Pathogenic Roles of Ion Channels and Transporters: Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β1a subunit (CACNB1)

摘要

Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of β1a subunit of the dihydropyridine receptor (DHPR), a channel essential for Ca²⁺ regulation in skeletal muscle. Here we evaluate the effect of the mutant variant V156A on the structure/function of DHPR β1a subunit and assess its role on Ca²⁺ 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 β1a subunit. Expression of the variant subunit in β1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of β1a-V156A-expressing myotubes revealed a −2 mV shift in voltage dependence of channel activation, but no changes in Ca²⁺ conductance, current kinetics, or sarcoplasmic reticulum Ca²⁺ load were observed. Measurement of resting free Ca²⁺ and Na⁺ concentrations shows that both cations were significantly elevated in β1a-V156A-expressing myotubes and that these changes were linked to increased rates of plasmalemmal Ca²⁺ entry through Na⁺/Ca²⁺ exchanger and/or transient receptor potential canonical channels. Overall, our data show that mutant variant V156A results in instability of protein subdomains of β1a subunit leading to a phenotype of Ca²⁺ dysregulation that partly resembles that of other MH-linked mutations of DHPR α1S subunit. These data prove that homozygous expression of variant β1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.