MOLECULAR PHYSIOLOGY OF NEUROMUSCULAR DISEASES

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Many inherited disorders of muscle are caused by an abnormality in the electrical excitability of the sarcolemmal hyperkalemic periodic paralysis (HPP) episodes of weakness occur in association with an elevation in extracellular potassium. During an attack, muscles are depolarized and electrically inexcitable. A related disorder, paramyotonia congenita (PMC), is characterized by localized cold-induced stiffness (myotonia) and mild weakness. Myotonia arises from repetitive after-discharges that originate in affected muscle independent from neuronal input. A combination of physiologic and genetic evidence has established that HPP, PMC and an equine form of periodic paralysis are all caused by mutations in the alpha subunit of the adult skeletal muscle isoform of the sodium channel. We have shown previously that the primary functional defect in HPP is a disruption of Na current inactivation. The loss of inactivation in human HPP myotubes was enhanced by raised extracellular [K]. This provided an explanation for the episodic nature of the attacks, but was unexpected biophysically. A major aim of this proposal is to determine whether extracellular K directly alters gating in mutant channels, to explore how K exerts its influence, and to elucidate the kinetic basis for the persistent Na current. Gating behavior of Na channels has never been measured in the temperature-sensitive phenotype, PMC. The functional defects produced by PMC mutations will be defined by heterologous expression of mutant cDNAs in mammalian cells. Many PMC mutations occur in the III-IV cytoplasmic loop, and additional site- directed mutagenesis will be performed to define how this domain participates in the process of inactivation. The aberrant Na channel behaviors in HPP and PMC will be incorporated into both a computer simulation and an animal model to explore the pathophysiologic basis for the dominant expression of these phenotypes. In equine periodic paralysis all affected animals have the same point mutation in the alpha subunit, and the frequency of attacks is reduced by phenytoin. Unitary Na currents will be recorded from equine myotubes to define the functional defect and to measure the effects of phenytoin on aberrant channel gating. The proposed studies are designed to provide a complete understanding of the molecular physiologic basis of two human neuromuscular diseases. In addition, these results will further our understanding of Na channel function at the molecular level, will provide insights from which to design rational therapy for these diseases, and will serve as a model system for understanding other disorders of altered electrical excitability (epilepsy, cardiac dysrhythmias).