Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β 1a subunit (CACNB1).

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.