Functional and structural characterization of a novel malignant hyperthermia-susceptible variant of DHPR-β_1a subunit (CACNB1)

Abstract

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

Protein Transduction System Based on Tryptophan-zipper against Intracellular Infections via Inhibiting Ferroptosis of Macrophages

“…However, PP-turn and triglycine, in which the hydrophilic surface area was concentrated in the range of 0–1.0 nm, were deeply embedded in the hydrophobic core (Figures E,F and S5). The hydrophilic properties of PTDs ensure the flexibility of the skeleton structure as well as its nondegradable properties, because excessive hydrophobicity reduces the stability of the protein . The highly hydrophobic PP-turn provided a prerequisite for the amino acid zipper formation of the β-hairpin.…”

“…The hydrophilic properties of PTDs ensure the flexibility of the skeleton structure as well as its nondegradable properties, because excessive hydrophobicity reduces the stability of the protein. 18 The highly hydrophobic PP-turn provided a prerequisite for the amino acid zipper formation of the β-hairpin. Therefore, we extracted the secondary structure information on all the residues within a time step of 100 ns.…”

A reconstituted depolarization-induced Ca2+ release platform for validation of skeletal muscle disease mutations and drug discovery

“…This has been performed using myotubes from mice that lack DICR machinery components, for example, knockout mice of Cav1.1 ( Tanabe et al, 1988 ), β1a ( Gregg et al, 1996 ), and Stac3 ( Nelson et al, 2013 ). While this approach has evaluated several disease-causing mutations in the DICR components ( Weiss et al, 2004 ; Pirone et al, 2010 ; Eltit et al, 2012 ; Polster et al, 2016 ; Perez et al, 2018 ), it has certain limitations. For example, it is expensive and labor intensive to maintain the mouse lines.…”