SODIUM-CHANNEL INACTIVATION IS IMPAIRED IN EQUINE HYPERKALEMIC PERIODIC PARALYSIS

1. Equine hyperkalemic periodic paralysis (E-HPP) is a dominantly inherited 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 defined physiologically by recording unitary currents from cell-attached patches on normal and affected equine myotubes. 2. The presence of the mutation was confirmed in our cell line by restriction digest of polymerase 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 primary physiological defect in mutant Na channels was an impairment of inactivation. This defect was manifest as bursts of persistent activity during which the channel closed and reopened throughout a maintained depolarization. Disrupted inactivation slowed the decay of the ensemble-averaged current and produced an eightfold increase in the steady-state open probability measured at the end of a 40-ms pulse. This point mutation identifies a new region of the alpha subunit that is important for rapid inactivation of the channel. 4. The persistent Na current was produced by a distinct mode of gating. Failure of a mutant channel to inactivate was infrequent and occurred in groups of consecutive trials. Furthermore, the open time distributions for mutant Na channels 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 modes of inactivation: rapid versus noninactivating. Although the variance was high, for mutant channels there was a trend toward more frequent bursts of noninactivating behavior when extracellular K+ was increased. For Na channels in E-HPP myotubes, the ratio of steady-state to peak 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 peak P P-open was 0.003 and invariant with [K+](o).