Mechanisms of cold sensitivity of paramyotonia congenita mutation R1448H and overlap syndrome mutation M1360V

Missense mutations of the human skeletal muscle voltage-gated Na⁺ channel (hSkM1) cause a variety of neuromuscular disorders. The mutation R1448H results in paramyotonia congenita and causes cold-induced myotonia with subsequent paralysis. The mutation M1360V causes an overlapping syndrome with both K⁺-induced muscle weakness and cold-induced myotonia. The molecular mechanisms of the temperature dependence of these disorders are not well understood. Therefore we investigated physiological parameters of these Na⁺ channel mutations at different temperatures. Channel proteins were recombinantly expressed in human embryonic kidney cells and studied electrophysiologically, using the whole-cell patch-clamp technique. We compared the wild-type (WT) channel with both mutants at different temperatures. Both mutations had slower inactivation and faster recovery from inactivation compared to WT channels. This effect was more pronounced at the R1448H mutation, leading to a larger depolarization of the cell membrane causing myotonia and paralysis. The voltage dependence of activation of R1448H was shifted to more negative membrane potentials at lower temperature but not at the M1360V mutation or in the WT. The window current by mutation R1448H was increased at lower temperatures. The results of this study may explain the stronger cold-induced clinical symptoms resulting from the R1448H mutation in contrast to the M1360V mutation.     

Effects of temperature and mexiletine on the F1473S Na+ channel mutation causing paramyotonia congenita

 The F1473S mutation of the adult human skeletal muscle Na⁺ channel causes paramyotonia congenita, a disease characterized by muscle stiffness sometimes followed by weakness in a cold environment. The symptoms are relieved by the local anaesthetic mexiletine. This mutation, which resides in the cytoplasmic S4-S5 loop in domain IV of the α-subunit, was studied by heterologous expression in HEK293 cells using standard patch-clamp techniques. Compared to wild-type (WT) channels, those with the F1473S mutation exhibit a twofold slowing of fast inactivation, an increased persistent Na⁺ current, a +18-mV shift in steady-state inactivation and a fivefold acceleration of recovery from fast inactivation; slow inactivation was similar for both clones. Single-channel recordings for the F1473S mutation revealed a prolonged mean open time and an increased number of channel reopenings that increased further upon cooling. The pharmacological effects of mexiletine on cells expressing either WT, F1473S or G1306E channels were studied. G1306E is a myotonia-causing mutation located within the inactivation gate that displays similar but stronger inactivation defects than F1473S. The hyperpolarizing shift in steady-state inactivation induced by mexiletine was almost identical for all three clones. In contrast, this agent had a reduced effectiveness on the phasic (use-dependent) block of Na⁺ currents recorded from the mutants: the relative order of block was WT>F1473S>G1306E. We suggest that the relative effectiveness of mexiletine is associated with the degree of abnormal channel inactivation and that the relative binding affinity of mexiletine is not substantially different between the mutations or the WT. Received: 13 January 1998 / Received after revision: 11 May 1998 / Accepted: 19 May 1998     

A Korean family with Arg1448Cys mutation of SCN4A channel causing paramyotonia congenita: electrophysiologic,histopathologic, and molecular genetic studies

A family with paramyotonia congenita (PC) is presented. At least 10 family members were affected in an autosomal dominant inheritance pattern. The proband had cold-sensitive muscle stiffness, paradoxical myotonia, and intermittent muscle weakness since childhood. The serum level of creatine kinase was mildly elevated and short exercise test with cooling revealed a drastic reduction of compound muscle action potentials with repetitive discharges. Muscle biopsy revealed marked variation in the fiber size and increased internal nuclei. The molecular biological study revealed a common missense mutation (Arg1448Cys) at the voltage-gated sodium channel gene (SCN4A). The repetitive CMAP discharges during short exercise test with cooling observed in the proband has not been reported previously. This observation needs to be confirmed among PC patients with different mutations. This is the first report on a PC family confirmed by the molecular biological technique in Korea.     

Functional expression and properties of the human skeletal muscle sodium channel

Full-length deoxyribonucleic acid, complementary (cDNA) constructs encoding the-subunit of the adult human skeletal muscle Na⁺ channel, hSkM1, were prepared. Functional expression was studied by electrophysiological recordings from cRNA-injectedXenopus oocytes and from transiently transfected tsA201 cells. The Na⁺ currents of hSkM1 had abnormally slow inactivation kinetics in oocytes, but relatively normal kinetics when expressed in the mammalian cell line. The inactivation kinetics of Na⁺ currents in oocytes, during a depolarization, were fitted by a weighted sum of two decaying exponentials. The time constant of the fast component was comparable to that of the single component observed in mammalian cells. The block of hSkM1 Na⁺ currents by the extracellular toxins tetrodotoxin (TTX) and -conotoxin (CTX) was measured. The IC₅₀ values were 25 nM (TTX) and 1.2 M (CTX) in oocytes. The potency of TTX is similar to that observed for the rat homolog rSkM1, but the potency of CTX is 22-fold lower in hSkM1, primarily due to a higher rate of toxin dissociation in hSkM1. Single-channel recordings were obtained from outside-out patches of oocytes expressing hSkM1. The single-channel conductance, 24.9 pS, is similar to that observed for rSkM1 expressed in oocytes.     

Expression and functional characterization of the cardiac L-type calcium channel carrying a skeletal muscle DHP-receptor mutation causing hypokalaemic periodic paralysis

A histidine substitution for the outermost arginine in II/S4 of the ₁ subunit of the human skeletal muscle dihydropyridine (DHP) receptor has been reported to cause hypokalaemic periodic paralysis (HypoPP). This mutation shifts the voltage dependence of L-type Ca current inactivation in myotubes from HypoPP patients by –40 mV without affecting activation. Based on the strong homology of II/S4 in cardiac and skeletal muscle ₁, we introduced the corresponding mutation into the rabbit cardiac ₁ subunit (R650H). Wild type (WT) and mutant constructs were transiently transfected in HEK cells together with and ₂ subunits and Ca and Ba currents were studied using the whole-cell patch-clamp technique. In contrast to the results obtained from human myotubes, R650H produced a small (–5 mV) but significant shift of both the steady-state activation and inactivation curves. When external pH was increased from 7.4 to 8.4 in order to favour deprotonization of H650, the only difference between WT and mutant channels was a slightly reduced steepness of the inactivation curve. Additional cotransfection of the subunit which is only found in skeletal but not in heart muscle, shifted the inactivation curves of both WT and R650H by –20 mV. We conclude that R650 plays a different role in voltage-dependent gating of the cardiac L-type Ca channel than the corresponding residue in the human skeletal muscle L-type channel, since a distinct and selective effect on the midpoint voltage of steady-state inactivation could not be found for R650H.     

The genomic structure of the human skeletal muscle sodium channel gene.

Electrical excitability of neurons and muscle cells reflects the actions of a family of structurally related sodium channels. Mutations in the adult skeletal muscle sodium channel have been associated with the inherited neuromuscular disorders paramyotonia congenita (PMC) and hyperkalemic periodic paralysis (HPP). We have deciphered the entire genomic structure of the human skeletal muscle sodium channel gene and developed a restriction map of the locus. SCN4A consists of 24 exons spanning 35 kb of distance on chromosome 17q. We describe the sequence of all intron/exon boundaries, the presence of several polymorphisms in the coding sequence, and the locations within introns of two dinucleotide repeat polymorphisms. This is the first sodium channel for which the entire genomic structure has been resolved. The organization of the SCN4A exons relative to the proposed protein structure is presented and represents a foundation for functional and evolutionary comparisons of sodium channels. Knowledge of the exon structure and flanking intron sequences for SCN4A will permit a systematic search for mutations in PMC and HPP.     

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.     

Conservation of pH sensitivity in the epithelial sodium channel (ENaC) with Liddle's syndrome mutation

Gain-of-function mutations of the epithelial Na+ channel (ENaC) cause a rare form of hereditary hypertension, Liddle's syndrome. How these mutations lead to increased channel activity is not yet fully understood. Since wild-type ENaC (wt-ENaC) is highly pH-sensitive, we wondered whether an altered pH-sensitivity of ENaC might contribute to the hyperactivity of ENaC with Liddle's syndrome mutation (Liddle-ENaC). Using Xenopus laevis oocytes as an expression system, we compared the pH-sensitivity of wt-ENaC (alphabetagammarENaC) and Liddle-ENaC (alphabeta(R564stop)gammarENaC). Oocytes were assayed for an amiloride-sensitive (2 microM) inward current (deltaIami) at -60 mV holding potential and cytosolic pH was altered by changing the extracellular pH in the presence of 60 mM sodium acetate. Alternatively, cytosolic acidification was achieved by proton loading the cells using a proton-coupled oligopeptide transporter (PepT-1) co-expressed in the oocytes together with ENaC. Cytosolic but not extracellular acidification substantially reduced deltaIami while cytosolic alkalinisation had a stimulatory effect. This pH-sensitivity was largely preserved in oocytes expressing Liddle-ENaC. The inhibition of wt-ENaC and Liddle-ENaC by cytosolic acidification was independent of so-called sodium-feedback inhibition, since it was not associated with a concomitant increase in intracellular Na+ concentration estimated from the reversal potential of deltaIami. In addition C-terminal deletions in the alpha or gamma subunits or in all three subunits of ENaC did not abolish the inhibitory effect of cytosolic acidification. We conclude that ENaC's pH-sensitivity is not mediated by its cytoplasmic C-termini and that an altered pH-sensitivity of ENaC does not contribute to the pathophysiology of Liddle's syndrome.     

A1152D mutation of the Na+ channel causes paramyotonia congenita and emphasizes the role of DIII/S4–S5 linker in fast inactivation

Missense mutations in the human skeletal muscle Na⁺ channel α subunit (hSkM1) are responsible for a number of muscle excitability disorders. Among them, paramyotonia congenita (PC) is characterized by episodes of muscle stiffness induced by cold and aggravated by exercise. We have identified a new PC-associated mutation, which substitutes aspartic acid for a conserved alanine in the S4–S5 linker of domain III (A1152D). This residue is of particular interest since its homologue in the rat brain type II Na⁺ channel has been suggested as an essential receptor site for the fast inactivation particle. To identify the biophysical changes induced by the A1152D mutation, we stably expressed hSkM1 mutant or wild-type (WT) channels in HEK293 (human embryonic kidney) cells, and recorded whole-cell Na⁺ currents with the patch-clamp technique. Experiments were performed both at 21 and 11°C to better understand the sensitivity to cold of paramyotonia. The A1152D mutation disrupted channel fast inactivation. In comparison to the WT, mutant channels inactivated with slower kinetics and displayed a 5 mV depolarizing shift in the voltage dependence of the steady-state. The other noticeable defect of A1152D mutant channels was an accelerated rate of deactivation from the inactivated state. Decreasing temperature by 10°C amplified the differences in channel gating kinetics between mutant and WT, and unveiled differences in both the sustained current and channel deactivation from the open state. Overall, cold-exacerbated mutant defects may result in a sufficient excess of Na⁺ influx to produce repetitive firing and myotonia. In the light of previous reports, our data point to functional as well as phenotypic differences between mutations of conserved S4–S5 residues in domains II and III of the human skeletal muscle Na⁺ channel.     

Identification of a novel CNTNAP1 mutation causing arthrogryposis multiplex congenita with cerebral and cerebellar atrophy

Arthrogryposis multiplex congenital, the occurrence of multiple joint contractures at birth, can in some cases be accompanied by insufficient myelination of peripheral nerves, muscular hypotonia, reduced tendon reflexes, and respiratory insufficiency. Recently mutations in the CASPR/CNTN1 complex have been associated with similar severe phenotypes and CNTNAP1 gene mutations, causing loss of the CASPR protein, were shown to cause severe, prenatal onset arthrogryposis multiplex congenita in four unrelated families. Here we report a consanguineous Arab family from Qatar with three children having an early lethal form of arthrogryposis multiplex congenita and a novel frameshift mutation in CNTNAP1. We further expand the existing CNTNAP1-associated phenotype to include profound cerebral and cerebellar atrophy.     

Preferential block of skeletal muscle sodium channels by geographutoxin II,a new peptide toxin fromConus geographus

The effects of geographutoxin II (GTX II), a novel polypeptide toxin isolated from the marine snailConus geographus, on nerves and muscles were studied by current clamp and voltage clamp techniques. GTX II (5×10^–7 M) abolished the action potential of the guinea pig skeletal muscle without change in the resting potential. However, action potentials of the crayfish giant axon, mouse neuroblastoma N1E-115 cell and guinea pig cardiac muscle were not affected by GTX II even at concentrations higher than 1×10^–6 M. In the voltage clamped bullfrog skeletal muscle fiber, sodium currents were almost completely blocked by GTX II (1×10^–6 M), and slowly recovered after washout. The time course of sodium currents was not appreciably altered by GTX II. These results suggest that GTX II selectively blocks skeletal muscle sodium channels in much the same way as tetrodotoxin.     

A gain-of-function mutation in the sodium channel gene Scn2a results in seizures and behavioral abnormalities

The GAL879-881QQQ mutation in the cytoplasmic S4-S5 linker of domain 2 of the rat brain IIA sodium channel (Na(v)1.2) results in slowed inactivation and increased persistent current when expressed in Xenopus oocytes. The neuron-specific enolase promoter was used to direct in vivo expression of the mutated channel in transgenic mice. Three transgenic lines exhibited seizures, and line Q54 was characterized in detail. The seizures in these mice began at two months of age and were accompanied by behavioral arrest and stereotyped repetitive behaviors. Continuous electroencephalogram monitoring detected focal seizure activity in the hippocampus, which in some instances generalized to involve the cortex. Hippocampal CA1 neurons isolated from presymptomatic Q54 mice exhibited increased persistent sodium current which may underlie hyperexcitability in the hippocampus. During the progression of the disorder there was extensive cell loss and gliosis within the hippocampus in areas CA1, CA2, CA3 and the hilus. The lifespan of Q54 mice was shortened and only 25% of the mice survived beyond six months of age. Four independent transgenic lines expressing the wild-type sodium channel were examined and did not exhibit any abnormalities.The transgenic Q54 mice provide a genetic model that will be useful for testing the effect of pharmacological intervention on progression of seizures caused by sodium channel dysfunction. The human ortholog, SCN2A, is a candidate gene for seizure disorders mapped to chromosome 2q22-24.     

Insulin sensitivity in skeletal muscle regulated by a hepatic hormone, HISS.

The current state of the HISS (hepatic insulin sensitizing substance) hypothesis is briefly outlined. In the postmeal absorptive state, 50-60% of the glucose storage action of insulin is accounted for by the actions of HISS released from the liver and acting on skeletal muscle. Hepatic parasympathetic nerves permissively regulate the ability of a pulse of insulin to release HISS, thereby potentiating the impact of insulin in the fed state. HISS release in response to insulin decreases progressively with fasting to create a physiological state of HISS-dependent insulin resistance. HISS release is regulated by parasympathetic nerves via muscarinic receptors and nitric oxide, and insulin resistance of skeletal muscle produced by hepatic denervation is reversed by intraportal but not intravenous acetylcholine or a nitric oxide donor. It is suggested that HISS-dependent insulin resistance occurs in animal models including sucrose-fed rats, spontaneously hypertensive rats, chronic liver disease, fetal alcohol effect in the adult offspring, and type 2 diabetes.