Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels I: Wild-type skeletal muscle NaV1.4

The number of voltage-gated sodium (Na_V) channels available to generate action potentials in muscles and nerves is adjusted over seconds to minutes by prior electrical activity, a process called slow inactivation (SI). The basis for SI is uncertain. Na_V channels have four domains (DI–DIV), each with a voltage sensor that moves in response to depolarizing stimulation over milliseconds to activate the channels. Here, SI of the skeletal muscle channel Na_V1.4 is induced by repetitive stimulation and is studied by recording of sodium currents, gating currents, and changes in the fluorescence of probes on each voltage sensor to assess their movements. The magnitude, voltage dependence, and time course of the onset and recovery of SI are observed to correlate with voltage-sensor movements 10,000-fold slower than those associated with activation. The behavior of each voltage sensor is unique. Development of SI over 1–160 s correlates best with slow immobilization of the sensors in DI and DII; DIII tracks the onset of SI with less fidelity. Showing linkage to the sodium conduction pathway, pore block by tetrodotoxin affects both SI and immobilization of all the sensors, with DI and DII significantly suppressed. Recovery from SI correlates best with slow restoration of mobility of the sensor in DIII. The findings suggest that voltage-sensor movements determine SI and thereby mediate Na_V channel availability.     

A mutation in segment I-S6 alters slow inactivation of sodium channels.

Slow inactivation occurs in voltage-gated Na+ channels when the membrane is depolarized for several seconds, whereas fast inactivation takes place rapidly within a few milliseconds. Unlike fast inactivation, the molecular entity that governs the slow inactivation of Na+ channels has not been as well defined. Some regions of Na+ channels, such as mu1-W402C and mu1-T698M, have been reported to affect slow inactivation. A mutation in segment I-S6 of mu1 Na+ channels, N434A, shifts the voltage dependence of activation and fast inactivation toward the depolarizing direction. The mutant Na+ current at +50 mV is diminished by 60-80% during repetitive stimulation at 5 Hz, resulting in a profound use-dependent phenomenon. This mutant phenotype is due to the enhancement of slow inactivation, which develops faster than that of wild-type channels (tau = 0.46 +/- 0.01 s versus 2.11 +/- 0.10 s at +30 mV, n = 9). An oxidant, chloramine-T, abolishes fast inactivation and yet greatly accelerates slow inactivation in both mutant and wild-type channels (tau = 0.21 +/- 0.02 s and 0.67 +/- 0.05 s, respectively, n = 6). These findings together demonstrate that N434 of mu1 Na+ channels is also critical for slow inactivation. We propose that this slow form of Na+ channel inactivation is analogous to the "C-type" inactivation in Shaker K+ channels.     

Ion permeation and block of the gating pore in the voltage sensor of NaV1.4 channels with hypokalemic periodic paralysis mutations

Hypokalemic periodic paralysis and normokalemic periodic paralysis are caused by mutations of the gating charge–carrying arginine residues in skeletal muscle Na_V1.4 channels, which induce gating pore current through the mutant voltage sensor domains. Inward sodium currents through the gating pore of mutant R666G are only ∼1% of central pore current, but substitution of guanidine for sodium in the extracellular solution increases their size by 13- ± 2-fold. Ethylguanidine is permeant through the R666G gating pore at physiological membrane potentials but blocks the gating pore at hyperpolarized potentials. Guanidine is also highly permeant through the proton-selective gating pore formed by the mutant R666H. Gating pore current conducted by the R666G mutant is blocked by divalent cations such as Ba²⁺ and Zn²⁺ in a voltage-dependent manner. The affinity for voltage-dependent block of gating pore current by Ba²⁺ and Zn²⁺ is increased at more negative holding potentials. The apparent dissociation constant (K_d) values for Zn²⁺ block for test pulses to −160 mV are 650 ± 150 µM, 360 ± 70 µM, and 95.6 ± 11 µM at holding potentials of 0 mV, −80 mV, and −120 mV, respectively. Gating pore current is blocked by trivalent cations, but in a nearly voltage-independent manner, with an apparent K_d for Gd³⁺ of 238 ± 14 µM at −80 mV. To test whether these periodic paralyses might be treated by blocking gating pore current, we screened several aromatic and aliphatic guanidine derivatives and found that 1-(2,4-xylyl)guanidinium can block gating pore current in the millimolar concentration range without affecting normal Na_V1.4 channel function. Together, our results demonstrate unique permeability of guanidine through Na_V1.4 gating pores, define voltage-dependent and voltage-independent block by divalent and trivalent cations, respectively, and provide initial support for the concept that guanidine-based gating pore blockers could be therapeutically useful.     

Residue-specific effects on slow inactivation at V787 in D2-S6 of Na(v)1.4 sodium channels

Slow inactivation in voltage-gated sodium channels (NaChs) occurs in response to depolarizations of seconds to minutes and is thought to play an important role in regulating membrane excitability and action potential firing patterns. However, the molecular mechanisms of slow inactivation are not well understood. To test the hypothesis that transmembrane segment 6 of domain 2 (D2-S6) plays a role in NaCh slow inactivation, we substituted different amino acids at position V787 (valine) in D2-S6 of rat skeletal muscle NaCh mu(1) (Na(v)1.4). Whole-cell recordings from transiently expressed NaChs in HEK cells were used to study and compare slow inactivation phenotypes between mutants and wild type. V787K (lysine substitution) showed a marked enhancement of slow inactivation. V787K enters the slow-inactivated state approximately 100x faster than wild type (tau(1) approximately 30 ms vs. approximately 3 s), and occurs at much more hyperpolarized potentials than wild type (V(1/2) of s(infinity) curve approximately -130 mV vs. approximately -75 mV). V787C (cysteine substitution) showed a resistance to slow inactivation, i.e., opposite to that of V787K. Entry into the slow inactivation state in V787C was slower (tau(1) approximately 5 s), less complete, and less voltage-dependent (V(1/2) of s(infinity) curve approximately -50 mV) than in wild type. Application of the cysteine modification agent methanethiosulfonate ethylammonium (MTSEA) to V787C demonstrated that the 787 position undergoes a relative change in molecular conformation that is associated with the slow inactivation state. Our results suggest that the V787 position in Na(v)1.4 plays an important role in slow inactivation gating and that molecular rearrangement occurs at or near residue V787 in D2-S6 during NaCh slow inactivation.     

Floxed allele for conditional inactivation of the voltage-gated sodium channel Scn8a (NaV1.6)

The sodium channel gene Scn8a encodes the channel NaV1.6, which is widely distributed in the central and peripheral nervous system. NaV1.6 is the major channel at the nodes of Ranvier in myelinated axons. Mutant alleles of mouse Scn8a result in neurological disorders including ataxia, tremor, paralysis, and dystonia. We generated a floxed allele of Scn8a by inserting loxP sites around the first coding exon. The initial targeted allele containing the neo-cassette was a severe hypomorph. In vivo deletion of the neo-cassette by Flp recombinase produced a floxed allele that generates normal expression of NaV1.6 protein. Ubiquitous deletion of the floxed exon by Cre recombinase in ZP3-Cre transgenic mice produced the Scn8a(del) allele. The null phenotype of Scn8a(del) homozygotes confirms the in vivo inactivation of Scn8a. Conditional inactivation of the floxed allele will make it possible to circumvent the lethality that results from complete loss of Scn8a in order to investigate the physiologic role of NaV1.6 in subpopulations of neurons.     

A sodium channel defect in hyperkalemic periodic paralysis: potassium-induced failure of inactivation.

Hyperkalemic periodic analysis (HPP) is an autosomal dominant disorder characterized by episodic weakness lasting minutes to days in association with a mild elevation in serum K+. In vitro measurements of whole-cell currents in HPP muscle have demonstrated a persistent, tetrodotoxin-sensitive Na+ current, and we have recently shown by linkage analysis that the Na+ channel alpha subunit gene may contain the HPP mutation. In this study, we have made patch-clamp recordings from cultured HPP myotubes and found a defect in the normal voltage-dependent inactivation of Na+ channels. Moderate elevation of extracellular K+ favors an aberrant gating mode in a small fraction of the channels that is characterized by persistent reopenings and prolonged dwell times in the open state. The Na+ current, through noninactivating channels, may cause the skeletal muscle weakness in HPP by depolarizing the cell, thereby inactivating normal Na+ channels, which are then unable to generate an action potential. Thus the dominant expression of HPP is manifest by inactivation of the wild-type Na+ channel through the influence of the mutant gene product on membrane voltage.     

Potassium-dependent slow inactivation of Kir1.1 (ROMK) channels

The ROMK (Kir1.1) family of epithelial K channels can be inactivated by a combination of low internal pH and low external K, such that alkalization does not reopen the channels unless external K is elevated. Previous work suggested that this inactivation results from an allosteric interaction between an inner pH gate and an outer K sensor, and could be described by a simple three-state kinetic model. In the present study, we report that a sustained depolarization slowly inactivated (half-time = 10-15 min) ROMK channels that had been engineered for increased affinity to internal polyamines. Furthermore, this inactivation occurred at external [K] < or =1 mM in ROMK mutants whose inner pH gate was constitutively open (ROMK2-K61M mutation). Both pH and voltage inactivation depended on external K in a manner reminiscent of C-type inactivation, but having a much slower time course. Replacement of ROMK extracellular loop residues by Kir2.1 homologous residues attenuated or abolished this inactivation. These results are consistent with the hypothesis that there are (at least) two separate closure processes in these channels: an inner pH-regulated gate, and an outer (inactivation) gate, where the latter is modulated by both voltage and external [K].     

A superfamily of voltage-gated sodium channels in bacteria

NaChBac, a six-alpha-helical transmembrane-spanning protein cloned from Bacillus halodurans, is the first functionally characterized bacterial voltage-gated Na(+)-selective channel. As a highly expressing ion channel protein, NaChBac is an ideal candidate for high resolution structural determination and structure-function studies. The biological role of NaChBac, however, is still unknown. In this report, another 11 structurally related bacterial proteins are described. Two of these functionally expressed as voltage-dependent Na(+) channels (Na(V)PZ from Paracoccus zeaxanthinifaciens and Na(V)SP from Silicibacter pomeroyi). Na(V)PZ and Na(V)SP share approximately 40% amino acid sequence identity with NaChBac. When expressed in mammalian cell lines, both Na(V)PZ and Na(V)SP were Na(+)-selective and voltage-dependent. However, their kinetics and voltage dependence differ significantly. These single six-alpha-helical transmembrane-spanning subunits constitute a widely distributed superfamily (Na(V)Bac) of channels in bacteria, implying a fundamental prokaryotic function. The degree of sequence homology (22-54%) is optimal for future comparisons of Na(V)Bac structure and function of similarity and dissimilarity among Na(V)Bac proteins. Thus, the Na(V)Bac superfamily is fertile ground for crystallographic, electrophysiological, and microbiological studies.     

Domain III S4 in closed-state fast inactivation: Insights from a periodic paralysis mutation

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.     

Domain III S4 in closed-state fast inactivation: Insights from a periodic paralysis mutation

Heterologous expression of sodium channel mutations in hypokalemic periodic paralysis reveals 2 variants on channel dysfunction. Charge-reducing mutations of voltage sensing S4 arginine residues alter channel gating as typically studied with expression in mammalian cells. These mutations also produce leak currents through the voltage sensor module, as typically studied with expression in Xenopus oocytes. DIIIS4 mutations at R3 in the skeletal muscle sodium channel produce gating defects and omega current consistent with the phenotype of reduced excitability. Here, we confirm DIIIS4 R3C gating defects in the oocyte expression system for fast inactivation and its recovery. We provide novel data for the effects of the cysteine mutation on voltage sensor movement, to further our understanding of sodium channel defects in hypokalemic periodic paralysis. Gating charge movement and its remobilization are selectively altered by the mutation at hyperpolarized membrane potential, as expected with reduced serum potassium.     

Gating pore currents in DIIS4 mutations of NaV1.4 associated with periodic paralysis: saturation of ion flux and implications for disease pathogenesis

S4 voltage–sensor mutations in CaV1.1 and NaV1.4 channels cause the human muscle disorder hypokalemic periodic paralysis (HypoPP). The mechanism whereby these mutations predispose affected sarcolemma to attacks of sustained depolarization and loss of excitability is poorly understood. Recently, three HypoPP mutations in the domain II S4 segment of NaV1.4 were shown to create accessory ionic permeation pathways, presumably extending through the aqueous gating pore in which the S4 segment resides. However, there are several disparities between reported gating pore currents from different investigators, including differences in ionic selectivity and estimates of current amplitude, which in turn have important implications for the pathological relevance of these aberrant currents. To clarify the features of gating pore currents arising from different DIIS4 mutants, we recorded gating pore currents created by HypoPP missense mutations at position R666 in the rat isoform of Nav1.4 (the second arginine from the outside, at R672 in human NaV1.4). Extensive measurements were made for the index mutation, R666G, which created a gating pore that was permeable to K⁺ and Na⁺. This current had a markedly shallow slope conductance at hyperpolarized voltages and robust inward rectification, even when the ionic gradient strongly favored outward ionic flow. These characteristics were accounted for by a barrier model incorporating a voltage-gated permeation pathway with a single cation binding site oriented near the external surface of the electrical field. The amplitude of the R666G gating pore current was similar to the amplitude of a previously described proton-selective current flowing through the gating pore in rNaV1.4-R663H mutant channels. Currents with similar amplitude and cation selectivity were also observed in R666S and R666C mutant channels, while a proton-selective current was observed in R666H mutant channels. These results add support to the notion that HypoPP mutations share a common biophysical profile comprised of a low-amplitude inward current at the resting potential that may contribute to the pathological depolarization during attacks of weakness.     

Implication of the C-terminal region of the alpha-subunit of voltage-gated sodium channels in fast inactivation

The α-subunit of both the human heart (hH1) and human skeletal muscle (hSkM1) sodium channels were expressed in a mammalian expression system. The channels displayed slow (hH1) and fast (hSkM1) current decay kinetics similar to those seen in native tissues. Hence, the aim of this study was to identify the region on the α-subunit involved in the differences of these current-decay kinetics. A series of hH1/hSkM1 chimeric sodium channels were constructed with the focus on the C-terminal region. Sodium currents of chimeric channels were recorded using the patch-clamp technique in whole-cell configuration. Chimeras where the C-terminal region had been exchanged between hH1 and hSkM1 revealed that this region contains the elements that cause differences in current decay kinetics between these sodium channel isoforms. Other biophysical characteristics (steady-state activation and inactivation and recovery from inactivation) were similar to the phenotype of the parent channel. This indicates that the C-terminus is exclusively implicated in the differences of current decay kinetics. Several other chimeras were constructed to identify a specific region of the C-terminus causing this difference. Our results showed that the first 100-amino-acid stretch of the C-terminal region contains constituents that could cause the differences in current decay between the heart and skeletal muscle sodium channels. This study has uncovered a direct relationship between the C-terminal region and the current-decay of sodium channels. These findings support the premise that a novel regulatory component exists for fast inactivation of voltage-gated sodium channels. Received: 1 March 2001/Revised: 18 May 2001     

Structural determinants of slow inactivation in human cardiac and skeletal muscle sodium channels.

Skeletal and heart muscle excitability is based upon the pool of available sodium channels as determined by both fast and slow inactivation. Slow inactivation in hH1 sodium channels significantly differs from slow inactivation in hSkM1. The beta(1)-subunit modulates fast inactivation in human skeletal sodium channels (hSkM1) but has little effect on fast inactivation in human cardiac sodium channels (hH1). The role of the beta(1)-subunit in sodium channel slow inactivation is still unknown. We used the macropatch technique on Xenopus oocytes to study hSkM1 and hH1 slow inactivation with and without beta(1)-subunit coexpression. Our results indicate that the beta(1)-subunit is partly responsible for differences in steady-state slow inactivation between hSkM1 and hH1 channels. We also studied a sodium channel chimera, in which P-loops from each domain in hSkM1 sodium channels were replaced with corresponding regions from hH1. Our results show that these chimeras exhibit hH1-like properties of steady-state slow inactivation. These data suggest that P-loops are structural determinants of sodium channel slow inactivation, and that the beta(1)-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the beta(1)-subunit indicate possible interactions among the beta(1)-subunit, P-loops, and the slow inactivation gate in sodium channels.     

Interaction of scorpion alpha-toxins with cardiac sodium channels: binding properties and enhancement of slow inactivation

The effects of the scorpion alpha-toxins Lqh II, Lqh III, and LqhalphaIT on human cardiac sodium channels (hH1), which were expressed in human embryonic kidney (HEK) 293 cells, were investigated. The toxins removed fast inactivation with EC(50) values of <2.5 nM (Lqh III), 12 nM (Lqh II), and 33 nM (LqhalphaIT). Association and dissociation rates of Lqh III were much slower than those of Lqh II and LqhalphaIT, such that Lqh III would not dissociate from the channel during a cardiac activation potential. The voltage dependence of toxin dissociation from hH1 channels was nearly the same for all toxins tested, but it was different from that found for skeletal muscle sodium channels (muI; Chen et al. 2000). These results indicate that the voltage dependence of toxin binding is a property of the channel protein. Toxin dissociation remained voltage dependent even at high voltages where activation and fast inactivation is saturated, indicating that the voltage dependence originates from other sources. Slow inactivation of hH1 and muI channels was significantly enhanced by Lqh II and Lqh III. The half-maximal voltage of steady-state slow inactivation was shifted to negative values, the voltage dependence was increased, and, in particular for hH1, slow inactivation at high voltages became more complete. This effect exceeded an expected augmentation of slow inactivation owing to the loss of fast inactivation and, therefore, shows that slow sodium channel inactivation may be directly modulated by scorpion alpha-toxins.     

Fast and slow inactivation of sodium channels: effects of photodynamic modification by methylene blue.

Illumination of crayfish giant axons, during internal perfusion with 0.5 mM methylene blue (MB), produces photodynamic effects that include (i) reduction in total sodium conductance, (ii) shifting of the steady-state inactivation curve to the right along the voltage axis, (iii) reduction in the effective valence of steady-state inactivation and, (iv) potentially complete removal of fast inactivation. Additionally, the two kinetic components of fast inactivation in crayfish axons are differentially affected by MB+light. The intercept of the faster component (tau h1) is selectively reduced at shorter MB+light exposure times. Neither tau h1 nor the slower (tau h2) process was protected from MB+light by prior steady-state inactivation of sodium channels. However, carotenoids provide differing degrees of protection against each of the photodynamic actions listed above, suggesting that the four major effects of MB+light are mediated by changes occurring within different regions of the sodium channel molecule.     

A model of the interaction between N-type and C-type inactivation in Kv1.4 channels

Kv1.4 channels are Shaker-related voltage-gated potassium channels with two distinct inactivation mechanisms. Fast N-type inactivation operates by a ball-and-chain mechanism. Slower C-type inactivation is not so well defined, but involves intracellular and extracellular conformational changes of the channel. We studied the interaction between inactivation mechanisms using two-electrode voltage-clamp of Kv1.4 and Kv1.4ΔN (amino acids 2–146 deleted to remove N-type inactivation) heterologously expressed in Xenopus oocytes. We manipulated C-type inactivation by introducing a lysine-tyrosine point mutation (K532Y, equivalent to Shaker T449Y) that diminishes C-type inactivation. We used experimental data to develop a comprehensive computer model of Kv1.4 channels to determine the interaction between activation and N- and C-type inactivation mechanisms needed to replicate the experimental data. C-type inactivation began at lower voltage preactivated states, whereas N-type inactivation was coupled directly to the open state. A model with distinct N- and C-type inactivated states was not able to reproduce experimental data, and direct transitions between N- and C-type inactivated states were required, i.e., there is coupling between N- and C-type inactivated states. C-type inactivation is the rate-limiting step determining recovery from inactivation, so understanding C-type inactivation, and how it is coupled to N-type inactivation, is critical in understanding how channels act to repetitive stimulation.     

Being there: cellular targeting of voltage-gated sodium channels in the heart

Voltage-gated sodium (Na_v) channels in cardiomyocytes are localized in specialized membrane domains that optimize their functions in propagating action potentials across cell junctions and in stimulating voltage-gated calcium channels located in T tubules. Mutation of the ankyrin-binding site of Na_v1.5, the principal Na_v channel in the heart, was previously known to cause cardiac arrhythmia and the retention of Na_v1.5 in an intracellular compartment in cardiomyocytes. Conclusive evidence is now provided that direct interaction between Na_v1.5 and ankyrin-G is necessary for the expression of Na_v1.5 at the cardiomyocyte cell surface.     

Solution structure of delta-Am2766: a highly hydrophobic delta-conotoxin from Conus amadis that inhibits inactivation of neuronal voltage-gated sodium channels

The three-dimensional (3D) NMR solution structure (MeOH) of the highly hydrophobic delta-conotoxin delta-Am2766 from the molluscivorous snail Conus amadis has been determined. Fifteen converged structures were obtained on the basis of 262 distance constraints, 25 torsion-angle constraints, and ten constraints based on disulfide linkages and H-bonds. The root-mean-square deviations (rmsd) about the averaged coordinates of the backbone (N, C(alpha), C) and (all) heavy atoms were 0.62+/-0.20 and 1.12+/-0.23 A, respectively. The structures determined are of good stereochemical quality, as evidenced by the high percentage (100%) of backbone dihedral angles that occupy favorable and additionally allowed regions of the Ramachandran map. The structure of delta-Am2766 consists of a triple-stranded antiparallel beta-sheet, and of four turns. The three disulfides form the classical 'inhibitory cysteine knot' motif. So far, only one tertiary structure of a delta-conotoxin has been reported; thus, the tertiary structure of delta-Am2766 is the second such example. Another Conus peptide, Am2735 from C. amadis, has also been purified and sequenced. Am2735 shares 96% sequence identity with delta-Am2766. Unlike delta-Am2766, Am2735 does not inhibit the fast inactivation of Na+ currents in rat brain Na(v)1.2 Na+ channels at concentrations up to 200 nM.