SODIUM-CHANNEL INACTIVATION IS IMPAIRED IN EQUINE HYPERKALEMIC PERIODIC PARALYSIS

1. Equine hyperkalemic periodic paralysis (E-HPP) is a dominantly inhe rited disorder of muscle that causes recurrent episodes of stiffness ( myotonia) and weakness in association with elevated serum K+. Affected horses carry a mutant allele of the skeletal muscle isoform of the Na channel alpha-subunit. To understand how this mutation may cause the disease phenotype, the functional defect in Na channel behavior was de fined physiologically by recording unitary currents from cell-attached patches on normal and affected equine myotubes. 2. The presence of th e mutation was confirmed in our cell line by restriction digest of pol ymerase chain reaction (PCR)-amplified genomic DNA. Myotubes from the affected horse were heterozygous for the point mutation that codes for a Phe to Leu substitution in S-3 of domain IV. This assay provides a rapid technique to screen for the mutation in horses at risk. 3. The p rimary physiological defect in mutant Na channels was an impairment of inactivation. This defect was manifest as bursts of persistent activi ty during which the channel closed and reopened throughout a maintaine d depolarization. Disrupted inactivation slowed the decay of the ensem ble-averaged current and produced an eightfold increase in the steady- state open probability measured at the end of a 40-ms pulse. This poin t mutation identifies a new region of the alpha subunit that is import ant for rapid inactivation of the channel. 4. The persistent Na curren t was produced by a distinct mode of gating. Failure of a mutant chann el to inactivate was infrequent and occurred in groups of consecutive trials. Furthermore, the open time distributions for mutant Na channel s contained a second, slow component (tau(s) = 1-2 ms) in addition to a fast component (tau(f) = 0.4 ms), which by itself was sufficient to represent the distribution in normal channels. These observations are consistent with the notion that channels slowly switch between two mod es of inactivation: rapid versus noninactivating. Although the varianc e was high, for mutant channels there was a trend toward more frequent bursts of noninactivating behavior when extracellular K+ was increase d. For Na channels in E-HPP myotubes, the ratio of steady-state to pea k open probability increased threefold (0.024 from 0.008) in 10 versus 0 mM [K+](o). Conversely, in normal Na channels the steady-state to p eak P P-open was 0.003 and invariant with [K+](o).