Theoretical reconstruction of myotonia and paralysis caused by incomplete inactivation of sodium channels.

Muscle fibers from individuals with hyperkalemic periodic paralysis generate repetitive trains of action potentials (myotonia) or large depolarizations and block of spike production (paralysis) when the extracellular K+ is elevated. These pathologic features are thought to arise from mutations of the sodium channel alpha subunit which cause a partial loss of inactivation (steady-state Popen approximately 0.02, compared to < 0.001 in normal channels). We present a model that provides a possible mechanism for how this small persistent sodium current leads to repetitive firing, why the integrity of the T-tubule system is required to produce myotonia, and why paralysis will occur when a slightly larger proportion of channels fails to inactivate. The model consists of a two-compartment system to simulate the surface and T-tubule membranes. When the steady-state sodium channel open probability exceeds 0.0075, trains of repetitive discharges occur in response to constant current injection. At the end of the current injection, the membrane potential may either return to the normal resting value, continue to discharge repetitive spikes, or settle to a new depolarized equilibrium potential. This after-response depends on both the proportion of noninactivating sodium channels and the magnitude of the activity-driven K+ accumulation in the T-tubular space. A reduced form of model is presented in which a two-dimensional phase-plane analysis shows graphically how this diversity of after-responses arises as extracellular [K+] and the proportion of noninactivating sodium channels are varied.     

Slow inactivation in human cardiac sodium channels.

The available pool of sodium channels, and thus cell excitability, is regulated by both fast and slow inactivation. In cardiac tissue, the requirement for sustained firing of long-duration action potentials suggests that slow inactivation in cardiac sodium channels may differ from slow inactivation in skeletal muscle sodium channels. To test this hypothesis, we used the macropatch technique to characterize slow inactivation in human cardiac sodium channels heterologously expressed in Xenopus oocytes. Slow inactivation was isolated from fast inactivation kinetically (by selectively recovering channels from fast inactivation before measurement of slow inactivation) and structurally (by modification of fast inactivation by mutation of IFM1488QQQ). Time constants of slow inactivation in cardiac sodium channels were larger than previously reported for skeletal muscle sodium channels. In addition, steady-state slow inactivation was only 40% complete in cardiac sodium channels, compared to 80% in skeletal muscle channels. These results suggest that cardiac sodium channel slow inactivation is adapted for the sustained depolarizations found in normally functioning cardiac tissue. Complete slow inactivation in the fast inactivation modified IFM1488QQQ cardiac channel mutant suggests that this impairment of slow inactivation may result from an interaction between fast and slow inactivation.     

Gating defects of a novel Na+ channel mutant causing hypokalemic periodic paralysis

Hypokalemic periodic paralysis type 2 (hypoPP2) is an inherited skeletal muscle disorder caused by missense mutations in the SCN4A gene encoding the alpha subunit of the skeletal muscle Na+ channel (Nav1.4). All hypoPP2 mutations reported so far target an arginine residue of the voltage sensor S4 of domain II (R672/G/H/S). We identified a novel hypoPP2 mutation that neutralizes an arginine residue in DIII-S4 (R1132Q), and studied its functional consequences in HEK cells transfected with the human SCN4A cDNA. Whole-cell current recordings revealed an enhancement of both fast and slow inactivation, as well as a depolarizing shift of the activation curve. The unitary Na+ conductance remained normal in R1132Q and in R672S mutants, and cannot therefore account for the reduction of Na+ current presumed in hypoPP2. Altogether, our results provide a clear evidence for the role of R1132 in channel activation and inactivation, and confirm loss of function effects of hypoPP2 mutations leading to muscle hypoexcitability.     

Slow inactivation of tetrodotoxin-insensitive Na+ channels in neurons of rat dorsal root ganglia

Summary Whole-cell patch-clamp experiments were performed with neurons cultured from rat dorsal root ganglia (DRG). Two types of Na⁺ currents were identified on the basis of sensitivity to tetrodotoxin. One type was blocked by 0.1 nm tetrodotoxin, while the other type was insensitive to 10 m tetrodotoxin. The peak amplitude of the tetrodotoxin-insensitive Na⁺ current gradually decreased after depolarization of the membrane. The steady-state value of the peak amplitude was attained several minutes after the change of holding potential. Such a slow inactivation was not observed in tetrodotoxin-sensitive Na⁺ current. The slow inactivation of the tetrodotoxin-insensitive Na⁺ current was kinetically distinct from the ordinary short-time steady-state inactivation. The voltage dependence of the slow inactivation could be described by a sigmoidal function, and its time course had a double-exponential process. A decrease of external pH partially antagonized the slow inactivation, probably through an increased diffusion potential across the membrane. However, the slow inactivation was not due to change in surface negative charges, since a shift of the kinetic parameters along the voltage axis was not observed during the slow inactivation. Due to the slow inactivation, the inactivation curves for the tetrodotoxininsensitive Na⁺ current were shifted in the negative direction as the prepulse duration was increased. Consequently, the window current activated at potentials close to the resting membrane potential was markedly reduced. Thus, the slow inactivation may be involved in the long-term regulation of the excitability of sensory neurons.We thank Prof. Hirosi Kuriyama for his support and advice and Dr. M. Yoshii for helpful discussions. This study was supported by the Japanese Ministry of Education (Scientific Research 02670090).     

Accelerated inactivation in a mutant Na(+) channel associated with idiopathic ventricular fibrillation

Idiopathic ventricular fibrillation (IVF) can cause sudden death in both adults and children. One form of IVF (Brugada syndrome), characterized by S-T segment elevation (STE) in the electrocardiogram, has been linked to mutations of SCN5A, the gene encoding the voltage-gated cardiac Na(+) channel. A missense mutation of SCN5A that substitutes glutamine for leucine at codon 567 (L567Q, in the cytoplasmic linker between domains I and II) is identified with sudden infant death and Brugada syndrome in one family. However, neither the functional effect of the L567Q mutation nor the molecular mechanism underlying the pathogenicity of the mutation is known. Patch-clamp analysis of L567Q channels expressed in human embryonic kidney cells revealed a marked acceleration and a negative shift in the voltage dependence of inactivation. Unlike other Brugada mutations, this phenotype was expressed independently of temperature or auxiliary beta(1)-subunits. These results support a proposed linkage between Brugada syndrome and some instances of sudden infant death and the hypothesis that reduced Na(+) conductance is the primary cause of IVF with STE.     

Impaired slow inactivation in mutant sodium channels.

Hyperkalemic periodic paralysis (HyperPP) is a disorder in which current through Na+ channels causes a prolonged depolarization of skeletal muscle fibers, resulting in membrane inexcitability and muscle paralysis. Although HyperPP mutations can enhance persistent sodium currents, unaltered slow inactivation would effectively eliminate any sustained currents through the mutant channels. We now report that rat skeletal muscle channels containing the mutation T698M, which corresponds to the human T704M HyperPP mutation, recover very quickly from prolonged depolarizations. Even after holding at -20 mV for 20 min, approximately 25% of the maximal sodium current is available subsequent to a 10-ms hyperpolarization (-100 mV). Under the same conditions, recovery is less than 3% in wild-type channels and in the F1304Q mutant, which has impaired fast inactivation. This effect of the T698M mutation on slow inactivation, in combination with its effects on activation, is expected to result in persistent currents such as that seen in HyperPP muscle.     

MiRP2 forms potassium channels in skeletal muscle with Kv3.4 and is associated with periodic paralysis

The subthreshold, voltage-gated potassium channel of skeletal muscle is shown to contain MinK-related peptide 2 (MiRP2) and the pore-forming subunit Kv3.4. MiRP2-Kv3.4 channels differ from Kv3.4 channels in unitary conductance, voltage-dependent activation, recovery from inactivation, steady-state open probability, and block by a peptide toxin. Thus, MiRP2-Kv3.4 channels set resting membrane potential (RMP) and do not produce afterhyperpolarization or cumulative inactivation to limit action potential frequency. A missense mutation is identified in the gene for MiRP2 (KCNE3) in two families with periodic paralysis and found to segregate with the disease. Mutant MiRP2-Kv3.4 complexes exhibit reduced current density and diminished capacity to set RMP. Thus, MiRP2 operates with a classical potassium channel subunit to govern skeletal muscle function and pathophysiology.     

Effects of local anesthetics on Na+ channels containing the equine hyperkalemic periodic paralysis mutation

We examined theability of local anesthetics to correct altered inactivation propertiesof rat skeletal muscle Na⁺channels containing the equine hyperkalemic periodic paralysis (eqHPP)mutation when expressed in Xenopusoocytes. Increased time constants of current decay in eqHPP channelscompared with wild-type channels were restored by 1 mM benzocaine butwere not altered by lidocaine or mexiletine. Inactivation curves, which were determined by measuring the dependence of the relative peak current amplitude after depolarization to 10 mV on conditioning prepulse voltages, could be shifted in eqHPP channels back toward thatobserved for wild-type (WT) channels using selected concentrations ofbenzocaine, lidocaine, and mexiletine. Recovery from inactivation at80 mV (50-ms conditioning pulse) in eqHPP channels followed amonoexponential time course and was markedly accelerated compared withwild-type channels (_WT = 10.8 ± 0.9 ms; _eqHPP = 2.9 ± 0.4 ms). Benzocaine slowed the time course of recovery(_eqHPP,ben = 9.6 ± 0.4 msat 1 mM) in a concentration-dependent manner. In contrast, the recoveryfrom inactivation with lidocaine and mexiletine had a fast component(_fast,lid = 3.2 ± 0.2 ms;_fast,mex = 3.1 ± 0.2 ms),which was identical to the recovery in eqHPP channels without drug, anda slow component (_slow,lid = 1,688 ± 180 ms; _slow,mex = 2,323 ± 328 ms). The time constant of the slow component of therecovery from inactivation was independent of the drug concentration,whereas the fraction of current recovering slowly depended on drugconcentrations and conditioning pulse durations. Our results show thatlocal anesthetics are generally incapable of fully restoring normal WTbehavior in inactivation-deficient eqHPP channels.

     

Slow inactivation does not block the aqueous accessibility to the outer pore of voltage-gated Na channels

Slow inactivation of voltage-gated Na channels is kinetically and structurally distinct from fast inactivation. Whereas structures that participate in fast inactivation are well described and include the cytoplasmic III-IV linker, the nature and location of the slow inactivation gating mechanism remains poorly understood. Several lines of evidence suggest that the pore regions (P-regions) are important contributors to slow inactivation gating. This has led to the proposal that a collapse of the pore impedes Na current during slow inactivation. We sought to determine whether such a slow inactivation-coupled conformational change could be detected in the outer pore. To accomplish this, we used a rapid perfusion technique to measure reaction rates between cysteine-substituted side chains lining the aqueous pore and the charged sulfhydryl-modifying reagent MTS-ET. A pattern of incrementally slower reaction rates was observed at substituted sites at increasing depth in the pore. We found no state-dependent change in modification rates of P-region residues located in all four domains, and thus no change in aqueous accessibility, between slow- and nonslow-inactivated states. In domains I and IV, it was possible to measure modification rates at residues adjacent to the narrow DEKA selectivity filter (Y401C and G1530C), and yet no change was observed in accessibility in either slow- or nonslow-inactivated states. We interpret these results as evidence that the outer mouth of the Na pore remains open while the channel is slow inactivated.     

Enhanced slow inactivation by V445M: a sodium channel mutation associated with myotonia.

Over 20 different missense mutations in the alpha subunit of the adult skeletal muscle Na channel have been identified in families with either myotonia (muscle stiffness) or periodic paralysis, or both. The V445M mutation was recently found in a family with myotonia but no weakness. This mutation in transmembrane segment IS6 is novel because no other disease-associated mutations are in domain I. Na currents were recorded from V445M and wild-type channels transiently expressed in human embryonic kidney cells. In common with other myotonic mutants studied to date, fast gating behavior was altered by V445M in a manner predicted to increase excitability: an impairment of fast inactivation increased the persistent Na current at 10 ms and activation had a hyperpolarized shift (4 mV). In contrast, slow inactivation was enhanced by V445M due to both a slower recovery (10 mV left shift in beta(V)) and an accelerated entry rate (1.6-fold). Our results provide additional evidence that IS6 is crucial for slow inactivation and show that enhanced slow inactivation cannot prevent myotonia, whereas previous studies have shown that disrupted slow inactivation predisposes to episodic paralysis.     

Defective fast inactivation recovery and deactivation account for sodium channel myotonia in the I1160V mutant.

The skeletal muscle sodium channel mutant I1160V cosegregates with a disease phenotype producing myotonic discharges (observed as muscle stiffness) that are worsened by elevated K+ levels but unaffected by cooling. The I1160V alpha-subunit was co-expressed with the beta1-subunit in Xenopus oocytes. An electrophysiological characterization was undertaken to examine the underlying biophysical characteristics imposed by this mutation. Two abnormalities were found. 1) The voltage dependence of steady-state fast inactivation was reduced in I1160V, which resulted in faster rates of closed-state fast inactivation onset and recovery in I1160V compared with wild-type channels. 2) The rates of deactivation were slower in I1160V than in wild-type channels. Using a computer-simulated model, the combination of both defects elicited myotonic runs under conditions of elevated K+, consistent with the observed phenotype of the mutant.     

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.     

Charged tetracaine as an inactivation enhancer in batrachotoxin-modified Na+ channels.

Two distinct types of local anesthetics (LAs) have previously been found to block batrachotoxin (BTX)-modified Na+ channels: type 1 LAs such as cocaine and bupivacaine interact preferentially with open channels, whereas type 2 LAs, such as benzocaine and tricaine, with inactivated channels. Herein, we describe our studies of a third type of LA, represented by tetracaine as a dual blocker that binds strongly with closed channels but also binds to a lesser extent with open channels when the membrane is depolarized. Enhanced inactivation of BTX-modified Na+ channels by tetracaine was determined by steady-state inactivation measurement and by the dose-response curve. The 50% inhibitory concentration (IC50) was estimated to be 5.2 microM at -70 mV, where steady-state inactivation was maximal, with a Hill coefficient of 0.98 suggesting that one tetracaine molecule binds with one inactivated channel. Tetracaine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 microM at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was found to be the primary active form in the blocking of inactivated channels. In addition, external Na+ ions appeared to antagonize the tetracaine block of inactivated channels. Consistent with these results, N-butyl tetracaine quaternary ammonium, a permanently charged tetracaine derivative, remained a strong inactivation enhancer. Another derivative of tetracaine, 2-(di-methylamino) ethyl benzoate, which lacked a 4-butylamino functional group on the phenyl ring, elicited block that was approximately 100-fold weaker than that of tetracaine. We surmise that 1) the binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation enhancers by electrostatic repulsion, 3) the 4-butylamino functional group on the phenyl ring is critical for block and for the enhancement of inactivation, and 4) there are probably overlapping binding sites for both inactivation enhancers and open-channel blockers within the Na+ pore.     

Parametric resonance and amplification of periodic disturbances in membranes containing ion channels with inactivation

Parametric resonance and amplification of periodic perturbations in the membrane transport of ions through channels with inactivation was studied in computational experiments. It has been shown that a periodic change in the membrane capacitance or in the applied electric current with a frequency approximately 2 omega 0 (omega 0--the own angular frequency of the membrane) may excite stable self-oscillations in the membrane with a frequency of approximately omega 0. For this to occur, the degree of the capacitance modulation m or the amplitude of the applied current i0 must exceed some critical values mcr and i0cr. Excitation of self-oscillations by alternating electric current of the frequency approximately 2 omega 0 has the characteristics of parametric resonance. This can be explained by the fact that the equivalent membrane inductance depends on ionic current and displays periodic changes with a frequency approximately 2 omega 0, as also does the current. Small-amplitude periodic changes in the capacitance (m less than mcr) with frequencies approximately 2 omega 0 may result in significant amplification of periodic perturbations with frequencies approximately omega 0.     

Gating of Na channels. Inactivation modifiers discriminate among models

Macroscopic Na currents were recorded from N18 neuroblastoma cells by the whole-cell voltage-clamp technique. Inactivation of the Na currents was removed by intracellular application of proteolytic enzymes, trypsin, alpha-chymotrypsin, papain, or ficin, or bath application of N-bromoacetamide. Unlike what has been reported in squid giant axons and frog skeletal muscle fibers, these treatments often increased Na currents at all test pulse potentials. In addition, removal of inactivation gating shifted the midpoint of the peak Na conductance-voltage curve in the negative direction by 26 mV on average and greatly prolonged the rising phase of Na currents for small depolarizations. Polypeptide toxins from Leiurus quinquestriatus scorpion and Goniopora coral, which slow inactivation in adult nerve and muscle cells, also increase the peak Na conductance and shift the peak conductance curve in the negative direction by 7-10 mV in neuroblastoma cells. Control experiments argue against ascribing the shifts to series resistance artifacts or to spontaneous changes of the voltage dependence of Na channel kinetics. The negative shift of the peak conductance curve, the increase of peak Na currents, and the prolongation of the rise at small depolarization after removal of inactivation are consistent with gating kinetic models for neuroblastoma cell Na channels, where inactivation follows nearly irreversible activation with a relatively high, voltage-independent rate constant and Na channels open only once in a depolarization. As the same kind of experiment does not give apparent shifting of activation and prolongation of the rising phase of Na currents in adult axon and muscle membranes, the Na channels of these other membranes probably open more than once in a depolarization.     

Identification and functional characterization of Kir2.6 mutations associated with non-familial hypokalemic periodic paralysis

Hypokalemic periodic paralysis (hypoKPP) is characterized by episodic flaccid paralysis of muscle and acute hypokalemia during attacks. Familial forms of hypoKPP are predominantly caused by mutations of either voltage-gated Ca(2+) or Na(+) channels. The pathogenic gene mutation in non-familial hypoKPP, consisting mainly of thyrotoxic periodic paralysis (TPP) and sporadic periodic paralysis (SPP), is largely unknown. Recently, mutations in KCNJ18, which encodes a skeletal muscle-specific inwardly rectifying K(+) channel Kir2.6, were reported in some TPP patients. Whether mutations of Kir2.6 occur in other patients with non-familial hypoKPP and how mutations of the channel predispose patients to paralysis are unknown. Here, we report one conserved heterozygous mutation in KCNJ18 in two TPP patients and two separate heterozygous mutations in two SPP patients. These mutations result in V168M, R43C, and A200P amino acid substitution of Kir2.6, respectively. Compared with the wild type channel, whole-cell currents of R43C and V168M mutants were reduced by ～78 and 43%, respectively. No current was detected for the A200P mutant. Single channel conductance and open probability were reduced for R43C and V168M, respectively. Biotinylation assays showed reduced cell surface abundance for R43C and A200P. All three mutants exerted dominant negative inhibition on wild type Kir2.6 as well as wild type Kir2.1, another Kir channel expressed in the skeletal muscle. Thus, mutations of Kir2.6 are associated with SPP as well as TPP. We suggest that decreased outward K(+) current from hypofunction of Kir2.6 predisposes the sarcolemma to hypokalemia-induced paradoxical depolarization during attacks, which in turn leads to Na(+) channel inactivation and inexcitability of muscles.     

Voltage-sensor movements describe slow inactivation of voltage-gated sodium channels II: A periodic paralysis mutation in NaV1.4 (L689I)

In skeletal muscle, slow inactivation (SI) of Na_V1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in Na_V1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in Na_V1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1–160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of Na_V1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (∼10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX.