Charged S4 Segments Research Articles

Different Roles of IS4 and IIS4 Segments in CaV1.2 Gating

Membrane depolarisation and upward movement of charged S4 segments initiates the opening of voltage gated calcium channels while repolarisation and S4 downward movement facilitates channel closure. The individual impact of segements IS4-IVS4 in CaV1.2 is largely unknown. CaV1.2 with a neutral IIS4 (IIS4N) activate and deactivate similar to wild type. A role of IIS4 in gating becames evident in CaV1.2 carrying positional specific mutations of glycines or alanines on one of the S6 gates (G432/A780/G1193/A1503, GAGA-mutations). In these slowly gating mutant channels neutralisation of IIS4N causes a paradoxical shift of the activation curve to the right, accelerates channel gating and thus partially rescues wild type gating.Here we make use of GAGA-S6-mutants to study the role of IS4 and IIS4 and the impact of individual S4 charges. Introduction of a single charge into neutralized IS4N (IS4N+R276) accelerated channel gating and reduced the the slope of the activation curve. IIS4N+R662, also accelerated gating and shifted the activation curve to more depolirized voltages but without significantly affecting its slope.Segments IS4 and IIS4 carrying either one or four charges at different positions all accelerated curent kinetics. Our data suggest different roles and impacts of IS4 and IIS4 in channel activation. IS4 appears to be more important for “unlocking” the closed resting gate while IIS4 contributes predominantly to stabilisation of the gate in the open conformation. A second finding was the different impact of partially and fully charged S4 segments on CaV1.2 gating. We hypothesise that the evolutionary design of CaV1.2 requires fife charges in IS4 and in IIS4 for voltage-dependent movements to their final up and down positions. Our data support a gating model of CaV1.2 where IS4 and IIS4 modulate all four cooperatively interacting S6 gates in a domain specific manner.

How to open a proton pore-more than S4?

Pioneering studies in voltage-gated potassium channels have described movement of the voltage-sensing domain (VSD) S4 helix across the membrane electric field in molecular detail, but much less is known regarding opening of the intrinsic proton pore within VSDs of voltage-dependent proton channels. By systematically probing local kinematics, a new study reveals that movements in helix S1 correlate with pore opening and are distinct from voltage-sensing movements of the charged S4 segment.

Offsetting the Electric Field Sensed by KV Channels through Residue Substitutions on Top of S1

Kv channel subunits consist of 6 transmembrane segments (S1-S6) whereby the S1 through S4 segments assemble into a voltage sensing domain (VSD) that detects the membrane electric field. The positively charged S4 segment forms the main component of the VSD and undergoes the largest reorientations upon a membrane de- or hyperpolarization, generating a transient gating current. The S1-S3 segments surround the S4 and facilitate the latter's movement across the hydrophobic plasma-membrane. A positive (lysine) and negative (aspartate) charge substitution scan at the extracellular end of the S1 segment in the Shaker-type Kv1.5 channel indicated that this region is sufficiently close to the S4 segment such that it modulates the local membrane electric field. At positions E268, E272, F273 and E276 a charge substitution or charge introduction exerted a surface charge effect and shifted the voltage dependence of channel opening accordingly. Surprisingly, these residues, which modulated the electric field, did not face the S4 in a predicted 3D structure of the Kv1.5 channel in the open state (homology model based on the crystal structure of the Kv2.1/Kv1.2 chimera). This suggests that the introduced charges affect the electrical field around the S4 segment in the closed state only. In conclusion, residues at the top of the S1 segment can state-dependently offset (polarize) the electric field sensed by the S4 segment, supporting that both segments are in close proximity. (This research was supported by fellowship CONACyT #203936 to EMM & grant FWO-G.0449.11N to DJS.)

S1–S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1–S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1–S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNaV1.4. These show that side chains of putative countercharges in hNaV1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I–III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.

The Conserved Phenylalanine in the K+ Channel Voltage-Sensor Domain Creates a Barrier with Unidirectional Effects

Voltage-gated ion channels are crucial for regulation of electric activity of excitable tissues such as nerve cells, and play important roles in many diseases. During activation, the charged S4 segment in the voltage sensor domain translates across a hydrophobic core forming a barrier for the gating charges. This barrier is critical for channel function, and a conserved phenylalanine in segment S2 has previously been identified to be highly sensitive to substitutions. Here, we have studied the kinetics of Kv1-type potassium channels (Shaker and Kv1.2/2.1 chimera) through site-directed mutagenesis, electrophysiology, and molecular simulations. The F290L mutation in Shaker (F233L in Kv1.2/2.1) accelerates channel closure by at least a factor 50, although opening is unaffected. Free energy profiles with the hydrophobic neighbors of F233 mutated to alanine indicate that the open state with the fourth arginine in S4 above the hydrophobic core is destabilized by ∼17 kJ/mol compared to the first closed intermediate. This significantly lowers the barrier of the first deactivation step, although the last step of activation is unaffected. Simulations of wild-type F233 show that the phenyl ring always rotates toward the extracellular side both for activation and deactivation, which appears to help stabilize a well-defined open state.

Intracellular Ions Impede Voltage Sensor Return in Kv1.2 Channels

Voltage sensing in Kv channels originates from the coupling of movement between the charged S4 segment and the activation gate at the cytoplasmic region of the pore domain. The voltage sensor moves prior to the pore opening and the pore must shut before the voltage sensor returns to its resting state. However, gating current recordings from Kv channels indicate that frequently voltage sensors return more slowly after depolarisations that populate open states, indicating that the open pore exerts a resistance to S4 return. This process of pore closure therefore intrinsically regulates the deactivation kinetics of Kv channels. We observed slow voltage sensor return (IgOFF) in WT-Kv1.2 channels under non-permeant ionic conditions after depolarisations to voltages that caused channel openings. Using TEA+ and NMG+ internal solutions resulted in a slower IgOFF than internal Cs+, suggesting that the intracellular ionic composition was modulating IgOFF. A mutation in the pore lining S6 segment to enlarge the inner cavity (Kv1.2-I402C) removed the slowing of IgOFF in the presence of internal NMG+, suggesting that NMG+ interacted within the inner cavity of the WT channel to prevent pore closure through a ‘foot in the door’ mechanism. Gating currents of a non-conducting, P-type inactivated channel (Kv1.2-W366F, V381T), in the presence of intracellular K+ ions also displayed a slowing of IgOFF after depolarisations that would open the channel pore. These results suggest that internal K+ ions bound in the inner cavity can also slow activation gate closure. We propose that internal ions in the cavity of Kv1.2 allosterically regulate the voltage sensor deactivation kinetics by preventing pore closure and thus rate limiting the return of voltage sensors.

Phe233 in the Voltage-Sensor is Rate Limiting for Channel Closure but not for the Opening

During activation, the charged S4 segment in the voltage sensor domain (VSD) of voltage-gated ion channels is required to translate across a hydrophobic zone. This constitutes a thin aperture clearly separating the open/relaxed and closed/resting states. The nature of this barrier is critical for channel function, and has been the focus of much attention. Previously, we identified the single preserved residue F233 (F290 in Shaker) as a structural barrier for the gating charges, that uniquely determines intermediate state and substitutions modulate the deactivation barrier. A fundamental understanding of the S4 activation/deactivation barrier as well as kinetics is an important remaining challenge to decipher gating. Here, we study the free barrier and kinetics of the VSD through combining in-vitro and in-silico experiments. We used site-directed mutagenesis and measured the voltage-dependence in-vitro to study the effect of F290L compared to the wild-type (WT). In parallel, molecular dynamics simulations allowed us to identify residues interacting with the phenyl ring. Through in-silico mutations we show their impact on the barrier, as well as the structural and kinetics effects for the phenyl ring orientation during the first step of deactivation and the (reverse) last step of the activation. Strikingly, the channel closing transition shows a huge speedup from the F290L mutant (1ms, WT 10ms), in contrast to the opening that is completely unaffected. This indicates that F233 is only rate-limiting for the channel closure, but not opening and suggests different kinetics for the activation/deactivation barriers. Additionally the ring is clearly stabilized through vdW interactions with surrounding hydrophobic residues, and appears to always open by upward rotation both for activation/deactivation. This upward rotation suggests a model where the closing is possibly a mostly entropic process, while opening would be largely enthalpic.

Exploring the Energetics of Membrane Protein Stability using a Deformable Model of the Membrane

Experimental and computational studies have shown that cellular membranes deform to stabilize the inclusion of transmembrane (TM) proteins. Recent analysis suggests that membrane bending helps to expose charged and polar amino acids to the aqueous environment and polar phospholipid headgroups. Our previous study used elasticity theory to characterize the distortions of a membrane in the presence of a TM peptide. Here, we extend our method through the implementation of a search algorithm that identifies the membrane shape that minimizes the free energy of helix insertion. We show that our model robustly identifies the hydrophobic stretch of a TM protein and allows for large deformations to embed these stretches in the membrane. When applied to the MscL stretch activated channel, our model predicts local membrane thinning at the protein boundary that is corroborated by fluorescence and cysteine scanning experiments. We go on to show that the insertion energy for proteins containing multiple charged residues is non-additive, and the model provides a clear physical mechanism for this non-additivity, which is absent in other continuum membrane models. We predict that some highly charged S4 segments from voltage-gated K+ channels are stable in the bilayer in accordance with previous experiments and molecular dynamics simulations. Our method thus captures essential aspects of protein-membrane interactions at a small fraction of the cost of traditional molecular simulations.

Mode Shift of the Voltage Sensors in Shaker K + Channels is Caused by Energetic Coupling to the Pore Domain

The voltage sensors (S1-S4) of Kv channels are known to undergo a conformational change when triggered by membrane depolarisation that subsequently leads to opening of the central pore (S5-S6) and ion conduction. The electromechanical coupling between these domains is mediated by two well conserved regions: the C-terminus of the S6 segment and the S4-S5 linker. Upon change in the membrane potential, the structural rearrangements induced by the charged S4 segment can be measured as gating currents. During prolonged depolarization, voltage-gated ion channels present a behaviour called the “mode-shift”. This shift causes the QV to change towards negative potentials and essentially affects the energy required by the system to bring all sensors back to their resting state. To understand the structural cause of this process, we investigated the coupling between the pore and voltage sensor and its influence on the development of the mode shift. In our approach we used the cut-open voltage clamp fluorometry method to simultaneously measure gating or ionic currents and conformational changes of the S4 segment. We identified mutations that fully uncouple voltage sensors from the pore domain thus eliminating the mutual influence of these two domains on one another. The results show that, when eliminating the coupling between these domains, the voltage sensors require less energy to move. At the same time the mode shift occurring during prolonged depolarization is abolished. By instead preventing open state stabilization, voltage sensors are kept in the opposite mode. We also found that the mode shift is accompanied by a conformational change in the S4 segment. We propose that the pore influences the voltage sensor by the presence of a ‘’mechanical load’’ and allosterically induces a conformational change in the S4.

Sodium Channel as a Gate for Molecular Arrhythmology: A Historical Overview

Ions carry transsarcolemmal currents that contribute importantly to electrical activity in cardiac myocytes, where the influx of Naþ ions plays an especially unique and critical role. The sodium current (INa), is not only responsible for initial cardiac action-potential depolarization, but also the influx of Naþ ions into the cytoplasm, which helps to generate the prolonged depolarization characteristics of the action-potential plateau. The magnitude and kinetics of INa are controlled in a complex manner by both membrane potential and intracellular metabolites. Such Naþ-preferring voltage-dependent channels are present in a wide variety of electrically excitable membranes of cardiac and skeletal muscles, nerves, and other tissues. The voltage-gated cardiac Naþ channel has been extensively studied by electrophysiological approaches, so that we have accumulated a large amount of information on the biophysical properties of this channel during the last thirty years. Mutations of the human voltage-gated cardiac Naþ-channel gene that cause several forms of inherited diseases have been identified. The underlying defects in the Naþ-channel-related disease state include aberrant regulation of channel function, resulting in lethal ventricular tachyarrhythmias and so on. This review article will cover certain aspects involved in the structure and regulation of the cardiac sarcolemmal voltage-gated Naþ channel in physiological and particularly in pathophysiological conditions. In 1984, in order to clarify the primary structure of the Naþ channel, Numa and colleagues used partial sequences from purified eel electroplax Naþ channels to clone the cDNA for the eel Naþ-channel -subunit. Using this probe, they cloned three Naþ-channel -subunits from the rat brain. The rat heart isoform (rHt) and its human counterpart (hHt) were cloned thereafter. The -subunit of the cardiac Naþ channel has been reported to be 240 kDa. A schematic diagram of the proposed membrane topology for the -subunit of the cardiac Naþ channel is shown in Figure 1. The -subunit consists of four large homologous membrane domains (I-IV). Each domain is comprised of six putative -helices (S1-S6). The channel’s pore is assumed to extend between segments S5 and S6. Studies with molecular-biological techniques, particularly site-directed mutagenesis of Naþ-channel proteins and functional expression mutant channels have pinpointed regions that are involved in different functions of the channel. Between each of the six transmembrane segments and particularly between each of the four larger domains are extensive regions that fold out from the membrane. Highly charged S4 segments of each domain transduce the transmembrane electrical voltage by moving across the transmembrane electrical field. This movement causes conformational changes that result in an opening or gating of the Naþ-conducting pathway. The opening of the channel, also called ‘‘activation’’, allows Naþ ions to passively enter the cell down the Naþconcentration gradient to depolarize the membrane. The Nand C-termini, as well as the linkers between segments alternate between intracellular and extracellular surfaces. The ball and chain mechanism was first proposed for the explanation of the sodium channel inactivation (Figure 1). The idea of the ball and chain inactivation in the Naþ channel is simple: when the channel is open, a peptide ball (composed of amino acid residues), tethered on the membrane by a chain, wanders randomly around the channel pore. Further studies have shown that the inactivation of sodium channels involves rather a hinged lid than a ball on chain, but this did not decrease the significance of the model.

Estimating Conformational Changes of KCNQ1 Channels During Gating Using Molecular Dynamics Simulations

Although the structure of an ion-channel protein may be known through crystallography or homology modeling, measuring its conformational changes during gating is extremely difficult if not impossible. Alternatively, molecular simulations is a powerful tool for estimating conformational changes. Movement of the positively charged S4 segment is a known conformational change of voltage-gated ion-channels, observed by measuring the accessibility of different amino-acid residues.A previously determined structure of KCNQ1 was used. Movement of the S4-S3 complex with 3 translational and 2 rotational degrees of freedom was assumed to be the major conformational change during gating. More than 1 million conformations were considered. This expanded configuration space (compared to 1 translational and 1 rotational degree of freedom and about 2000 conformations used previously) enabled more accurate analysis of stable conformations. Conformations with steric overlap were eliminated. The electrostatic energies of the remaining conformations for various membrane potentials were computed and used to determine the probability of the ion-channel residing in each conformation (state residency). Conformations with small state residencies were eliminated.Two regions in the configuration space with high state residency were identified: one associated with open conformations (outward position of S4-S3) and the other with closed conformations (inward position of S4-S3). These regions were separated by a narrow region with low state residency (an energy barrier). The open state consists of a large number of conformations while the energy barrier and its adjacent closed state (intermediate closed) consist of only few conformations. Channel conformations may branch from the intermediate closed conformation into two trajectories toward different subsets of closed conformations (deep closed). The simulated steady state open probabilities at various membrane potentials were consistent with experimental channel activation curves.

Focused Electric Field across the Voltage Sensor of Potassium Channels

Voltage-gated ion channels respond to changes in membrane potential by movement of their voltage sensors across the electric field between cytoplasmic and extracellular solutions. The principal voltage sensors in these proteins are positively charged S4 segments. The absolute magnitude of S4 movement discriminates two competing classes of gating models. In one class, the movement is <10 Angstrom due to the fact that the electric field is focused by aqueous crevices in the channel protein. In an alternative model, based in part on the crystal structure of a potassium channel, the side chains of S4 arginines move their charges across the bilayer's electric field, a distance of >25 Angstrom. Here, using tethered charges attached to an S4 segment, we provide evidence that the electric field falls across a distance of <4 Angstrom, supporting a model in which the relative movement between S4 and the electric field is very small.

S4 movement in a mammalian HCN channel.

Hyperpolarization-activated, cyclic nucleotide–gated ion channels (HCN) mediate an inward cation current that contributes to spontaneous rhythmic firing activity in the heart and the brain. HCN channels share sequence homology with depolarization-activated Kv channels, including six transmembrane domains and a positively charged S4 segment. S4 has been shown to function as the voltage sensor and to undergo a voltage-dependent movement in the Shaker K+ channel (a Kv channel) and in the spHCN channel (an HCN channel from sea urchin). However, it is still unknown whether S4 undergoes a similar movement in mammalian HCN channels. In this study, we used cysteine accessibility to determine whether there is voltage-dependent S4 movement in a mammalian HCN1 channel. Six cysteine mutations (R247C, T249C, I251C, S253C, L254C, and S261C) were used to assess S4 movement of the heterologously expressed HCN1 channel in Xenopus oocytes. We found a state-dependent accessibility for four S4 residues: T249C and S253C from the extracellular solution, and L254C and S261C from the internal solution. We conclude that S4 moves in a voltage-dependent manner in HCN1 channels, similar to its movement in the spHCN channel. This S4 movement suggests that the role of S4 as a voltage sensor is conserved in HCN channels. In addition, to determine the reason for the different cAMP modulation and the different voltage range of activation in spHCN channels compared with HCN1 channels, we constructed a COOH-terminal–deleted spHCN. This channel appeared to be similar to a COOH-terminal–deleted HCN1 channel, suggesting that the main functional differences between spHCN and HCN1 channels are due to differences in their COOH termini or in the interaction between the COOH terminus and the rest of the channel protein in spHCN channels compared with HCN1 channels.

Evidence for intersubunit interactions between S4 and S5 transmembrane segments of the Shaker potassium channel.

Voltage-gated potassium channels are transmembrane proteins made up of four subunits, each comprising six transmembrane (S1-S6) segments. S1-S4 form the voltage-sensing domain and S5-S6 the pore domain with its central pore. The sensor domain detects membrane depolarization and transmits the signal to the activation gates situated in the pore domain, thereby leading to channel opening. An understanding of the mechanism by which the sensor communicates the signal to the pore requires knowledge of the structure of the interface between the voltage-sensing and pore domains. Toward this end, we have introduced single cysteine mutations into the extracellular end of S4 (positions 356 and 357) in conjunction with a cysteine in S5 (position 418) of the Shaker channel and expressed the mutants in Xenopus oocytes. We then examined the propensity of each pair of engineered cysteines to form a metal bridge or a disulfide bridge, respectively, by examining the effect of Cd2+ ions and copper phenanthroline on the K+ conductance of a whole oocyte. Both reagents reduced currents through the S357C,E418C double mutant channel, presumably by restricting the movements necessary for coupling the voltage-sensing function to pore opening. This inhibitory effect was seen in the closed state of the channel and with heteromers composed of S357C and E418C single mutant subunits; no effect was seen with homomers of any of the single mutant channels. These data indicate that the extracellular end of S4 lies in close proximity to the extracellular end of the S5 of the neighboring subunit in closed channels.

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation.

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.

Movement and crevices around a sodium channel S3 segment.

Voltage sensing is due mainly to the movement of positively charged S4 segments through the membrane electric field during changes of membrane potential. The roles of other transmembrane segments are under study. The S3 segment of domain 4 (D4/S3) in the sodium channel Nav1.4 carries two negatively charged residues and has been implicated in voltage-dependent gating. We substituted cysteines into nine putative “high impact” sites along the complete length of D4/S3 and evaluated their accessibilities to extracellular sulfhydryl reagents. Only the four outermost substituted cysteines (L1433C, L1431C, G1430C, and S1427C) are accessible to extracellular sulfhydryl reagents. We measured the voltage-dependent modification rates of the two cysteines situated at the extreme ends of this accessible region, L1433C and S1427C. Independent of the charge on the sulfhydryl reagents, depolarization increases the reactivity of both of these residues. Thus, the direction of the voltage dependence is opposite to that expected for a negatively charged voltage sensor, namely an inward translational movement in response to depolarization. Intrinsic electrostatic potentials were probed by charged sulfhydryl reagents and were either negative or positive, respectively, near L1433C and S1427C. The magnitude of the electrostatic potential near S1427C decreases with depolarization, suggesting that the extracellular crevice next to it widens during depolarization. S1427C experiences 44% of the electric field, as probed by charged cysteine reagents. To further explore movements around D4/S3, we labeled cysteines with the photoactivatable cross-linking reagent benzophenone-4-carboxamidocysteine methanethiosulfonate and examined the effects of UV irradiation on channel gating. After labeling with this reagent, all accessible cysteine mutants show altered gating upon brief UV irradiation. In each case, the apparent insertion efficiency of the photoactivated benzophenone increases with depolarization, indicating voltage-dependent movement near the extracellular end of D4/S3.

Independence and cooperativity in rearrangements of a potassium channel voltage sensor revealed by single subunit fluorescence.

Voltage-gated potassium channels are composed of four subunits. Voltage-dependent activation of these channels consists of a depolarization-triggered series of charge-carrying steps that occur in each subunit. These major charge-carrying steps are followed by cooperative step(s) that lead to channel opening. Unlike the late cooperative steps, the major charge-carrying steps have been proposed to occur independently in each of the channel subunits. In this paper, we examine this further. We showed earlier that the two major charge-carrying steps are associated with two sequential outward transmembrane movements of the charged S4 segment. We now use voltage clamp fluorometry to monitor these S4 movements in individual subunits of heterotetrameric channels. In this way, we estimate the influence of one subunit's S4 movement on another's when the energetics of their transmembrane movements differ. Our results show that the first S4 movement occurs independently in each subunit, while the second occurs cooperatively. At least part of the cooperativity appears to be intrinsic to the second S4 charge-carrying rearrangement. Such cooperativity in gating of voltage-dependent channels has great physiological relevance since it can affect both action potential threshold and rate of propagation.

Structure and function of the cardiac sodium channels.

The coincident cloning of the voltage-gated Na channel from the electric eel electroplax and development of patch-clamp methodology has allowed an explosive phase of investigation into the structural basis of cardiac Na channel function. Recognizing the importance of structural motifs that underlie gating (charged S4 segments, III-IV linker) and permeation (P-loops) have complemented new molecular information surrounding inherited cardiac arrhythmias, such as the chromosome 3-linked form of the long QT syndrome. Although the proarrhythmic potential recognized in the CAST trial [1] slowed the development of class I antiarrhythmic agents, our emerging understanding of the molecular pharmacology of Na channels may motivate strategies for Na-channel drug discovery that involve targeting particular structural domains.

Structure and function of cardiac potassium channels.

Recent advances in molecular biology have had a major impact on our understanding of the biophysical and molecular properties of ion channels. This review is focused on cardiac potassium channels which, in general, serve to control and limit cardiac excitability. Approximately 60 K+ channel subunits have been cloned to date. The (evolutionary) oldest potassium channel subunits consist of two transmembrane (Tm) segments with an intervening pore-loop (P). Channels formed by four 2Tm-1P subunits generally function as inwardly rectifying K(+)-selective channels (KirX.Y): they conduct substantial current near the resting potential but carry little or no current at depolarized potentials. The inward rectifier IK1 and the ligand-gated KATP and KACh channels are composed of such subunits. The second major class of K+ channel subunits consists of six transmembrane segments (S1-S6). The S5-P-S6 section resembles the 2Tm-1P subunit, and the additional membrane-spanning segments (especially the charged S4 segment) endow these 6Tm-1P channels with voltage-dependent gating. For both major families, four subunits assemble into a homo- or heterotetrameric channel, subject to specific subunit-subunit interactions. The 6Tm-1P channels are closed at the resting potential, but activate at different rates upon depolarization to carry sustained or transient outward currents (the latter due to inactivation by different mechanisms). Cardiac cells typically display at least one transient outward current and several delayed rectifiers to control the duration of the action potential. The molecular basis for each of these currents is formed by subunits that belong to different Kvx.y subfamilies and alternative splicing can contribute further to the diversity in native cells. These subunits display distinct pharmacological properties and drug-binding sites have been identified. Additional subunits have evolved by concatenation of two 2Tm-1P subunits (4Tm-2P); dimers of such subunits yield voltage-independent leak channels. A special class of 6Tm-1P subunits encodes the 'funny' pacemaker current which activates upon hyperpolarization and carries both Na+ and K+ ions. The regional heterogeneity of K+ currents and action potential duration is explained by the heterogeneity of subunit expression, and significant changes in expression occur in cardiac disease, most frequently a reduction. This electrical remodelling may also be important for novel antiarrhythmic therapeutic strategies. The recent crystallization of a 2Tm-1P channel enhances the outlook for more refined molecular approaches.

Independent versus coupled inactivation in sodium channels. Role of the domain 2 S4 segment.

The voltage sensor of the sodium channel is mainly comprised of four positively charged S4 segments. Depolarization causes an outward movement of S4 segments, and this movement is coupled with opening of the channel. A mutation that substitutes a cysteine for the outermost arginine in the S4 segment of the second domain (D2:R1C) results in a channel with biophysical properties similar to those of wild-type channels. Chemical modification of this cysteine with methanethiosulfonate-ethyltrimethylammonium (MTSET) causes a hyperpolarizing shift of both the peak current-voltage relationship and the kinetics of activation, whereas the time constant of inactivation is not changed substantially. A conventional steady state inactivation protocol surprisingly produces an increase of the peak current at -20 mV when the 300-ms prepulse is depolarized from -190 to -110 mV. Further depolarization reduces the current, as expected for steady state inactivation. Recovery from inactivation in modified channels is also nonmonotonic at voltages more hyperpolarized than -100 mV. At -180 mV, for example, the amplitude of the recovering current is briefly almost twice as large as it was before the channels inactivated. These data can be explained readily if MTSET modification not only shifts the movement of D2/S4 to more hyperpolarized potentials, but also makes the movement sluggish. This behavior allows inactivation to have faster kinetics than activation, as in the HERG potassium channel. Because of the unique properties of the modified mutant, we were able to estimate the voltage dependence and kinetics of the movement of this single S4 segment. The data suggest that movement of modified D2/S4 is a first-order process and that rate constants for outward and inward movement are each exponential functions of membrane potential. Our results show that D2/S4 is intimately involved with activation but plays little role in either inactivation or recovery from inactivation.