Electrophysiological Analysis of Two Malignant Hyperthermia-Linked Mutations in the 1,4-Dihydropyridine Receptor α1S Subunit

Malignant hyperthermia (MH) is a pharmacogenetic Ca2+-handling disorder of skeletal muscle, for which three identified causative mutations reside in the α1S subunit of the skeletal muscle 1,4-dihydropyridine receptor (DHPR). In this study, we investigated the effects of two of these mutations (R174W, R1086C) on the ability of the DHPR to function as an L-type Ca2+ channel. To do so, we introduced the respective mutations into α1S subunits amino-terminally tagged with yellow fluorescent protein (YFP-α1S), and expressed the fusion proteins in dysgenic (α1S null) myotubes. Confocal imaging of the YFP tags indicated that both YFP-R174W and YFP-R1086C were efficiently targeted to plasma membrane junctions with the sarcoplasmic reticulum. Measurement of intramembrane charge movements showed no significant difference in YFP-R174W or YFP-R1086C expression relative to YFP-α1S (p > 0.05, ANOVA). However, L-type Ca2+ currents in YFP-R1086C-expressing myotubes were substantially reduced relative to myotubes expressing YFP-α1S (p < 0.05, t-test). Strikingly, YFP-R174W failed to produce inward Ca2+ current during 200-ms test depolarizations under standard recording conditions (n = 7; p < 0.005, t-test) or in the presence of the L-type channel agonist ±Bay K 8644 (10 μM; n = 2; p < 0.05, t-test). In addition, exogenous expression of YFP-R174W reduced (∼85%) L-type currents in myotubes cultured from normal mice, indicating that the R174W mutation may potentially interfere with functional expression of the wild-type allele. Supported by NIH AR44750 and MDA 4319 to K.G.B.,and MDA 4155 to R.A.B.