism, it does not represent the exact movement of S4 segments in the VSDs of all voltage-dependent proteins (Li et al. Reference Li, Wanderling, Paduch, Medovoy, Singharoy, Mcgreevy, Villalba-Galea, Hulse, Roux, Schulten, Kossiakoff and Perozo2014). Indeed, a larger movement of the S4 segment would be needed to account for the required gating charge in VGICs (Li et al. Reference Li, Wanderling, Paduch, Medovoy, Singharoy, Mcgreevy, Villalba-Galea, Hulse, Roux, Schulten, Kossiakoff and Perozo2014).
2.3. The gating charge transfer center (GCTC)
The movement of the S4 segment has been studied extensively since it was identified. The immediate environment of the S4 segment has recently been the focus of more in-depth investigations. It is now known that the S1, S2, and S3 segments create an environment that enables the S4 segment to move in response to changes in voltage.
The first indication that some residues are more important than others in the creation of this environment came from the crystal structure of the chimeric Kv1.2–Kv2.1 channel (Long et al. Reference Long, Tao, Campbell and Mackinnon2007). The determination of previous crystal structures provided some clues about conformational changes in VSDs (Jiang et al. Reference Jiang, Lee, Chen, Ruta, Cadene, Chait and Mackinnon2003; Long et al. Reference Long, Campbell and Mackinnon2005), but did not agree with previous arginine accessibility studies (Chahine et al. Reference Chahine, Bennett, George and Horn1994; Starace et al. Reference Starace, Stefani and Bezanilla1997; Yang & Horn, Reference Yang and Horn1995; Yang et al. Reference Yang, George and Horn1996). Other crystal structures have since provided insights into the structural arrangements of four TM proteins such as Hv1 and Ci-VSP (Li et al. Reference Li, Wanderling, Paduch, Medovoy, Singharoy, Mcgreevy, Villalba-Galea, Hulse, Roux, Schulten, Kossiakoff and Perozo2014; Takeshita et al. Reference Takeshita, Sakata, Yamashita, Fujiwara, Kawanabe, Kurokawa, Okochi, Matsuda, Narita, Okamura and Nakagawa2014). Crystal structures have also offered valuable perspectives on PDs as well as on the activated conformation and the inactivation process of bacterial Nav channels (McCusker et al. Reference Mccusker, Bagneris, Naylor, Cole, D'Avanzo, Nichols and Wallace2012; Payandeh et al. Reference Payandeh, Scheuer, Zheng and Catterall2011, Reference Payandeh, Gamal El-Din, Scheuer, Zheng and Catterall2012).
The crystal structure of the chimeric channel revealed that negative residues in VSDs cluster into two groups separated by a phenylalanine in the middle of the S2 segment (Long et al. Reference Long, Tao, Campbell and Mackinnon2007). This F233 residue corresponds to the F290 residue in the Shaker K+ channel and, according to Long et al. (Reference Long, Tao, Campbell and Mackinnon2007), is the most conserved amino acid outside the selectivity filter in Kv channels. This residue (F1 in Fig. 2) is highly conserved in the VSD protein superfamily. In the crystal structure, the F233 residue is in close proximity to the K5 residue which, under depolarized conditions, moves but does not cross the GCTC (Long et al. Reference Long, Tao, Campbell and Mackinnon2007). It thus contributes only half a charge to the gating current (Aggarwal & MacKinnon, Reference Aggarwal and Mackinnon1996; Seoh et al. Reference Seoh, Sigg, Papazian and Bezanilla1996), indicating that the crystal structure represents the VSD in its fully activated state and that the F233 residue is involved in the stabilization of this state.
Other studies have confirmed the role of this conserved phenylalanine (Pless et al. Reference Pless, Galpin, Niciforovic and Ahern2011; Tao et al. Reference Tao, Lee, Limapichat, Dougherty and Mackinnon2010). Tao et al. reported that introducing a lysine at the R1 position and an arginine at the K5 position stabilized the resting state of the VSD via a stronger interaction with the phenylalanine residue (Tao et al. Reference Tao, Lee, Limapichat, Dougherty and Mackinnon2010). These authors used natural and unnatural amino acid substitutions to test the hypothesis that F290 in the Shaker channel forms a cation-π interaction with the positively charged residues of the S4 segment. They found no correlation between the electrostatic potential of the aromatic ring and the affinity of the channel for the activated state, indicating that the cyclic ring on the phenylalanine residue plays a greater functional role than its aromaticity. However, this has been contested by Pless et al., who noted a correlation between the electrostatic potential of the aromatic ring and shifts in the G–V curves of the Shaker channel (Pless et al. Reference Pless, Galpin, Niciforovic and Ahern2011). Moreover, the role of the phenylalanine in the GCTC may be different for different channels since substitutions of this residue in the Kv7.2 channel result in much less marked changes in the biophysical properties of the channel than similar substitutions in the Shaker channel (Li et al. Reference Li, Chen, Xu, Sun, Li, Liu, Yang, Gao, Jiang and Li2013; Tao et al. Reference Tao, Lee, Limapichat, Dougherty and Mackinnon2010). Further studies are clearly warranted to determine how phenylalanine interacts with the S4 segment.
The role of the negatively charged residues clustered on the intracellular side of the phenylalanine has also been under scrutiny recently. Tao et al. proposed that the assembly of the conserved phenylalanine, glutamate, and aspartate residues (Figs 2 and 4) constitute the GCTC as they seem to interact with K5 (Tao et al. Reference Tao, Lee, Limapichat, Dougherty and Mackinnon2010). These residues correspond to F290, E293, and D316 in Shaker channel. Pless et al. used unnatural amino acid substitutions to show that E293 does indeed interact with the positively charged residues of the S4 segment (Pless et al. Reference Pless, Galpin, Niciforovic and Ahern2011). However, they also reported that the E283 (located above the F290) and D316 residues do not interact with S4, but may be involved in the formation of the water crevices.
GCTC structure. The structure of the VSD of DI of Nav1.4 in its resting state is depicted in gray. The positively charged residues of the S4 segment are shown in blue. The negatively charged residues and the conserved aromatic amino acid of the GCTC are shown in red and orange, respectively. The hourglass-shaped water accessible volume is shown in light blue. For purposes of clarity, the S1 segment has been removed in (b and c). To emphasize the hydrophobic septum in (c), the water accessible volume is depicted in a darker shade.
Other approaches have been used to probe VSDs. Early reports showed that suppressing the interaction of the S4 segment with its environment in a given state leads to cation permeation (Starace & Bezanilla, Reference Starace and Bezanilla2004; Tombola et al. Reference Tombola, Pathak and Isacoff2005). The identification of the GCTC as the interaction partner of the S4 segment led to the observation that this interaction creates a hydrophobic septum in the center of the VSD (Fig. 4) (Delemotte et al. Reference Delemotte, Klein and Tarek2012) that is maintained during the movement of S4 since the positively charged residues of S4 interact sequentially with the GCTC, resulting in metastable intermediate states (Amaral et al. Reference Amaral, Carnevale, Klein and Treptow2012; Delemotte et al. Reference Delemotte, Tarek, Klein, Amaral and Treptow2011). When the S4–GCTC interaction is suppressed, the hydrophobic septum is disrupted, enabling the water crevices to communicate. This results in a cation leak through a dedicated permeation pathway known as the gating pore. This permeation pathway is the physiological pore of Hv1 (Berger & Isacoff, Reference Berger and Isacoff2011; Ramsey et al. Reference Ramsey, Mokrab, Carvacho, Sands, Sansom and Clapham2010).
Gating pore currents have been used to probe the hydrophobic septa of Nav channel VSDs (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a), explore the permeation pathway of Hv1 (Berger & Isacoff, Reference Berger and Isacoff2011; Musset et al. Reference Musset, Smith, Rajan, Morgan, Cherny and Decoursey2011), and investigate the biophysical mechanisms of various diseases (Francis et al. Reference Francis, Rybalchenko, Struyk and Cannon2011; Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahine2012b; Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007; Struyk & Cannon, Reference Struyk and Cannon2007). Interestingly, not all VSDs generate gating pore currents via single-residue substitutions (Gamal El-Din et al. Reference Gamal El-Din, Heldstab, Lehmann and Greeff2010; Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a). The VSD of the DIV of Nav1.4 requires three arginine substitutions to be permeable to cations (Capes et al. Reference Capes, Arcisio-Miranda, Jarecki, French and Chanda2012). This can be explained by the fact that the septum of this domain is larger than the septa of other domains (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a). Such differences in septum depth could explain why the activation kinetics of the VSD of this channel differs from those of other VSDs (Chanda & Bezanilla, Reference Chanda and Bezanilla2002). In fact, the larger the hydrophobic septum, the higher the free energy required to induce conformational changes in VSDs. The positive charge of the S4 arginine residues makes it difficult to cross larger hydrophobic septa.
The Shaker channel requires two arginine substitutions to generate an omega current when residues other than R1 are targeted (Gamal El-Din et al. Reference Gamal El-Din, Heldstab, Lehmann and Greeff2010). This indicates that the septum in the Shaker channel is larger than that of the first three domains of the Nav1.4 channel. Again, this would make conformation changes slower for this VSD than for the VSDs of Nav channels. These differences could explain why Kv channels typically activate after Nav channels, as suggested by Lacroix et al. (Reference Lacroix, Campos, Frezza and Bezanilla2013).
2.4. The kinetic pathway of the S4 segment
Changes in voltage induce conformational rearrangements of the VSD that can be tracked by the Q–V curve. The kinetic pathway of the S4 segment can be defined as its propensity to react to changes in free energy. In addition, the kinetic pathway of the charge movement of S4 may be different for the activation and deactivation processes, i.e., Q–V activation (from resting to activated state) differs from Q–V deactivation (from activated to resting state) (Armstrong & Bezanilla, Reference Armstrong and Bezanilla1977). The primary function of VSDs is to modulate the activity of voltage-dependent proteins. VSDs are thus usually part of a larger protein structure and, as such, the structural rearrangement of the protein influences the kinetic pathway of the S4 segment (Batulan et al. Reference Batulan, Haddad and Blunck2010). After a short depolarization, the deactivation kinetic pathway of the charge movement is similar to the activation kinetic pathway (Cha et al. Reference Cha, Ruben, George, Fujimoto and Bezanilla1999). Longer depolarization periods will impact the entire protein structure since the VGIC enters into an inactivated state. This process may be responsible for the modification of the kinetic pathway. These differences have been observed in several ion channels, including the Kv, Nav, Cav, and HCN channels (Kuzmenkin et al. Reference Kuzmenkin, Bezanilla and Correa2004; Mannikko et al. Reference Mannikko, Pandey, Larsson and Elinder2005; Olcese et al. Reference Olcese, Latorre, Toro, Bezanilla and Stefani1997; Piper et al. Reference Piper, Varghese, Sanguinetti and Tristani-Firouzi2003; Shirokov et al. Reference Shirokov, Levis, Shirokova and Rios1992; Villalba-Galea et al. Reference Villalba-Galea, Sandtner, Starace and Bezanilla2008). In the case of Shaker channels, the Q–V deactivation curve is shifted much more to the left for inactivated channels than for non-inactivated channels (Olcese et al. Reference Olcese, Latorre, Toro, Bezanilla and Stefani1997). This indicates that inactivation causes S4 charge immobilization, which is also known as S4 segment freezing. At hyperpolarized voltages, most of the S4 segments of non-inactivated channels would thus be in a downward configuration while most of the S4 segments of inactivated channels would be in an upward configuration. In other words, depending on the duration of the depolarization, the S4 segments of VSDs would be in an upward conformation at hyperpolarized voltages (Fig. 5). Site-directed mutagenesis of Shaker channels has revealed that charge immobilization can be explained by interactions between the S4–S5 linker of one subunit and the S6 of an adjacent subunit (Batulan et al. Reference Batulan, Haddad and Blunck2010). This interaction, which occurs after the activation of the S4 segment, causes a conformational modification, leading to a change in the S4 kinetic pathway (Batulan et al. Reference Batulan, Haddad and Blunck2010; Goodchild & Fedida, Reference Goodchild and Fedida2012).
S4 segment freezing due to a long depolarization period. Schematic representation of VSDs following depolarization. WT and mutated VSDs can be found in three different states: resting, activated, and immobilized (top section: left, middle, and right, respectively). During short depolarization periods, WT and mutated VSDs appear to adopt the resting and activated states only (bottom left section). After long depolarization periods, the deactivation voltage dependence of WT VSDs differs from the voltage dependence of activation. This may be due to S4 freezing as shown in the lower section. Although the voltage returns to hyperpolarized values, the VSDs do not immediately return to their resting conformation. The mechanisms of S4 freezing are different for WT and mutated VSDs.
Cha et al. used a fluorescence labeling approach to show that the charge immobilization of DIII and DIV of the S4 segment of Nav1.4 channels takes 0.9–8 ms (Cha et al. Reference Cha, Ruben, George, Fujimoto and Bezanilla1999), which is much longer than that for DI and DII. This difference may be related to different conformational rearrangements between Nav domains. Given that the inactivation process is different for Nav and Kv channels, the exact mechanism leading to charge immobilization may be different for these two channels.
S4 freezing shows that voltage sensing is mediated by the direct environment of VSDs. The structural conformation of the surrounding protein appears to be crucial for determining the voltage dependence and kinetics of the charge movement inside VSDs.
In VGICs, S4 freezing appears to be directly related to the conformational rearrangement of the PD mediated by the inactivation process. In the case of voltage-sensitive phosphatases, which do not have a PD, S4 freezing would thus not likely occur. Surprisingly, charge freezing after long depolarizations (from hundreds of milliseconds to seconds) has also been observed with Ci-VSP (Villalba-Galea et al. Reference Villalba-Galea, Sandtner, Starace and Bezanilla2008). Villalba-Galea et al. observed S4 charge freezing in Ci-VSP mutants lacking the phosphatase domain, indicating that voltage-independent conformational rearrangements also occur on the VSD motif alone. These authors concluded that the protein environment of the VSD may modulate this intrinsic phenomenon, thus explaining the effect of channel inactivation on S4 charge freezing. These findings led to a new hypothesis about the structural conformations of VSDs. During the two main states of the VSD (i.e. resting and activated), the S4 sensor can be found in three principal configurations: resting (downward), activated (upward), and relaxed (Fig. 6) (Villalba-Galea et al. Reference Villalba-Galea, Sandtner, Starace and Bezanilla2008). Villalba-Galea et al. suggested that the helix making up the S4 segment may be partially in a 310 conformation during the transition from the resting to the activated state. Under depolarized conditions and after reaching the activated state, the S4 segment would undergo a voltage-insensitive conformational change toward a more stable relaxed conformation. This conformational change would result in the transformation of the S4 segment from a 310 conformation (three residues/helical turn, 2 Å between each residue) to an α-helix (3.6 residues/helical turn, 1.5 Å between each residue) (Fig. 6) (Villalba-Galea et al. Reference Villalba-Galea, Sandtner, Starace and Bezanilla2008). The recently crystallized VSD of Ci-VSP (in both the resting and activated states) exhibits an α-helical S4 segment after long periods of 0 mV depolarization that are inherent to the crystallization technique (Li et al. Reference Li, Wanderling, Paduch, Medovoy, Singharoy, Mcgreevy, Villalba-Galea, Hulse, Roux, Schulten, Kossiakoff and Perozo2014). The α-helix conformation is the predicted relaxed configuration of the S4 segment (Villalba-Galea et al. Reference Villalba-Galea, Sandtner, Starace and Bezanilla2008). The crystal structures of KvAP, the Kv1.2–2.1 chimera, NavRh, and Hv1 might also support the relaxed conformation since the S4 forms an α-helix (Jiang et al. Reference Jiang, Lee, Chen, Ruta, Cadene, Chait and Mackinnon2003; Long et al. Reference Long, Tao, Campbell and Mackinnon2007; Takeshita et al. Reference Takeshita, Sakata, Yamashita, Fujiwara, Kawanabe, Kurokawa, Okochi, Matsuda, Narita, Okamura and Nakagawa2014; Zhang et al. Reference Zhang, Ren, Decaen, Yan, Tao, Tang, Wang, Hasegawa, Kumasaka, He, Wang, Clapham and Yan2012). Several crystal structures have also revealed that the portion of S4 that interacts with the GCTC is a 310 helix (Long et al. Reference Long, Tao, Campbell and Mackinnon2007; Takeshita et al. Reference Takeshita, Sakata, Yamashita, Fujiwara, Kawanabe, Kurokawa, Okochi, Matsuda, Narita, Okamura and Nakagawa2014; Zhang et al. Reference Zhang, Ren, Decaen, Yan, Tao, Tang, Wang, Hasegawa, Kumasaka, He, Wang, Clapham and Yan2012). This strengthens the hypothesis that the S4 segment is in a 310 conformation during the transition between the resting and activated states.
Changes in S4 conformation may underlie S4 segment freezing. Schematic representation of the different states of the S4 segment in response to depolarized voltages. During the transition from the resting to the activated state, the S4 segment or a part of the segment adopts a 310 helix structure. During prolonged depolarization periods, the S4 segment reaches a relaxed state through a voltage-insensitive change of conformation. The change of conformation results in the transition of the S4 segment or a part of the segment from a 310 to an α-helix. For the purposes of clarity, the complete S4 segment of each VSD is represented in a single conformation. However, the 310 conformation of the S4 segment might be found only in the GCTC. Similarly, the relaxation process may not involve the complete S4 segment.
Unlike other crystal structures, NavAb does not support the transition of the S4 segment to a relaxed state given that the stretch from R1 to R4 is in a 310 conformation (Payandeh et al. Reference Payandeh, Scheuer, Zheng and Catterall2011, Reference Payandeh, Gamal El-Din, Scheuer, Zheng and Catterall2012). These results suggest that, although VSDs in general have conserved structures, they may also have important structural differences.
3. Gating pores
3.1. Gating
3.1.1. Voltage dependence
As previously mentioned, gating pores are created by the disruption of interactions between S4 and the GCTC in a given state. Gating pores can thus be created in the resting, intermediate, or activated states. Mutating the outermost S4 arginines creates a permeant gating pore in the resting state (Fig. 7). The voltage dependence of such pores is expressed by 1 minus the Q–V curve. At hyperpolarized voltages, gating pores are open (Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014). As the probability of finding a VSD in its resting state decreases, the probability of finding a VSD in a conformation where the mutated residue is in the GCTC also decreases (Fig. 7). In contrast, the voltage dependence of gating pores due to mutations of the innermost S4 residues follows the Q–V curve. As such, the probability of finding the mutated residue in the GCTC increases with the probability of finding a VSD in its activated configuration (Fig. 7). H+-specific transporters display intermediate behavior. Their maximal current is reached when VSDs have a higher probability of moving, i.e., at V 1/2 of the Q–V curve (Starace & Bezanilla, Reference Starace and Bezanilla2004).
Gating pores are permeant at hyperpolarized or depolarized voltages. The Q–V curve and the activation of gating pores are intimately related. Gating pores are created by the disruption of the S4–GCTC interaction in a given state (resting or activated). Mutations of the outermost S4 arginines lead to the disruption of the S4–GCTC interaction in the resting state (blue). The newly formed permeation pathway is open at hyperpolarized voltages. The inward rectification is caused by the decreasing probability of finding the VSD in its resting state due to depolarization. On the other hand, mutations of the innermost S4 arginines lead to the disruption of the S4–GCTC interaction when VSDs are in the activated state. This leads to permeation through gating pores at depolarized voltages (green). In this case, outward rectification is caused by the increasing probability of finding the VSD in the activated state due to depolarization.
The intimate relationship between S4 movement and gating pore voltage dependence has proven to be a powerful tool for investigating the structure–function of VSDs. Gating pore currents have been used as a read-out of voltage sensor movement (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a). In addition to providing more information on the activation sequence of VSDs in Nav channels, this study also highlights major differences between the VSDs of Nav channels.
3.1.2. S4 segment freezing
Mutating S4 arginines on the intracellular side leads to the creation of gating pores that are activated in depolarized conditions (Fan et al. Reference Fan, Lehmann-Horn, Weber, Bednarz, Groome, Jonsson and Jurkat-Rott2013; Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2008). At hyperpolarized voltages, the mutated residues are located below the GCTC and do not open the gating pore permeation pathway. Interestingly, after a long depolarization, gating pores become permeant at hyperpolarized voltages (Fan et al. Reference Fan, Lehmann-Horn, Weber, Bednarz, Groome, Jonsson and Jurkat-Rott2013; Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2008), which means that the mutated residue remains in the GCTC. According to the WT Q–V curve, the VSD would be in its resting state and the S4 segment in a downward position. The presence of gating pore currents at hyperpolarized potentials led to the hypothesis that the S4 segment remains temporarily trapped in an upward configuration (Fig. 5). Gating pore currents recorded under hyperpolarized conditions are the consequences of S4 freezing (Fan et al. Reference Fan, Lehmann-Horn, Weber, Bednarz, Groome, Jonsson and Jurkat-Rott2013; Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2008). These examples also show that mutations leading to the creation of gating pores likely alter the probability of finding a defined population of VSDs in various states at a given voltage. The voltage dependence of gating pores always follows the Q–V curve of the mutated VSD. S4 mutations likely induce a hyperpolarized shift in the Q–V deactivation curve compared with the Q–V activation curve. This leads to different voltage-dependent activation and deactivation. It should be noted that the mechanism responsible for S4 freezing of mutated channels may be different in WT channels where the freezing may be caused by a conformational rearrangement of the entire protein discussed above. The freezing of mutated channels could be attributed to the charge neutralization. The voltage-sensing mechanism in the GCTC of mutated VSDs may thus be affected and cause a delay or a change in voltage dependence.
This freezing process underlies the pathological mechanisms of several mutations causing K+-sensitive NormoPP (Fan et al. Reference Fan, Lehmann-Horn, Weber, Bednarz, Groome, Jonsson and Jurkat-Rott2013; Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2008). In these pathological conditions, S4 immobilization thus leads to the appearance of a pathological Na+ leak at hyperpolarized voltages (Fan et al. Reference Fan, Lehmann-Horn, Weber, Bednarz, Groome, Jonsson and Jurkat-Rott2013; Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2008). More studies are warranted to elucidate the role of Na+ leaks in these pathologies.
3.1.3. S1–S3 segments
Although S4 segments in VSDs have been the subject of numerous studies, the surrounding S1–S3 segments likely also play a primordial role as a static base for the movement of S4 segments (Banerjee & MacKinnon, Reference Banerjee and Mackinnon2008; Tao et al. Reference Tao, Lee, Limapichat, Dougherty and Mackinnon2010). Recent studies have shown that the S1–S3 segments also move albeit to a far lesser extent than the S4 segment. The recent determination of the crystal structure of NavAb (Payandeh et al. Reference Payandeh, Gamal El-Din, Scheuer, Zheng and Catterall2012) showed that the complete VSD including the S1–S3 segments moves following activation. This process may also play a role in charge immobilization.
The creation of a Zn2+ bridge between the S2 and S3 residues in Shaker channels dramatically delays the activation kinetics of the channels without disturbing the maximal conductance (Lin et al. Reference Lin, Hsieh, Mock and Papazian2011). The creation of a Zn2+ bridge considerably increases the rigidity of the VSD structure, which appears to alter the gating process. This indicates that only small conformational rearrangements of S1–S3 segments may be needed for the normal activation process.
Campos et al. have reported the creation of two unusual gating pores. These results may suggest the movement of the S1–S3 segments (Campos et al. Reference Campos, Chanda, Roux and Bezanilla2007). Although the vast majority of gating pores result from the mutations of positively charged S4 residues, substituting isoleucine for histidine on the S1 or S2 segment has also been reported to create an H+-selective gating pore (Campos et al. Reference Campos, Chanda, Roux and Bezanilla2007). As mentioned previously, the GCTC is composed of two negatively charges acidic residues on the S2 and S3 segments and one aromatic amino acid (Fig. 4). It is thus surprising that mutations of residues outside the GCTC can cause the appearance of gating pores. H+-selective gating pores caused by the I241H and I287H mutations on Shaker channels are activated by hyperpolarized voltages and exhibit significant inward rectification (Campos et al. Reference Campos, Chanda, Roux and Bezanilla2007). In the case of S4 mutations, inward rectification of gating pore current has been associated to the large upward movement of this segment. Gating pores would be expected to form under both depolarized and hyperpolarized conditions following mutations on the S1–S3 segments, usually considered as a relatively rigid structure. If the S4 segment is the only moving part, then its position would not have an impact on the current. However, inward rectification of the gating pore current also means that, at depolarized voltages, the S1–S3 segments undergo a conformational rearrangement sufficient to disrupt the communication of the water crevices, resulting in the closure of the gating pore.
Lastly, mutations of GCTC residues have never been shown to lead to the creation a gating pore current. While such mutations might be expected to lead to the communication of the water crevices, the behavior of such gating pores remains completely unexplored. It would likely be very challenging to record such gating pore currents. If the movement of the S1–S3 segments in the VSD is not sufficient to close the newly created gating pore, then the gating pore current may be linear and, as such, indistinguishable from the non-specific leak inherent to the recording method.
3.2. Permeation
3.2.1. Ionic selectivity
Most gating pores appear to be cation selective (Francis et al. Reference Francis, Rybalchenko, Struyk and Cannon2011; Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007; Tombola et al. Reference Tombola, Pathak and Isacoff2005). This selectivity has been attributed to an excess of negatively charged residues in the GCTC due to the neutralization of S4 charges. In addition to opening a new permeation pathway, this neutralization leads to a charge imbalance in the GCTC, which may explain the selectivity against anions (Delemotte et al. Reference Delemotte, Treptow, Klein and Tarek2010; Khalili-Araghi et al. Reference Khalili-Araghi, Tajkhorshid, Roux and Schulten2012). Cations could bind to the negatively charged residues of the GCTC to cross the membrane.
Most studies have reported that gating pores are typically more permeable to large cations (Fig. 8) (Francis et al. Reference Francis, Rybalchenko, Struyk and Cannon2011; Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007; Tombola et al. Reference Tombola, Pathak and Isacoff2005). The selectivity sequences appear to converge toward Eisenmann's first or second sequences (Eisenman, Reference Eisenman1962). First and second sequences represent selectivity due to the weak binding sites of narrow pores without dedicated selectivity filters, i.e., gating pores.
Ionic selectivity. The selectivity sequences reported in several studies are summarized. In general, large ions are more permeant than small ions. K+ is generally more permeant than Na+ or Li+, whereas Cs+ and guanidium are more permeant.
Interestingly, Francis et al. and Sokolov et al. both described a very low but measurable permeation of the large cation NMDG+ (Francis et al. Reference Francis, Rybalchenko, Struyk and Cannon2011; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007). A similar degree of permeation has also been observed for TEA+ (Fig. 8) (Sokolov et al. Reference Sokolov, Scheuer and Catterall2007). Large cations are not expected to flow through gating pores since the permeation pathways of gating pores created in Kv1.2 channel VSDs appear to be 3.5 Å in diameter (Khalili-Araghi et al. Reference Khalili-Araghi, Tajkhorshid, Roux and Schulten2012). Both NMDG+ and TEA+ would thus be larger than the permeation pathway, considerably reducing their permeation potential. Nevertheless, gating pores created in Nav1.4 VSDs may be larger. However, according to the supposed selectivity sequence, very large cations must either be small enough to flow unimpeded through VSDs or too large to transit VSDs. Interestingly both studies used a pH of 7.4 for their recordings. The gating pore currents recorded may in fact be due to the permeation of H+. Based on the radiuses of a number of ions and the estimated size of gating pores (3.5 Å), guanidium (CH6N3+) would be the most permeant ion, whereas NDMG+ and TEA+ would be non-permeant. With a radius of 2.1 Å (Marcus, Reference Marcus2012), guanidium might be large enough to permeate gating pores.
Two divergent selectivity sequences are depicted in Fig. 8. It is important to note that these selectivity sequences are derived from two atypical gating pores (Berger & Isacoff, Reference Berger and Isacoff2011; Klassen et al. Reference Klassen, Spencer and Gallin2008). These selectivity sequences were obtained from naturally conductive VSDs in Nat-Kv3.2 and Hv1 channels and, as such, these gating pores may differ slightly from other gating pores, which may explain the differences.
There are two types of gating pores. Although most gating pores are cation selective, gating pores with arginine-to-histidine substitutions are highly selective for H+ (Campos et al. Reference Campos, Chanda, Roux and Bezanilla2007; Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a, Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahineb; Starace & Bezanilla, Reference Starace and Bezanilla2004; Struyk & Cannon, Reference Struyk and Cannon2007). This high selectivity for H+ results from the pKa of the histidine. Since it has a pKa of 6.8, histidine can be easily protonated at pH 7.4. The conduction mechanism would, however, be different from other gating pores since H+ flows would occur via a Grotthus hopping mechanism using water wires on both sides of the membrane (Starace & Bezanilla, Reference Starace and Bezanilla2004). The Grotthus mechanism, which is also called the hop and turn mechanism, involves the hopping of solvated H+ ions from one water molecule to another. Protonated histidine would thus connect the extracellular and intracellular water crevices, leading to the creation of an H+ wire. Such a conduction mechanism may be similar to the mechanism regulating H+ conduction through Gramicidin-A channels (Pomes & Roux, Reference Pomes and Roux2002). Under pathophysiological conditions, this highly H+ selective permeation pathway leads to cellular acidification, which is probably responsible for the development of some pathologies (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahine2012b).
Surprisingly, gating pores created by arginine-to-histidine substitutions have been reported to be permeable to ions such as Na+, K+, Li+, and guanidium (Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007, Reference Sokolov, Scheuer and Catterall2010), which indicates that the characteristics of gating pores depend on the mutated VSD and the location of the mutation. However, Sokolov et al. observed that, with the exception of H+, guanidium may be the only ion capable of permeating the gating pores of R666H (R2H – DII/Nav1.4) mutant channels (Sokolov et al. Reference Sokolov, Scheuer and Catterall2010). Similar observations have been reported with Hv1-mutated channels (Berger & Isacoff, Reference Berger and Isacoff2011). In this case, the permeation of guanidium appears to be anomalous. According to Eisenmann's selectivity sequences and other gating pore studies, if large guanidium cations can permeate a gating pore, then other smaller cations such as Cs+, K+, or Na+ should also flow through the gating pore. However, guanidium alters the normal structure of proteins (England & Haran, Reference England and Haran2011). While a mutation is required to observe the permeation of guanidium, once guanidium has access to the permeation pathway, the mechanism by which it permeates may be related to its potential to denature proteins. Guanidium cations might thus make their way through VSDs rather than using the typical permeation pathway that would exclude this large cation (Decoursey, Reference Decoursey2013).
The H+ selectivity of gating pores appears to be associated with the presence of a histidine (replacing arginine). In Hv1 channels, H+ selectivity, which appears to be specific to these channels, has recently been attributed to the aspartate 112 residue (Musset et al. Reference Musset, Smith, Rajan, Morgan, Cherny and Decoursey2011). Musset et al. reported that the amino acid at position 112 plays a fundamental role in the exclusion of negatively charged ions. Indeed, the D112H mutation makes Hv1 channels permeable to anions (Musset et al. Reference Musset, Smith, Rajan, Morgan, Cherny and Decoursey2011).
A disruption of the S4–GCTC interaction is to be expected following S2 or S3 countercharge mutations. As mentioned above, an excess of negatively charged residues in the GCTC underlies the cation selectivity of gating pores. GCTC mutations may thus change the charged environment and, as a result, modify the cation selectivity of gating pores.
3.2.2. Conductance
Gating pores are generally small leak currents that usually amount to 1–6% of the alpha pore currents of mutated channels (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007) and represent unitary conductances ranging from a few femto Siemens (fS) to several hundred fS for gating pore currents caused by single mutations (34–1060 fS) (Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014). As expected, gating pore currents resulting from multiple mutations in VSDs exhibit larger unitary conductances (up to 3420 fS) (Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014; Tombola et al. Reference Tombola, Pathak, Gorostiza and Isacoff2007). H+ selective gating pores are a special case due to the different conduction mechanism (Grotthus mechanism). Nevertheless, reported conductances remain variable, and range from 0.17 to 420 fS (Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014).
The unitary conductances of gating pores appear to be closely related to the nature and location of the mutations causing the gating pores. This variability once again shows that while VSDs have similar structures they also display major variations that likely influence the size or shape of water crevices and, in turn, result in different conductances.
3.3. Blockers and modulators
3.3.1. Blockers
As shown in Fig. 9, blocking gating pores is a subject of some debate. Although blocking by divalent (Mg2+), trivalent (Y3+, Yb3+, Lu3+, Ti3+), and quadrivalent (Hf4+) cations is not in doubt (Sokolov et al. Reference Sokolov, Scheuer and Catterall2010; Tombola et al. Reference Tombola, Pathak and Isacoff2005), blocking by many other cations remains controversial (Berger & Isacoff, Reference Berger and Isacoff2011; Francis et al. Reference Francis, Rybalchenko, Struyk and Cannon2011; Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahine2012b; Klassen et al. Reference Klassen, Spencer and Gallin2008; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007; Struyk & Cannon, Reference Struyk and Cannon2007; Struyk et al. Reference Struyk, Markin, Francis and Cannon2008). Cd2+ and Li+ have both been reported to have no effect on gating pore currents (Fig. 9) (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahine2012b; Struyk & Cannon, Reference Struyk and Cannon2007). Gating pore blockers appear to be highly dependent on the nature of the mutation and its location and on experimental conditions. The lack of a universal gating pore blocker is an impediment to research since the inhibition of gating pore currents would be a powerful tool for characterizing these currents. Furthermore, the identification of a universal gating pore blocker would be of utmost importance for developing treatments for pathologies caused by the appearance of gating pores.
Gating pore blockers. The blocking effect of several cations and compounds is summarized. The gating pore blockers are shown in the left panel. The effect of these blockers is not in dispute. 2GBI derivatives have also been reported to block the Hv1 channels, whereas 2GBI is the most efficient (Hong et al. Reference Hong, Pathak, Kim, Ta and Tombola2013, Reference Hong, Kim and Tombola2014. There is some dispute concerning the blockers shown in the middle panel. Li+ or Cd2+ has no effect on gating pore currents (right panel). For the purposes of clarity, a single mutation is shown on the VSD. The data summarized here were obtained using different channels and mutations.
A number of researchers have suggested that guanidine derivatives could be effective gating pore blockers (Hong et al. Reference Hong, Pathak, Kim, Ta and Tombola2013, Reference Hong, Kim and Tombola2014; Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2010; Tombola et al. Reference Tombola, Pathak and Isacoff2005) based on the assumption that the arginines in WT VSDs naturally occlude the gating pore permeation pathway. Indeed, guanidine makes up the arginine side chain and is also reported to be highly permeant for several gating pores (Berger & Isacoff, Reference Berger and Isacoff2011; Groome et al. Reference Groome, Lehmann-Horn, Fan, Wolf, Winston, Merlini and Jurkat-Rott2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2010; Tombola et al. Reference Tombola, Pathak and Isacoff2005). Guanidine derivatives may thus turn out to be powerful, specific blockers of gating pores. However, published results to date are contradictory. While 1-(2,4-xylyl) guanidine-carbonate and 2-gunaidiniumbenzimidazole (2GBI) have been reported to block gating pores (Hong et al. Reference Hong, Pathak, Kim, Ta and Tombola2013, Reference Hong, Kim and Tombola2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2010), ethyl-guanidium does not (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahine2012b; Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2010; Tombola et al. Reference Tombola, Pathak and Isacoff2005). Here again, the block seems to depend on the nature of the VSD and the mutation and on the experimental conditions. Guanidine derivatives may also be potentially useful for developing therapeutics to treat gating pore-related pathologies. In addition, it is not anticipated that guanidium derivatives will affect the normal functioning of channels.
3.3.2. Modulators
Gating modifiers may also be of interest for modulating gating pores. NH29 (a diclofenac derivative), ztz240, β-scorpion toxins, β-spider toxins, and μ-Oconotoxins could be used to stabilize VSDs in a given state (Cestele & Catterall, Reference Cestele and Catterall2000; Leipold et al. Reference Leipold, Debie, Zorn, Borges, Olivera, Terlau and Heinemann2007; Li et al. Reference Li, Chen, Xu, Sun, Li, Liu, Yang, Gao, Jiang and Li2013; Peretz et al. Reference Peretz, Pell, Gofman, Haitin, Shamgar, Patrich, Kornilov, Gourgy-Hacohen, Ben-Tal and Attali2010; Stevens et al. Reference Stevens, Peigneur and Tytgat2011). Xiao et al. recently showed that toxins like Huwentoxin-IV (HWTX-IV) and ProTx-II may promote or inhibit gating pore currents by stabilizing the resting or activated state (Xiao et al. Reference Xiao, Blumenthal and Cummins2014). These authors also used gating pores for studying the mechanisms of action of several other toxins (Xiao et al. Reference Xiao, Blumenthal and Cummins2014). Based on the voltage dependence of gating pores, it is possible that gating modifiers could be used to prevent or modulate the opening or closing of gating pores. Interestingly, HWTX-IV shifts the voltage dependence of gating pores activated at hyperpolarized voltages and increases the amplitude of the currents (Xiao et al. Reference Xiao, Blumenthal and Cummins2014). The shift of the voltage dependence may be caused by the stabilization of the resting state of the Nav–DII VSD. However, the increased amplitude of the gating pore currents was not anticipated at hyperpolarized voltages since the S4 segment was expected to be in a stable downward configuration. These authors proposed that, even at hyperpolarized voltages in absence of toxins, the S4 segment may fluctuate between positions that enable or prevent the communication of water crevices, thus slightly decreasing the amplitude of the gating pore current. HWTX–IV may thus stabilize the S4 segment in a conformation in which the water crevices remain connected (Xiao et al. Reference Xiao, Blumenthal and Cummins2014).
A major drawback of this approach is that the use of gating modifiers may compromise the normal functions of the VGIC. Indeed, stabilizing the DII of Nav channels in its resting state while allowing the other domains to move prevents the activation of the channel and precludes normal Na+ conduction (Xiao et al. Reference Xiao, Blumenthal and Cummins2014).
4. Conclusions and perspectives
The notion that VSDs are features solely of ion channels has been transcended by the discovery that other proteins such as enzymes have acquired VSDs for specific functions in living organisms and that VSDs serve as built-in H+ channels. The recent crystallization of several mammalian ion channels and their bacterial homologs, including an isolated voltage sensor, has revealed that these VSDs are organized as hourglass-like structures with deep water crevices on both sides of the channel protein (Jiang et al. Reference Jiang, Lee, Chen, Ruta, Cadene, Chait and Mackinnon2003; Long et al. Reference Long, Tao, Campbell and Mackinnon2007; Payandeh et al. Reference Payandeh, Scheuer, Zheng and Catterall2011, Reference Payandeh, Gamal El-Din, Scheuer, Zheng and Catterall2012; Takeshita et al. Reference Takeshita, Sakata, Yamashita, Fujiwara, Kawanabe, Kurokawa, Okochi, Matsuda, Narita, Okamura and Nakagawa2014; Zhang et al. Reference Zhang, Ren, Decaen, Yan, Tao, Tang, Wang, Hasegawa, Kumasaka, He, Wang, Clapham and Yan2012). The narrowest region is a very hydrophobic structure and is maintained by the GCTC, which focuses the electric field and allows the movement of the S4 in response to changes in voltage. A single point mutation in which S4 arginines are replaced with neutral amino acids can cause the water cervices to communicate and allow cations to permeate through the gating pore. When the mutated residue is a histidine, this allows H+ to translocate from one side to another. We recently used histidine scanning mutagenesis of the S4 segment as a readout to study the specific movement of a given voltage sensor among the four domains of the Nav channel (Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a) and showed that the disruption of the S4–GCTC interaction leads to the creation of a gating pore. However, the effects of mutations in the GCTC are unknown. Such mutations may also disrupt the S4–GCTC interaction and lead to the creation of a gating pore. The voltage dependence and selectivity of these hypothetical gating pores remains to be investigated. In addition, two mutations on the S1 and S2 segments have been reported to cause the appearance of gating pores (Campos et al. Reference Campos, Chanda, Roux and Bezanilla2007). However, it is not known whether these residues participate in the GCTC. All these results indicate that the mutation of residues outside the GCTC or the S4 segment may result in the communication of the water crevices. Gating pores may thus appear not only after the disruption of the GCTC–S4 interaction but, more generally, when the integrity of the hydrophobic septum is altered.
VSDs are shared by a large number of proteins. However, while VSDs share similar structures, they also display substantial differences that modulate their biophysical characteristics. Elucidating the molecular basis of these differences would significantly improve our understanding of VSDs as functional entities.
Gating pore currents amount to 1–6% of alpha pore currents, depending on the channel, probably due to their low conductance, which is estimated to be in the fS range (Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014). More importantly, a growing number of naturally occurring mutations on the S4 have been shown to underlie cell excitability diseases (Fan et al. Reference Fan, Lehmann-Horn, Weber, Bednarz, Groome, Jonsson and Jurkat-Rott2013; Gosselin-Badaroudine et al. Reference Gosselin-Badaroudine, Delemotte, Moreau, Klein and Chahine2012a, Reference Gosselin-Badaroudine, Keller, Huang, Pouliot, Chatelier, Osswald, Brink and Chahineb; Reference Gosselin-Badaroudine, Moreau and Chahine2013; Moreau et al. Reference Moreau, Gosselin-Badaroudine and Chahine2014; Sokolov et al. Reference Sokolov, Scheuer and Catterall2007; Struyk & Cannon, Reference Struyk and Cannon2007). Owing to the large distribution of VSDs in living organisms, these pathologies affect a wide range of physiological functions, including cardiac, motor, and cognitive functions. To date, only a few ions and molecules have been shown to be effective gating pore blockers. Their effectiveness appears to be related to the nature of the mutation causing the gating pore. Future studies will be required to elucidate the molecular basis of these discrepancies and potentially lead to the discovery of new universal gating pore blockers.
Gating modifier molecules and toxins could also be used to modulate gating pores. In addition to modulating gating pores, these modifiers may have an impact on the gating of ion channels. Several naturally occurring mutations that cause pain or cardiac arrhythmia result from shifts as small as 5 mV in the G v curves of ion channels. These small gating modifications may have a major impact on cell excitability. We thus anticipate that VSDs will serve as targets for the development of novel future drugs that will essentially be gating modifiers. ztz240 has been shown to modulate the gating of Kv7.2 channels (Gao et al. Reference Gao, Zhang, Wu, Xiong, Sun, Zhang, Zu, Wang and Li2010). Li et al. recently showed that the action of ztz240 is mediated by a direct interaction with the GCTC (Li et al. Reference Li, Chen, Xu, Sun, Li, Liu, Yang, Gao, Jiang and Li2013) and that it prevents epileptic seizures. These studies on ztz240 have provided the first evidence that VSDs, and more specifically the GCTC, may be valid drug targets, and that such an approach shows potential for treating a number of several cell excitability diseases, including pain, epilepsy, and cardiac arrhythmias.
Gating pore currents and the structure and functioning of VSDs have been the focus of much attention in the last decade. The VSDs of voltage-sensitive proteins were thought to share a common structure. However, more recent findings have shown that they have many variations that modulate their structure and function and, consequently, the biophysical characteristics of gating pores. Future studies should thus focus on investigating these differences in order to increase our understanding of the structure, function, and dysfunction of VSDs.
5. Acknowledgements
This study was supported by the grants from the Heart and Stroke Foundation of Quebec (HSFQ) and the Canadian Institutes of Health Research (CIHR; MOP-111072 and MOP-130373); and Grant from the Natural Sciences and Engineering Research Council of Canada (RGPIN-2014-04609).
