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

ABSTRACT: Malignant hyperthermia (MH) susceptibility has been recently linked to a novel variant of ?1a 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 ?1a 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 ?1a subunit. Expression of the variant subunit in ?1-null mouse myotubes resulted in increased sensitivity to caffeine stimulation. Whole cell patch-clamp analysis of ?1a-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 ?1a-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 ?1a subunit leading to a phenotype of Ca2+ dysregulation that partly resembles that of other MH-linked mutations of DHPR ?1S subunit. These data prove that homozygous expression of variant ?1a-V156A has the potential to be a pathological variant, although it may require other gene defects to cause a full MH phenotype.

SUBMITTER: Perez CF 

PROVIDER: S-EPMC6335014 | biostudies-literature | 2018 Mar

REPOSITORIES: biostudies-literature