Abstract
Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β(1a) subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca(2+) regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β(1a) subunit and assess its role on Ca(2+) metabolism of cultured myotubes. Using differential scanning fluorimetry, we show that mutation V156A causes a significant reduction in thermal stability of the Src homology 3/guanylate kinase core domain of β(1a) subunit. Expression of the variant subunit in β(1)-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of β(1a)-V156A-expressing myotubes revealed a -2 mV shift in voltage dependence of channel activation, but no changes in Ca(2+) conductance, current kinetics, or sarcoplasmic reticulum Ca(2+) load were observed. Measurement of resting free Ca(2+) and Na(+) concentrations shows that both cations were significantly elevated in β(1a)-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca(2+) entry through Na(+)/Ca(2+) exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of β(1a) subunit leading to a phenotype of Ca(2+) dysregulation that partly resembles that of other MH-linked mutations of DHPR α(1S) subunit. These data prove that homozygous expression of variant β(1a)-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.