SUMMARY1. Three families with a form of myotonia (muscle stiffness due to membrane hyperexcitability) clinically distinct from previously classified myotonias were examined. The severity of the disease greatly differed among the families.2. Three dominant point mutations were discovered at the same nucleotide position of the SCN4A gene encoding the adult skeletal muscle Na+ channel asubunit. They predict the substitution of either glutamic acid, valine or alanine for glycine1306, a highly conserved residue within the supposed inactivation gate.Additional SCN4A mutations were excluded.3. Electrophysiological studies were performed on biopsied muscle specimens obtained for each mutation. Patch clamp recordings on sarcolemmal blebs revealed an increase in the time constant of fast Na+ channel inactivation, Th, and in late channel openings as compared to normal controls. Th was increased from 1-2 to 1-6-2-1 ms and the average late currents from 04 to 1-6 % of the peak early current.4. Intracellular recordings on resealed fibre segments revealed an abnormal tetrodotoxin-sensitive steady-state inward current, and repetitive action potentials. Since K+ and Cl-conductances were normal, only the increase in the number of non-inactivating Na+ channels has to be responsible for the membrane hyperexcitability.5. Length, ramification and charge of the side-chains of the substitutions correlated well with the Na+ channel dysfunction and the severity of myotonia, with alanine as the most benign and glutamic acid as the substitution with a major steric effect.6. Our electrophysiological and molecular genetic studies strongly suggest that these Na+ channel mutations cause myotonia. The naturally occurring mutants allowed us to gain further insight into the mechanism of Na+ channel inactivation. ¶ To whom correspondence should be addressed.MS 2411

1. Wild type (WT) and V1589M channels were expressed in human embryonic kidney (HEK293) cells for the study of the pathophysiology of the V1589M muscle Na+ channel mutation leading to K+-aggravated myotonia. 2. In comparison to WI, whole-cell recordings with V1589M channels showed an increased Na+ steady-state to peak current ratio (I/Ipeak) (3-15 + 0 70 vs. 0-87 + 0410%, at -15 mV) and a significantly faster recovery from inactivation. The recovery time constants, Trl and Tr2, were decreased from 1-28 + 0-12 to 0-92 + 0-08 ms and from 4-74 + 0 94 to 2-66 + 0-51 ms for the WT and mutant channels, respectively.3. Single-channel recordings with mutant channels showed higher probability of short isolated late openings (0 40 + 0 09 vs. 0-06 + 0-02, at -30 mV) and bursts of late openings (0-011 + 0 003 vs. 0 003 + 0 001, at -30 mV) compared to VVW.4. These results suggest that the mutation increases the probabilities for channel transitions from the inactivated to the closed and the opened states. 5. Increased extracellular concentrations of K+ had no effects on either V1589M or WI currents in HEK293 cells. The aggravation of myotonia seen in patients during increased serum K+ may arise from the associated membrane depolarization which favours the occurence of late openings in the mutant channel.

The single strand conformation polymorphism (SSCP) technique was used to screen genomic DNA of a family with myotonia aggravated by cold, potassium loading and suxamethonium, but without muscle weakness. An aberrant band was found in exon 24 of SCN4A, the gene encoding the adult skeletal muscle sodium channel alpha-subunit. DNA sequencing led to the detection of a G-to-A transition of cDNA nucleotide 4765 predicting a substitution of methionine for valine at position 1589 of the protein sequence. This amino acid is located within transmembrane segment S6 of channel repeat IV close to the cytoplasmic surface, a region which is supposed to act as acceptor of the inactivation gate of the channel. Four lines of evidence indicate that this mutation causes the disease: (i) the transition was only found for affected family members; (ii) no mutations were found in all other SCN4A exons; (iii) the affected gene region is conserved among various species; and (iv) an increase in the number of non-inactivating sodium channels had been revealed in earlier electrophysiological studies on an excised muscle specimen from the index patient. In addition, the close-by occurring substitution of valine for methionine at position 1592 known to cause hyperkalemic periodic paralysis was deduced for six families with the myotonic, non-dystrophic form of this disease.

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Recessive myotonia congenita (Becker) is genetically linked to HUMCLC, the gene encoding the muscular chloride channel, localized on chromosome 7q35. Three point mutations have so far been reported in HUMCLC, one causing recessive Becker-type myotonia, the others causing the clinically similar Thomsen-type myotonia, which is inherited as a dominant trait. We report a homozygous patient having a 4 base pair deletion in HUMCLC that shifts the reading frame and causes early stop codons, thus destroying the gene's coding potential for several membrane-spanning domains. In addition, we report a patient homozygous for a novel point mutation located at the extracellular side of the first membrane-spanning domain that causes removal of a negative charge (aspartic acid-136-glycine). Both mutations lead to the recessive type of myotonia congenita. Since the patient having the deletion presents less severe clinical myotonia than the patient carrying the missense mutation, it seems that the absence or truncation of the channel protein may disturb muscle fibre function less than the substitution of a single amino acid.