A high-conductance anion channel in adult amphibian skeletal muscle

Membrane patches were excised from enzymatically dissocfated frog toe muscle. High-conductance anion channels could be induced in previously quiet patches by 20–120 s depolarizations beyond +20 mV and then studied in the potential range from –80 to +60 mV for a long time. From reversal potentials the estimated permeability ratiosP _Cl/P _Na andP _Cl/P _glucuronate were near 3.5 and 4, respectively. There were probably 5 or more conductance levels (substates) for a single channel, the most common in symmetrical 110 mM NaCl being 260 and 70 pS at 10°C. Gating was complex, with rapid and slow events and several gating modes, including periods of rapid flickering. Channels closed reversibly at potentials more negative than –50 mV. The channel was blocked by application to the cytoplasmic face of tannic acid, gallic acid, and zinc but not of DIDS or 9-anthracene-carboxylic acid, and it was blocked by extracellular zinc.     

Single Ca channel currents in mammalian visceral smooth muscle cells

Recordings of single Ca channel currents in mammalian visceral smooth muscle cells were obtained using patch clamp techniques. Smooth muscle cells from guinea-pig taenia coli were prepared by enzymatic dispersion using 0.3% collagenase. The recordings were obtained from cell-attached membrane patches of isolated cells with a pipette filled with isotonic 50 mM Ba²⁺. When the membrane patch was depolarized, brief inward current pulses of unitary size and small amplitude were observed. The amplitude of these single channel currents decreased linearly with increasing depolarization in a voltage range from –20 mV to +50 mV about the resting potential. The slope conductance was estimated to be about 30 pS. The mean current reconstructed by averaging individual current responses showed kinetic behaviour with a rapid activation and a slower inactivation process similar to the macroscopic Ca²⁺ current observed in strips of guinea-pig taenia coli. The present study suggests that the inward current pulses of unitary size induced by voltage-clamp pulses were due to Ba ions passing through a single voltage dependent Ca channel.     

Ionic permeabilities ofl-glutamate activated,excitatory synaptic channel in crayfish muscle

Excitatory single channel currents triggered byl-glutamate were measured in outside-out excised patches of crayfish muscle membrane. If an intracellular solution was present in the pipette and normal extracellular solution with added glutamate (10^–3 M) passed the outside of the patch, the single channel currents,i ₁, had amplitudes of –8 pA at a patch potential of –70 mV. If in the extracellular solution Na⁺ was replaced by Li⁺ or Ca²⁺, the amplitudes of single channel currents were reduced by about 30%. Only about 20% of the channel current amplitude remained on replacement of Na⁺ by choline. Replacement of Na⁺ reduced the variance of channel amplitude distributions to the level of the baseline. Presence of Na⁺ thus induces an additional variance of open channel current. When the proportions of Na⁺/choline were varied, the resulting channel currents could be separated in Na⁺, Ca²⁺ and choline components. The amplitude of the Na⁺ component,i _1,Na, could be described by a constant channel permeability _Na = 110 10^–15 cm³ s^–1 according to the constant field equation. Ba²⁺ could replace Ca²⁺ without change in single channel current, while replacement of Ca²⁺ by Mg²⁺ reduced the channel currents by 20%. The following permeabilities of the single channel were estimated (in 10^–15 cm³ s^–1): _Na = 110, _K = 86, _Ca = 30, _Mg = 24, _Ba = 30, _Li = 84 and _choline = 11. These permeabilities were obtained inserting ionic concentrations. The respective permeabilities are listed also as calculated on the basis of ionic activities. In presence of high Na⁺ the single channel current amplitudes were not affected appreciably by a reduction of the extracellular Ca²⁺ from 13.5 to 1 mM, or by application of calcium channel blockers like La³⁺ or Cd²⁺.Supported by the Deutsche Forschungsgemeinschaft     

Sarcolemmal ion channels in dystrophin-deficient skeletal muscle fibres

Duchenne muscular dystrophy (DMD) is a genetic disease caused by mutations in the dystrophin gene and characterized by progressive skeletal muscle degeneration. A current hypothesis suggests that degeneration of dystrophin-deficient skeletal muscle results from a chronic intracellular Ca²⁺ overload. Ca²⁺ handling in skeletal muscle is tightly controlled by the membrane potential which is set by sarcolemmal ion channels activity. Also, with regard to the subsarcolemmal localization of dystrophin, it is reasonable to enquire if the distribution and function of ion channels might be affected by the absence of dystrophin. This paper briefly summarizes the current knowledge of the properties of sarcolemmal ion channels in fully differentiated dystrophin-deficient skeletal muscle fibres.     

Insulin modulation of ATP-sensitive K+ channel of rat skeletal muscle is impaired in the hypokalaemic state

 In the present work, we examined the effects of in vivo administration of insulin to rats made hypokalaemic by feeding a K⁺-free diet. The i.p. injection of insulin in the hypokalaemic rats provoked muscle paralysis within 3–5 h. Consistent with this observation, the skeletal muscle fibres of the paralysed rats were depolarized. In contrast, in the normokalaemic animals, insulin neither provoked paralysis nor produced significant fibre hyperpolarization. In the hypokalaemic rats, insulin almost completely abolished the sarcolemma adenosine triphosphate (ATP)-sensitive K⁺ currents without altering the sensitivity of the channels to ATP or glibenclamide. In contrast, in the normokalaemic rats, insulin enhanced ATP-sensitive K⁺ currents that became also resistant to ATP and glibenclamide. Our experiments indicate that the modulation of the sarcolemma ATP-sensitive K⁺ channels by insulin is impaired in the hypokalaemic state. This phenomenon appears to be related to the fibre depolarization and paralysis observed in the same animals. Received: 21 July 1998 / Received after revision: 17 September 1998 / Accepted: 25 September 1998     

The effect of the outermost basic residues in the S4 segments of the CaV3.1 T-type calcium channel on channel gating

The contribution of voltage-sensing S4 segments in domains I to IV of the T-type Ca_V3.1 calcium channel to channel gating was investigated by the replacement of the uppermost charged arginine residues by neutral cysteines. In each construct, either a single (R180C, R834C, R1379C or R1717C) or a double (two adjacent domains) mutation was introduced. We found that the neutralisation of the uppermost arginines in the IS4, IIS4 and IIIS4 segments shifted the voltage dependence of channel activation in a hyperpolarising direction, with the most prominent effect in the IS4 mutant. In contrast, the voltage dependence of channel inactivation was shifted towards more negative membrane potentials in all four single mutant channels, and these effects were more pronounced than the effects on channel activation. Recovery from inactivation was affected by the IS4 and IIIS4 mutations. In double mutants, the effects on channel inactivation and recovery from inactivation, but not on channel activation, were additive. Exposure of mutant channels to the reducing agent dithiothreitol did not alter channel properties. In summary, our data indicate that the S4 segments in all four domains of the Ca_V3.1 calcium channels contribute to voltage sensing during channel inactivation, while only the S4 segments in domains I, II and III play such role in channel activation. Furthermore, the removal of the outermost basic amino acids from the IVS4 and IIIS4 and, to a lesser extent, from IS4 segments stabilised the open state of the channel, whereas neutralization from that of IIS4 destabilised it. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Martina Kurejová and L’ubica Lacinová contributed equally to this work. An erratum to this article can be found at     

Alterations in calcium homeostasis reduce membrane excitability in amphibian skeletal muscle

The effects of alterations in intracellular calcium homeostasis on surface membrane excitability were investigated in resting Rana temporaria sartorius muscle. This was prompted by initial results from a fatiguing stimulation protocol study that demonstrated a fibre subpopulation in which action potential generation in response to a standard 1.5 V electrical stimulus failed despite mean membrane potentials [E _m, −69±2.3 mV (n=14)] compatible with spike firing in a control set of quiescent muscle fibres. Intracellular micro-electrode recordings showed a similar reversible loss of excitability, attributable to an increased threshold, despite only small (7.1±1.8 mV) positive changes in E _m after approximately 60-min exposures to nominally 0 Ca²⁺ Ringer solutions in which Ca²⁺ was replaced by Mg²⁺. This effect was not reproduced by addition of Mg²⁺ to the Ringer solution and persisted under conditions of Cl⁻ deprivation. The effects of three pharmacological agents, cyclopiazonic acid (CPA), caffeine and 4-chloro-m-cresol (4-CmC), each known to deplete store Ca²⁺ and increase cytosolic Ca²⁺ through contrasting mechanisms without influencing E _m, were then investigated. All three agents produced a more rapid, but nevertheless still reversible, loss of membrane excitability than in 0 Ca²⁺ Ringer solution alone. This reduction in membrane excitability persisted in fibres studied in solutions containing a normal [Ca²⁺] following prior depletion of store Ca²⁺ using CPA- and 4-CmC-containing solutions. These novel findings suggest that sarcoplasmic reticulum Ca²⁺ content profoundly influences surface membrane excitability, thereby providing a potential mechanism by which spike firing fails in well-polarised fibres during fatigue.The authors Usher-Smith and Xu were equal contributors to this paper.     

Polymyxin B as a highly effective gating modifier of high-conductance Ca2+-activated K+ channels in mouse skeletal muscle

Large-conductance Ca²⁺-activated K⁺ channels were studied in excised inside-out membrane patches from adult mouse skeletal muscle. The channels had a conductance of about 250 pS in symmetrical 155 mM KCl solutions. They showed gating characteristics similar to those described for this type of channel in rat and rabbit skeletal muscle. Polymyxin B, a cyclic polypeptide antibiotic, produced a voltage-dependent block, whereas polymyxin E was only slightly effective. The concentration at which half-maximal blockage occurred was very iow: 0.5 μg/ml at a voltage of + 30 mV. The blockage occurred with a Hill coefficient ofh=1.2. At negative membrane potentials, polymyxin B caused the appearance of a substate with a conductance of about 10% of the fully open state. The mode of blockage is discussed and compared to the effect of polymyxin B on glucose uptake into the muscle cell.     

Dependence of single channel properties of the N-methyl-d-aspartate ion channel on stereoisomer agonists

The cell-attached patch-clamp configuration has been used to determine the single channel properties of the N-methyl-D-aspartate (NMDA) ion channel with activation of the NMDA receptor by stereoisomer agonists. All of the agonists studied, including the L and d forms of N-methyl-aspartate and the L and d forms of homocysteate, activated a 42-pS conductance channel in cultured hippocampal neurons. For all agonists, the mean open times of the channel were diminished with increased patch hyperpolarization and exhibited an exponential dependence on potential over the range -40 mV to -120 mV. The mean open times, for patch potentials close to resting potential, and the mean frequencies of channel openings, at all patch potentials, were significantly different between each member of the stereoisomer pairs. For both L-homocysteate and NMLA, a fourfold increase in the patch pipette concentration caused an approximate quadrupling in the frequency of unitary events, with no significant change in mean open time. The open channel probability was used as a measure of agonist potency, and, at a concentration of 30 M, NMDA and L-homocysteate were significantly more potent (P open in excess of 1.5%) than the corresponding stereoisomer compounds NMLA and D-homocysteate (P open near 0.3%). The relative potencies of the stereoisomer pairs were in reasonable agreement with the potency ratios measured in binding studies.     

Changes of the biophysical properties of calcium-activated potassium channels of rat skeletal muscle fibres during aging

 In the present work, we have investigated the effects of the aging process on Ca²⁺-activated K⁺ channels (K_Ca2+) of rat skeletal muscle fibres. K_Ca2+ channels of adult (5–7 months old) and aged (24–26 months old) rats were surveyed by the patch-clamp technique. In aged rats, K_Ca2+ channels were routinely detected on the surface membrane of the fibres in both cell-attached and inside-out configurations. Conversely, in adult rat fibres, K_Ca2+ channels were rarely detected. In the cell-attached configuration, the open probability of the aged rat K_Ca2+ channel, measured in the range of potentials from –60 mV to +20 mV, was about 1.5–2 times higher than that of the adult one. The number of functional channels was abnormally increased by aging. An average of three channels per patch/area was counted in the inside-out patches of aged rat fibres, whereas no more than one open channel per patch/area was detected in the adult rat fibres. The frequency of finding channels in the patches also increased with aging, i.e. 11.5% and 30.1% in the adult and in the aged rat fibres, respectively. However, no significant change in the single-channel conductance has been observed with aging: it was 227 pS and 231 pS for adult and aged rat channels, respectively. In detached patches, both the adult and aged rat channels showed a similar voltage dependence of open probability and a similar sensitivity to Ca²⁺ ions. The aging process did not alter the response of the single channel to charybdotoxin, or its modulation by nucleotides, MgATP and adenosine 5’-O-(3-thiotriphosphate) (ATP[γ-S]). On the other hand, charybdotoxin reduced the abnormally high resting macroscopic K⁺ conductance of the aged rat fibres, recorded using the two-intracellular-microelectrode technique. These findings indicate that, in skeletal muscle, the activity of K_Ca2+ channels increases with advancing age. Received: 10 April 1997 / Received after revision and accepted: 4 June 1997     

Alterations in triad ultrastructure following repetitive stimulation and intracellular changes associated with exercise in amphibian skeletal muscle

This study used Rana temporaria sartorius muscles to examine the effect of fatiguing electrical stimulation on the gap between the T-tubular and sarcoplasmic reticular membranes (T-SR distance) and the T-tubule diameter and compared this with corresponding effects on resting fibres exposed to a range of extracellular conditions that each replicate one of the major changes associated with muscular activity: membrane depolarisation, isotonic volume increase, acidification and intracellular lactate accumulation. Following each treatment, muscles were immersed in isotonic fixative solution and processed for transmission electron microscopy (TEM). Mean T-SR distances were estimated from orthogonal intercepts to provide estimates of diffusion distances between T and SR membranes and T-tubule diameter was estimated by measuring its shortest axis in the sampled J-SR complexes. Measurements from muscles fatigued by low frequency intermittent stimulation showed significant (P << 0.05) reversible increases in both T-SR distance and T-tubule diameter from 15.97 ± 0.37 nm to 20.15 ± 0.56 nm and from 15.44 ± 0.60 nm to 22.26 ± 0.84 nm (n = 40, 30) respectively. Exposure to increasing concentrations of extracellular [K⁺] in the absence of Cl⁻ to produce membrane depolarisation without accompanying cell swelling reduced T-SR distance and increased T-tubule diameter, whilst comparable increases in [K⁺]_e in the presence of Cl⁻ suggested that isotonic cell swelling has the opposite effect. Acidification alone, produced by NH₄Cl addition and withdrawal, also decreased T-SR distance and T-tubule diameter. A similar reduction in T-SR distance occurred following exposure to extracellular Na-lactate where such acidification was accompanied by elevations of intracellular lactate, but these conditions produced a significant swelling of T-tubules attributable to movement of lactate from the cell into the T-tubules. This study thus confirms previous reports of significant increases in T-SR distance and T-tubule diameter following stimulation. However, of membrane depolarisation, isotonic cell swelling, intracellular acidification and lactate accumulation, only isotonic cell swelling increases T-SR distance whilst membrane depolarisation and intracellular lactate likely contribute to the observed increases in T-tubule diameter.     

Single-channel current/voltage relationships of two kinds of Na+ channel in vertebrate sensory neurons

The electrical signals of nerve and muscle are fundamentally dependent on the voltage-gated Na⁺ channel, which is responsible for the rising phase of the action potential. At least two kinds of Na⁺ channel are expressed in the membrane of frog dorsal root ganglion (DRG) cells: Na⁺ channels with fast kinetics that are blocked by tetrodotoxin (TTX) at high affinity, and Na⁺ channels with slower kinetics that are insensitive to TTX. Recordings of single-channel currents from frog DRG cells, under conditions favoring Na⁺ as the charge carrier, reveal two distinct amplitudes of single-channel events. With 300 mM external Na⁺, single-channel events that can be measured in the presence of 1 M TTX have a slope conductance 7.5 pS. In the absence of TTX, events with a slope conductance of 14.9 pS dominate. Ensemble averages of the smaller single-channel events display the slower kinetics characteristic of the macroscopic TTX-insensitive Na⁺ currents, and ensemble averages of the larger events display the faster kinetics characteristic of the TTX-sensitive currents. The results are consistent with the idea that the toxin-binding site is sufficiently close to the pore to influence ion permeation.     

Giga-seal formation alters properties of sodium channels of human myoballs

The influence of giga-seal formation on the properties of the Na⁺ channels within the covered membrane patch was investigated with a whole-cell pipette and a patch pipette applied to the same cell. Current kinetics, current/voltage relation and channel densities were determined in three combinations: (i) voltage-clamping and current recording with the whole-cell pipette, (ii) voltage-clamping with the whole-cell pipette and current recording with the patch pipette and, (iii) voltage-clamping and current recording with the patch pipette. The Hodgkin-Huxley (1952) parameters _m and _h were smaller for the patch currents than for the whole cell, and the h curve was shifted in the negative direction. The channel density was of the order of 10 times smaller. All effects were independent of the extracellular Ca²⁺ concentration. The capacitive current generated in the patch by the whole-cell Na⁺ current and its effect on the transmembrane voltage of the patch were evaluated. The kinetic parameters of the Na⁺ channels in the patch did not depend on whether the voltage was clamped with the whole-cell pipette or the patch pipette. Thus, the results are not due to spurious voltage.     

Expression of the muscular dystrophy-associated caveolin-3P104L mutant in adult mouse skeletal muscle specifically alters the Ca2+ channel function of the dihydropyridine receptor

Caveolins are plasma-membrane-associated proteins potentially involved in a variety of signalling pathways. Different mutations in CAV3, the gene encoding for the muscle-specific isoform caveolin-3 (Cav-3), lead to muscle diseases, but the underlying molecular mechanisms remain largely unknown. Here, we explored the functional consequences of a Cav-3 mutation (P104L) inducing the 1C type limb-girdle muscular dystrophy (LGMD 1C) in human on intracellular Ca²⁺ regulation of adult skeletal muscle fibres. A YFP-tagged human Cav-3^P104L mutant was expressed in vivo in muscle fibres from mouse. Western blot analysis revealed that expression of this mutant led to an ∼80% drop of the level of endogenous Cav-3. The L-type Ca²⁺ current density was found largely reduced in fibres expressing the Cav-3^P104L mutant, with no change in the voltage dependence of activation and inactivation. Interestingly, the maximal density of intramembrane charge movement was unaltered in the Cav-3^P104L-expressing fibres, suggesting no change in the total amount of functional voltage-sensing dihydropyridine receptors (DHPRs). Also, there was no obvious alteration in the properties of voltage-activated Ca²⁺ transients in the Cav-3^P104L-expressing fibres. Although the actual role of the Ca²⁺ channel function of the DHPR is not clearly established in adult skeletal muscle, its specific alteration by the Cav-3^P104L mutant suggests that it may be involved in the physiopathology of LGMD 1C. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Postnatal induction and neural regulation of inward rectifiers in mouse skeletal muscle

The whole-cell voltage-clamp technique was used to examine developmental changes of inward rectifier currents in fibres of the flexor digitorum brevis muscle acutely isolated from mice on postnatal day 0 (PO) to P36. Neither a steady-state component (I _s–s) nor a slowly activated component (I _rise) of inward rectifier currents were observed in fibres of P0 and P4 mice. Both I _s–s and I _rise became apparent between days P8 and PI6. The specific amplitudes of I _s–s and I _rise measured at a test-pulse potential of –100 mV at 20 mM extracellular K⁺ ([K⁺]₀) increased to their respective plateau values of –68 ±10 and –15 ±7 A/cm² at P20. In fibres denervated on day P4 the developmental increase of I _s–s was suppressed, its specific amplitude at P20 being one-tenth of that in the corresponding normal fibres. I _rise did not appear in P4-denervated fibres throughout the development. In muscle fibres denervated at P16 or P20, the specific amplitudes of I _s–s and I _rise decreased, reaching the levels of P4-denervated fibres in 2–4 days after denervation. We conclude that I _s–s and I _rise develop within 3 weeks after birth, and suggest that innervation plays a key role in their induction.     

Effect of repetitive stimulation on cell volume and its relationship to membrane potential in amphibian skeletal muscle

The effect of electrical stimulation on cell volume, V _c, and its relationship to membrane potential, E _m, was investigated in Rana temporaria striated muscle. Confocal microscope xz-plane scanning and histology of plastic sections independently demonstrated significant and reversible increases in V _c of 19.8±0.62% (n=3) and 27.1±8.62% (n=3), respectively, after a standard stimulation protocol. Microelectrode measurements demonstrated an accompanying membrane potential change, ΔE _m, of +23.6±0.98 mV (n=3). The extent to which this ΔE _m might contribute to the observed changes in V _c was explored in quiescent muscle exposed to variations in extracellular potassium concentration, [K⁺]_e. E _m and V _c varied linearly with log [K⁺]_e and [K⁺]_e, respectively, in the range 2.5–15 mM (R ²=0.99 and 0.96), and these results were used to reconstruct an approximately linear relationship between V _c and E _m (ΔV _c=0.85E _m+68.53; R ²=0.99) and hence derive the ΔV _c expected from the ΔE _m during stimulation. This demonstrated that both the time course and magnitude of the increase and recovery of V _c observed in active muscles could be reproduced by the corresponding [K⁺]_e-induced depolarisation in quiescent muscles, suggesting that the depolarisation associated with membrane activity makes a substantial contribution to the cell swelling during exercise. Furthermore, conditions of Cl⁻ deprivation abolished the relationship between E _m and V _c, supporting a mechanism in which the depolarisation of E _m drives a passive redistribution of Cl⁻ and hence cellular entry of Cl⁻ and K⁺ and an accompanying, osmotically driven, increase in V _c.     

Intracellular and extracellular amino acids that influence C-type inactivation and its modulation in a voltage-dependent potassium channel

The rate of C-type inactivation of the cloned voltage-gated potassium channel, Kv1.3, measured in membrane patches from Xenopus oocytes, increases when the patch is detached from the cell; the structural basis for this on-cell/off-cell change was examined. First, four serine and threonine residues, that are putative sites for phosphorylation by protein kinases A and C, were mutated to alanines. Mutating any one of these residues, or two or three of them simultaneously, does not eliminate the change in C-type inactivation. However, the basal rate of C-type inactivation in the cell-attached patch is markedly slower in the triple phosphorylation site mutant. Second, a homologous potassium channel, Kv 1.6, does not exhibit the on-cell/off-cell change. When an extracellular histidine at position 401 of Kv1.3 is replaced with tyrosine, the residue at the equivalent position (430) in Kv1.6, the resulting Kv1.3 H401Y mutant channel does not undergo the on-cell/off-cell change. The results indicate that several potentially phosphorylatable intracellular amino acids influence the basal rate of C-type inactivation, but are not essential for the on-cell/off-cell change in inactivation kinetics. In contrast, an extracellular amino acid is critical for this on-cell/off-cell change.     

Calcium channel effectors are potent non-competitive blockers of acetylcholine receptors

Nicardipine and other calcium channel effectors (CCEs) were studied for their effects on nicotinic acetylcholine receptor (nAChR) activity. While CCEs had no effect on frog (Rana pipiens) skeletal muscle contractions resulting from nerve stimulation or direct stimulation of the muscle, nicotinic agonist-induced contractures were blocked. Nicardipine did not affect nAChR single-channel open time or amplitude, corroborating data from endplate currents (EPCs); EPC amplitudes and decays were unaffected. All the CCEs tested, however, non-competitively blocked nAChRs. The block of nAChRs resulted in a shortened agonist-induced depolarization and thus a diminished contracture response. An increase in cultured mouse skeletal muscle (C-2) cell single-channel closed times was observed with the intracellular addition of nicardipine, verifying a direct block of nAChRs. Using single-channel analysis, nicardipine's site of action, or at least access to its site of action, was determined to be at the intracellular side of the receptor. A direct action of the CCEs on the nAChR was also shown by their ability to block phencyclidine (PCP) binding to Torpedo nobiliana membranes. All the CCEs blocked specific binding of [³H]-PCP to its binding site on the nAChR-channel complex, with bepridil and nicardipine being the most potent. These data are compatible with a model in which nicardipine and other CCEs, at concentrations which do not alter nAChR channel open time or conductance, block the effects of superfused nicotinic agonist on nAChRs either by stabilizing the formation of the nAChR desensitized state or by effecting a slow channel block.     

Genetic variation in LINGO-1 (rs9652490) and risk of Parkinson's disease: twelve studies and a meta-analysis

Mutations of the voltage gated sodium channel gene (SCN4A) are responsible for non-dystrophic myotonia including hyperkalemic periodic paralysis, paramyotonia congenita, and sodium channel myotonia, as well as congenital myasthenic syndrome. In vitro functional analyses have demonstrated the non-dystrophic mutants to show a gain-of-function defect of the channel; a disruption of fast inactivation, an enhancement of activation, or both, while the myasthenic mutation presents a loss-of function defect. This report presents a case of non-dystrophic myotonia that is incidentally accompanied with acquired myasthenia. The patient presented a marked warm-up phenomenon of myotonia but the repeated short exercise test suggested mutations of the sodium channel. The genetic analysis identified a novel mutation, G1292D, of SCN4A. A functional study of the mutant channel revealed marked enhancement of activation and slight impairment of fast inactivation, which should induce muscle hyperexcitability. The effects of the alteration of channel function to the myasthenic symptoms were explored by using stimulation of repetitive depolarization pulses. A use-dependent channel inactivation was reduced in the mutant in comparison to normal channel, thus suggesting an opposing effect to myasthenia.     

Sodium channel slow inactivation and the distribution of sodium channels on skeletal muscle fibres enable the performance properties of different skeletal muscle fibre types

Na⁺ currents (I_Na) and membrane capacitance were studied with the loose patch voltage clamp technique and action potential properties were studied with a two-electrode voltage clamp on the end-plate, at the end-plate border and on extrajunctional membrane of skeletal muscle fibres. Slow inactivation regulates the available I_Na and is operative at the resting potential of both rat and human fibres. At the resting potential, slow inactivation causes a greater reduction in I_Na in fast- than in slow-twitch fibres. The relative resistance of slow-twitch fibres to slow inactivation may enable slow-twitch fibres to remain tonically active. Na⁺ channel inactivation may provide a peripheral mechanism that limits the duration that fast-twitch fibres can fire at high rates to prevent injury associated with prolonged high-frequency contraction. Consequently, slow inactivation may enable fast-twitch fibres to operate phasically at high rates or slow-twitch fibres to fire continuously at lower rates. For both fast- and slow-twitch fibres, I_Na normalized to membrane area was greatest on the end-plate, intermediate on the end-plate border and smallest on extrajunctional membrane. When normalized to membrane capacitance, I_Na was the same on the end-plate and the end-plate border and smallest on extrajunctional membrane. For a given membrane region, I_Na was larger on fast- than on slow-twitch fibres. The higher density of Na⁺ channels near the end-plate increased the safety factor for neuromuscular transmission by lowering the action potential threshold and increasing the action potential rate of rise at the end-plate.