Human Skeletal Muscle Sodium Channel Research Articles

Block of voltage-operated sodium channels by 2,6-dimethylphenol, a structural analogue of lidocaine's aromatic tail.

1. The structural features that determine the state-dependent interaction of local anaesthetics with voltage-operated sodium channels are still a matter of debate. We have studied the blockade of sodium channels by 2,6-dimethylphenol, a phenol derivative which resembles the aromatic tail of lidocaine, etidocaine, and bupivacaine. 2. The effects of 2,6-dimethylphenol were studied on heterologously (HEK 293) expressed rat neuronal (rat brain IIA) and human skeletal muscle (hSkM1) sodium channels using whole-cell voltage-clamp experiments. 3. 2,6-Dimethylphenol was effective in blocking whole-cell sodium inward currents. Its potency was comparable to the potency of lidocaine previously obtained with similar protocols by others. The IC(50) at -70 mV holding potential was 150 and 187 microM for the skeletal muscle and the neuronal isoform, respectively. In both isoforms, the blocking potency increased with the fraction of inactivated channels at depolarized holding potentials. However, the block achieved at -70 mV with respect to -150 mV holding potential was significantly higher only in the skeletal muscle isoform. The estimated dissociation constant K(d) from the inactivated state was 25 microM and 28 microM in the skeletal muscle and the neuronal isoform, respectively. The kinetics of drug equilibration between resting and inactivated channel states were about 10 fold faster compared with lidocaine. 4. Our results show that the blockade induced by 2,6-dimethylphenol retains voltage-dependency, a typical feature of lidocaine-like local anaesthetics. This is consistent with the hypothesis that the 'aromatic tail' determines the state-dependent interaction of local anaesthetics with the sodium channel.

Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol

Background and objective: Thymol is a naturally occurring phenol derivative used in anaesthetic practice as a stabilizer and preservative of halothane, usually at a concentration of 0.01%. Although analgesic effects have long been described for thymol and its structural homologue menthol, a molecular basis for these effects is still lacking. We studied the blocking effects of thymol and menthol on voltage-activated sodium currents in vitro as possible molecular target sites. Methods: Whole cell sodium inward currents via heterologously (HEK293 cells) expressed rat neuronal (rat type IIA) and human skeletal muscle (hSkM1) sodium channels were recorded in the absence and presence of definite concentrations of either thymol or menthol. Results: When depolarizing pulses to 0 mV were started from a holding potential of −70 mV, half-maximum blocking concentrations (IC50) for the skeletal muscle and the neuronal sodium channel were 104 and 149 μmol for thymol and 376 and 571 μmol for menthol. The blocking potency of both compounds increased at depolarized holding potentials with the fraction of inactivated channels. The estimated dissociation constant Kd for thymol and menthol from the inactivated state was 22 and 106 μmol for the neuronal and 23 and 97 μmol for the skeletal muscle sodium channel, respectively. Conclusions: The results suggest that antinociceptive and local anaesthetic effects of thymol and menthol might be mediated via blockade of voltage-operated sodium channels with the phenol derivative thymol being as potent as the local anaesthetic lidocaine.

Voltage-dependent block of neuronal and skeletal muscle sodium channels by thymol and menthol.

Thymol is a naturally occurring phenol derivative used in anaesthetic practice as a stabilizer and preservative of halothane, usually at a concentration of 0.01%. Although analgesic effects have long been described for thymol and its structural homologue menthol, a molecular basis for these effects is still lacking. We studied the blocking effects of thymol and menthol on voltage-activated sodium currents in vitro as possible molecular target sites. Whole cell sodium inward currents via heterologously (HEK293 cells) expressed rat neuronal (rat type IIA) and human skeletal muscle (hSkM1) sodium channels were recorded in the absence and presence of definite concentrations of either thymol or menthol. When depolarizing pulses to 0 mV were started from a holding potential of -70 mV, half-maximum blocking concentrations (IC50) for the skeletal muscle and the neuronal sodium channel were 104 and 149 mumol for thymol and 376 and 571 mumol for menthol. The blocking potency of both compounds increased at depolarized holding potentials with the fraction of inactivated channels. The estimated dissociation constant Kd for thymol and menthol from the inactivated state was 22 and 106 mumol for the neuronal and 23 and 97 mumol for the skeletal muscle sodium channel, respectively. The results suggest that antinociceptive and local anaesthetic effects of thymol and menthol might be mediated via blockade of voltage-operated sodium channels with the phenol derivative thymol being as potent as the local anaesthetic lidocaine.

Impairment of slow inactivation as a common mechanism for periodic paralysis in DIIS4-S5.

Mutations in the human skeletal muscle sodium channels are associated with hyperKPP, hypoKPP, paramyotonia congenita, and potassium-aggravated myotonia. This article describes the clinical manifestations of a patient with hyperKPP carrying a mutation (L689I) occurring in the linker DIIS4-S5 and its functional expression in a mammalian system. To correlate the clinical manifestations of hyperkalemic periodic paralysis (hyperKPP) with the functional expression of a sodium channel mutation. The mutation was introduced into a mammalian expression vector and expressed in the human embryonic kidney 293 cells. The functional expression of the L689I and that of the wild-type channels was monitored using the whole cell voltage-clamp technique. There was no change in the kinetics of fast inactivation, and inactivation curves were indistinguishable from that of wild-type channels. However, the L689I mutation caused a hyperpolarizing shift in the voltage dependence of activation and the mutant channels showed an impaired slow inactivation process. In addition, the mutant channels have a larger persistent current at -40 mV where window current may occur. The L689I mutation has similar effects to the T704M mutation and causes hyperKPP in this family. Because both of these hyperKPP mutations cause episodic muscle weakness, and because patients harboring another mutation (I693T) also can have episodic weakness, it is hypothesized that mutations occurring in this region of the sodium channel may cause episodic weakness through an impaired slow inactivation process coupled with enhanced activation.

Gating of myotonic Na channel mutants defines the response to mexiletine and a potent derivative.

Myotonia and periodic paralysis caused by sodium channel mutations show variable responses to the anti-myotonic drug mexiletine. To investigate whether variability among sodium channel mutants results from differences in drug binding affinity or in channel gating. Whole-cell sodium currents (I(Na)) were recorded in tsA201 cells expressing human wild-type (WT) and mutant skeletal muscle sodium channels (A1156T, hyperkalemic periodic paralysis; R1448C, paramyotonia congenita; G1306E, potassium-aggravated myotonia). At a holding potential (hp) of -120 mV, mexiletine produced a tonic (TB, 0.33 Hz) and a use-dependent (UDB, 10 Hz) block of peak I(Na) with a potency following the order rank R1448C > WT approximately equal A1156T > G1306E. Yet, when assayed from an hp of -180 mV, TB and UDB by mexiletine were similar for the four channels. The different midpoints of channel availability curves found for the four channels track the half-maximum inhibitory value (IC50) measured at -120 mV. Thus differences in the partitioning of channels between the closed and fast-inactivated states underlie the different IC50 measured at a given potential. The mexiletine-derivative, Me7 (alpha-[(2-methylphenoxy)methyl]-benzenemethanamine), behaved similarly but was approximately 5 times more potent than mexiletine. Interestingly, the higher drug concentrations ameliorated the abnormally slower decay rate of myotonic I(Na). These results explain the basis of the apparent difference in block of mutant sodium channels by mexiletine and Me7, opening the way to a more rationale drug use and to design more potent drugs able to correct specifically the biophysical defect of the mutation in individual myotonic patients.

Structural requirements for voltage-dependent block of muscle sodium channels by phenol derivatives.

We have studied the effects of four different phenol derivatives, with methyl and halogen substituents, on heterologously expressed human skeletal muscle sodium channels, in order to find structural determinants of blocking potency. All compounds blocked skeletal muscle sodium channels in a concentration-dependent manner. The methylated phenol 3-methylphenol and the halogenated phenol 4-chlorophenol blocked sodium currents on depolarization from -100 mV to 0 mV with IC(50) values of 2161 and 666 microM respectively. Methylation of the halogenated compound further increased potency, reducing the IC(50) to 268 microM in 2-methyl-4-chlorophenol and to 150 microM in 3,5-dimethyl-4-chlorophenol. Membrane depolarization before the test depolarization increased sodium channel blockade. When depolarizations were started from -70 mV or when a 2.5 s prepulse was introduced before the test pulse inducing slow inactivation, the IC(50) was reduced more than 3 fold in all compounds. The values of K(D) for the fast-inactivated state derived from drug-induced shifts in steady-state availability curves were 14 microM for 3,5-dimethyl-4-chlorophenol, 19 microM for 2-methyl-4-chlorophenol, 26 microM for 4-chlorophenol and 115 microM for 3-methylphenol. All compounds accelerated the current decay during depolarization and slowed recovery from fast inactivation. No relevant frequency-dependent block after depolarizing pulses applied at 10, 50 and 100 Hz was detected for any of the compounds. All the phenol derivatives that we examined are effective blockers of skeletal muscle sodium channels, especially in conditions that are associated with membrane depolarization. Blocking potency is increased by halogenation and by methylation with increasing numbers of methyl groups.

A sodium channel mutation causing epilepsy in man exhibits subtle defects in fast inactivation and activation in vitro.

Generalized epilepsy with febrile seizures plus (GEFS+) is a benign epileptic syndrome of humans. It is characterized by febrile and afebrile generalized seizures that occur predominantly in childhood and respond well to standard antiepileptic therapy. A mutation in the b1-subunit of the voltage-gated sodium channel, linked to chromosome 19q13 (GEFS+ type 1) has been found in one family. For four other families, linkage was found to chromosome 2q21-33 (GEFS+ type 2) where three genes encoding neuronal sodium channel a-subunits are located (SCN1-3A). Recently, the first two mutations were identified in SCN1A. We introduced one of these mutations, which is highly conserved to SCN1A, into the cDNA of the gene SCN4A encoding the a-subunit of the human skeletal muscle sodium channel (hSkm1). The mutation is located in the S4 voltage sensor of domain IV, predicting substitution of histidine for the fifth of eight arginines (R1460H in hSkm1). Functional studies were performed by expressing the a-subunit alone in the mammalian tsA201 cell line using the whole-cell patch clamp technique. Compared to wild-type (WT), mutant R1460H channels showed small defects in fast inactivation. The time course of inactivation was slightly (1.5-fold) slowed and its voltage dependence reduced, and recovery from inactivation was accelerated 3-fold. However, there was no increase in persistent sodium current as observed for SCN4A mutations causing myotonia or periodic paralysis. The activation time course of R1460H channels was slightly accelerated. Slow inactivation was slightly but significantly stabilized, confirming the importance of this region for slow inactivation. The combination of activation and fast inactivation defects can explain the occurrence of epileptic seizures, but the effects were much more subtle than the inactivation defects described previously for mutations in SCN4A causing disease in skeletal muscle. Hence, with regard to pathological excitability, our results suggest a greater vulnerability of the central nervous system compared to muscle tissue.

Voltage-dependent blockade of normal and mutant muscle sodium channels by benzylalcohol.

1. We studied the effects of benzylalcohol on heterologously expressed wild type (WT), paramyotonia congenita (R1448H) and hyperkalaemic periodic paralysis (M1360V) mutant alpha-subunits of human skeletal muscle sodium channels. 2. Benzylalcohol blocked rested channels at -150 mV membrane potential, with an ECR(50) of 5.3 mM in wild type, 5.1 mM in R1448H, and 6.2 mM in M1360V. When blockade was assessed at -100 mV, the ECR(50) was reduced in R1448H (2 mM) compared with both wild type (4.3 mM; P<0.01) and M1360V (4.3 mM). 3. Membrane depolarization before the test depolarization significantly promoted benzylalcohol-induced sodium channel blockade. The values of K(D) for the fast-inactivated state derived from benzylalcohol-induced shifts in steady-state availability curves were 0.66 mM in wild type and 0.58 mM in R1448H. In the presence of slow inactivation induced by 2.5 s depolarizing prepulses, the ECI(50) for benzylalcohol-induced current inhibition was 0.59 mM in wild type and 0.53 mM in R1448H. 4. Recovery from fast inactivation was prolonged in the presence of drug in all clones. 5. Benzylalcohol induced significant frequency-dependent block at stimulating frequencies of 10, 50, and 100 Hz in all clones. 6. Our results clearly show that benzylalcohol is an effective blocker of muscle sodium channels in conditions that are associated with membrane depolarization. Mutants that enter voltage-dependent inactivation at more hyperpolarized membrane potentials compared with wild type are more sensitive to inhibitory effects at the normal resting potential.

Lack of sodium channel mutation in an Italian family with paramyotonia congenita.

To conduct the genotype-phenotype correlation in a family in which several individuals share clinical and electrophysiologic features of paramyotonia congenita (PC). PC, hyperkalemic periodic paralysis (HyperPP), and potassium-aggravated myotonias form the group of hereditary sodium channelopathies. Each of these disorders is associated with different point mutations in SCN4A, the gene encoding the alpha-subunit of the adult human skeletal muscle sodium channel. However, in HyperPP families, evidence of a causative gene different from SCN4A has been found. We conducted direct clinical examination, electrophysiologic (EMG/electroneurographic) and cardiologic studies, as well as laboratory screening in several affected and nonaffected members of the family. We performed the genotype-phenotype correlation by microsatellite linkage and cDNA-mutation analyses of the SCN4A gene. Affected members in this family showed clinical and electrophysiologic features typical of PC. The disease phenotype segregated with the chromosomal region that includes the SCN4A gene. Analysis of the entire cDNA sequence of the SCN4A gene in the index case disclosed a G3826A transition, which results in the Val1276Ile substitution. However, PCR-single-stranded confirmation polymorphism and direct sequencing analysis of the segment coding for Val-1276 on genomic DNA confirmed the G3826A transition in the index case but was negative in 11 affected members of the family; however, neither mutations nor aberrant splicings causative of the PC phenotype in this family were found on SCN4A. The existence of a second gene different from SCN4A that can give rise to a clinical PC phenotype can be speculated upon.

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 β 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 β 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 β 1-subunit coexpression. Our results indicate that the β 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 β 1-subunit modulates slow inactivation in hSkM1 but not hH1. Changes in slow inactivation time constants in sodium channels coexpressed with the β 1-subunit indicate possible interactions among the β 1-subunit, P-loops, and the slow inactivation gate in sodium channels.

Differential effects of homologous S4 mutations in human skeletal muscle sodium channels on deactivation gating from open and inactivated states.

1. The outermost charged amino acid of S4 segments in the alpha subunit of human skeletal muscle sodium channels was mutated to cysteine in domains I (R219C), II (R669C), III (K1126C), and IV (R1448C). Double mutations in DIS4 and DIVS4 (R219C/R1448C), DIIS4 and DIVS4 (R669C/R1448C), and DIIIS4 and DIVS4 (K1126C/R1448C) were introduced in other constructs. Macropatch recordings of mutant and wild-type (hSkM1-wt) skeletal muscle sodium channels expressed in Xenopus oocytes were used to measure deactivation kinetics from open or fast inactivated states. 2. Conductance (voltage) curves (G (V)) derived from current (voltage) (I (V)) relations indicated a right-shifted G (V) relationship for R669C and for R669C/R1448C, but not for other mutations. The apparent valency was decreased for all mutations. Time-to-peak activation at -20 mV was increased for R1448C and for double mutations. 3. Deactivation kinetics from the open state were determined from the monoexponential decay of tail currents. Outermost charge-to-cysteine mutations in the S4 segments of domains III and IV slowed deactivation, with the greatest effect produced by R1448C. The deactivation rate constant was slowed to a greater extent for the DIII/DIV double mutation than that calculated from additive effects of single mutations in each of these two domains. Mutation in DIIS4 accelerated deactivation from the open state, whereas mutation in DIS4 had little effect. 4. Delays in the onset to recovery from fast inactivation were determined to assess deactivation kinetics from the inactivated state. Delay times for R219C and R669C were not significantly different from those for hSkM1-wt. Recovery delay was increased for K1126C, and was accelerated for R1448C. 5. Homologous charge mutations of S4 segments produced domain-specific effects on deactivation gating from the open and from the fast inactivated state. These results are consistent with the hypothesis that translocations of S4 segments in each domain during deactivation are not identical and independent processes. Non-identical effects of these mutations raise several possibilities regarding deactivation gating; translocation of DIVS4 may constitute the rate-limiting step in deactivation from the open state, DIVS4 may be part of the immobilizable charge, and S4 translocations underlying deactivation in human skeletal muscle sodium channel may exhibit co-operativity.

Voltage Sensors in Domains III and IV, but Not I and II, Are Immobilized by Na + Channel Fast Inactivation

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.

State-Dependent Cocaine Block of Sodium Channel Isoforms, Chimeras, and Channels Coexpressed with the β1 Subunit

Cocaine block of human cardiac (hH1) and rat skeletal ( μ1) muscle sodium channels was examined under whole-cell voltage clamp in transiently transfected HEK293t cells. Low affinity block of resting μ1 and hH1 channels at −180 mV was the same, and high affinity block of inactivated channels at −70 mV was the same. Cocaine block of hH1 channels was greater than block of μ1 channels at voltages between −120 mV and −90 mV, suggesting that greater steady-state inactivation of hH1 channels in this voltage range makes them more susceptible to cocaine block. We induced shifts in the voltage dependence of steady-state inactivation at μ1 and hH1 channels by constructing μ1/hH1 channel chimeras or by coexpressing the wild-type channels with the rat brain β1 subunit. In contrast to several previous reports, coexpression of the rat brain β1 subunit with μ1 or hH1 produced large positive shifts in steady-state inactivation. Shifts in the voltage dependence of steady-state inactivation elicited linear shifts in steady-state cocaine block, yet these manipulations did not affect the cocaine affinity of resting or inactivated channels. These data, as well as simulations used to predict block, indicate that state-dependent cocaine block depends on both steady-state inactivation and channel activation, although inactivation appears to have the predominant role.

Voltage sensitivity and conformational change of isolated S4L45 fragments from sodium channels are tuned to proline.

Peptide fragments reproducing the sequences of S4 segments extended with L45 linkers from the four homologous domains of the electric eel sodium channel were chemically synthesized and purified to allow circular dichroism studies in various solvents and conductance assays in planar lipid bilayers. Repeats III (with proline) and IV (lacking proline) present the lowest and highest helicities, respectively. The conformational transition (from helix to beta-strand) shown to occur on an increase of solvent dielectric constant is broader with repeat III. Analytical ultracentrifugation (interference fringe pattern) is consistent with a monodispersion of the peptide. In macroscopic conductance experiments, the proline containing peptides (repeats I, II and especially III) display higher voltage-sensitivities than repeat IV. The apparent and averaged number of monomers per intramembrane conducting aggregate is 4-5. The influence of proline is confirmed in similar experiments carried out on homologous S4 segments of repeat IV of the human skeletal muscle sodium channel comparing the wild type and an analogue where the fourth arginine was substituted with a proline. Thus, both conformational switching and voltage-sensitivity appear correlated to the presence and position of a single proline residue. Since voltage sensors are likely to experience different polarity environments in the channel open and closed states, our results suggest an alternative gating mechanism, i.e. a voltage-driven conformational change of S4L45s. The data also implies a plausible functional asymmetry, namely a "three- or four-stroke" activation sequentially involving the four domains of the sodium channel.

Human sodium channel gating defects caused by missense mutations in S6 segments associated with myotonia: S804F and V1293I

1. Missense mutations in the alpha-subunit of the human skeletal muscle sodium channel (hSkM1) have been detected in some heritable forms of myotonia. By recording Na+ currents from cells transfected with cDNA encoding either wild-type or mutant hSkM1, we characterized the functional consequences of two myotonia-associated mutations that lie at the cytoplasmic end of the sixth transmembrane segment in domain II (S804F) or domain III (V1293I). 2. Both mutations caused modest, but unequivocal, alterations in the voltage-dependent gating behaviour of hSkM1. For S804F, the abnormalities were limited to fast inactivation: the persistent Na+ current at the end of a 50 ms depolarization was increased 3-fold, the rate of inactivation from the open state was slowed 2-fold, and the voltage dependence of fast inactivation (h) was shifted by +3 mV. V1293I also disrupted fast inactivation, as evidenced by a 3-fold faster rate of recovery at hyperpolarized potentials (-70 mV). Activation was altered as well for V1293I: the voltage dependence was shifted by -6 mV (hyperpolarized). 3. Slow inactivation was not altered by S804F or V1293I. 4. We conclude that S804F and V1293I are not benign polymorphisms. Either mutation causes detectable alterations in channel gating and, in model simulations, the magnitude of the defects is sufficient to produce runs of myotonic discharges.

Myotonic dystrophy kinase modulates skeletal muscle but not cardiac voltage-gated sodium channels

Altered modulation of skeletal muscle voltage-gated sodium channels by myotonic dystrophy kinase (DMPK) has been proposed as a possible mechanism underlying myotonia in this disease. We examined the effect of a recombinant mouse DMPK on the functional properties of human skeletal muscle (hSkM1) and cardiac (hH1) voltage-gated sodium channels in the Xenopus oocyte expression system. Co-expression of DMPK with hSkM1 in oocytes resulted in significantly lower peak sodium current amplitude as compared to cells expressing hSkM1 alone in agreement with a previous report. By contrast, DMPK had no effect on the level of expressed sodium current in cells expressing hH1. Similarly, there were no measurable effects of the kinase on the kinetics or steady-state properties of activation or inactivation. Our findings support the previous observations made with rat muscle sodium channels and demonstrate that the effect of DMPK on sodium channels is isoform specific despite conservation of a putative phosphorylation site between the two isoforms.

Human Na+ channel fast and slow inactivation in paramyotonia congenita mutants expressed in Xenopus laevis oocytes.

1. Paramyotonia congenita (PC) is a human hereditary disease caused by one or more amino acid substitutions in skeletal muscle sodium channels. Using macropatches, the effect of PC mutations R1448C and T1313M were compared with wild-type (WT) in Xenopus oocytes coinjected with both alpha- and beta-subunits of human skeletal muscle (SkM1) sodium channels. 2. Slow inactivation in either T1313M or R1448C was not different from WT. Fast inactivation in both PC mutants, however, was significantly altered. 3. Commonly used biophysical protocols (such as I-V curves, steady-state inactivation curves, and measurements of inactivation rates) did not uniformly indicate that hyperexcitability should result from T1313M or R1448C. In fact, the only alteration of fast inactivation common to T1313M and R1448C that predicted cellular hyperexcitability was slowed open-state inactivation, compared with WT. 4. To test whether this alteration was sufficient to cause the phenotypic hyperexcitability, we used a novel voltage command that simulated muscle membrane activity. With this protocol, we found that R1448C and T1313M were similar in that they maintained a significantly higher channel availability during high frequency activity, compared with WT.

Paramyotonia congenita and hyperkalemic periodic paralysis associated with a Met 1592 Val substitution in the skeletal muscle sodium channel α subunit — a large kindred with a novel phenotype

Paramyotonia congenita (PC) and Hyperkalemic periodic paralysis (HyperPP) are caused by amino acid substitutions in the α subunit of the human skeletal muscle sodium channel. One such substitution, methionine for valine at position 1592, has been associated with HyperPP with myotonia and cold sensitivity. We report clinical, electromyographic (EMG), genetic and pathological features of a large kindred with the Met1592Val substitution. Affected members were phenotypically heterogenous and had episodic potassium-sensitive paralysis, and stiffness and weakness induced by exercise and cold, which was confirmed by EMG studies. These features indicate a combined PC-HyperPP phenotype not previously described with this mutation.

Modulation of Human Muscle Sodium Channels by Intracellular Fatty Acids Is Dependent on the Channel Isoform

Free fatty acids (FFAs), including arachidonic acid (AA), are implicated in the direct and indirect modulation of a spectrum of voltage-gated ion channels. Skeletal muscle sodium channels can be either activated or inhibited by FFA exposure; the response is dependent on both FFA structure and site of exposure. Recombinant human skeletal muscle sodium channels (hSkM1) were transfected into heterologous human renal epithelium HEK293t cells. Cytoplasmic delivery of 5 microM AA augmented the voltage-activated sodium current of hSkM1 channels by 190% (+/-54 S.E., n = 7) over a 20-min period. Similar results were seen with 5 microM oleic acid. Sodium currents in HEK293t cells transfected with human cardiac muscle sodium channels (hH1) were insensitive to AA treatment, and exposure to oleic acid inhibited the hH1 currents over a 20-min period by 29% (+/-13 S.E., n = 5). The increase in hSkM1 current was not accompanied by shifts in voltage dependence of activation, steady-state inactivation, or markedly altered kinetics of inactivation of the macroscopic current. The FFA-induced increase in sodium currents was not dependent on protein kinase C activity. In contrast, both isoforms were reversibly inhibited by external application of unsaturated FFA. Thus, the differential effects of FFA on skeletal muscle sodium channels first noted in cultured muscle cells can be reproduced by expressing recombinant sodium channels in epithelial cells. Although the responses to applied FFAs could be direct or indirect, we suggest that: 1) SkM1 has two classes of response to FFA, one which produces augmentation of macroscopic currents with intracellular FFA, and a second which produces inhibition with extracellular FFA; 2) H1 has only one class of response, which produces inhibition with extracellular FFA. A testable hypothesis is that the presence or absence of each response is due to a specific structure in SkM1 or H1. These specific structures may directly interact with FFA or may interact with intermediate components.

Sea Anemone Toxin (ATX II) Modulation of Heart and Skeletal Muscle Sodium Channel α-Subunits Expressed in tsA201 Cells

We have expressed recombinant alpha-subunits of hH1 (human heart subtype 1), rSkM1 (rat skeletal muscle subtype 1) and hSkM1 (human skeletal muscle) sodium channels in human embryonic kidney cell line, namely the tsA201 cells and compared the effects of ATX II on these sodium channel subtypes. ATX II slows the inactivation phase of hH1 with little or no effect on activation. At intermediate concentrations of ATX II the time course of inactivation is biexponential due to the mixture of free (fast component, taufasth) and toxin-bound (slow component, tauslowh) channels. The relative amplitude of tauslowh allows an estimate of the IC50 values approximately 11 nM. The slowing of inactivation in the presence of ATX II is consistent with destabilization of the inactivated state by toxin binding. Further evidence for this conclusion is: (i) The voltage-dependence of the current decay time constants (tauh) is lost or possibly reversed (time constants plateau or increase at more positive voltages in contrast to these of untreated channels). (ii) The single channel mean open times are increased by a factor of two in the presence of ATX II. (iii) The recovery from inactivation is faster in the presence of ATX II. Similar effects of ATX II on rSkM1 channel behavior occur, but only at higher concentrations of toxin (IC50 = 51 nM). The slowing of inactivation on hSkM1 is comparable to the one seen with rSkM1. A residual or window current appears in the presence of ATX II that is similar to that observed in channels containing mutations associated with some of the familial periodic paralyses.