Identification and functional characterization of malignant hyperthermia mutation T1354S in the outer pore of the Cav alpha1S-subunit

To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent Ca2+ channel (CaV) alpha1S-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of CaV alpha 1S in all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged alpha1ST1354S in dysgenic (alpha1S-null) myotubes. Whole cell patch-clamp analysis revealed that alpha1ST1354S produced significantly faster activation of L-type Ca2+ currents upon 200-ms depolarizing test pulses compared with wild-type GFP-alpha1S (alpha1SWT). In addition, alpha1ST1354Sexpressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca2+ release and to larger action-potential-induced intracellular Ca2+ transients under low (< 2 mM) caffeine concentrations compared with alpha1SWT. Thus our data suggest that an additional influx of Ca2+ due to faster activation of the alpha1ST1354S L-type Ca2+ current, in concert with higher caffeine sensitivity of Ca2+ release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype