Regulation of the RYR1 and RYR2 Ca²⁺ Release Channel Isoforms by Ca²⁺-Insensitive Mutants of Calmodulin

Abstract

Calmodulin (CaM) may function as a regulatory subunit of ryanodine receptor (RYR) channels, modulating both channel activation and inhibition by Ca2+; however, mechanisms underlying differences in CaM regulation of the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2) are poorly understood. Here we use a series of CaM mutants deficient in Ca2+ binding to compare determinants of CaM regulation of the RYR1 and RYR2 isoforms. In submicromolar Ca2+, activation of the RYR1 isoform by each of the single-point CaM mutants was similar to that by wild-type apoCaM, whereas in micromolar Ca2+, RYR1 inhibition by Ca2+CaM was abolished by mutations targeting CaM's C-terminal Ca2+ sites. In contrast to the RYR1, no activation of the cardiac RYR2 isoform by wild-type CaM was observed, but rather CaM inhibited the RYR2 at all Ca2+ concentrations (100 nM to 1 mM). Consequently, whereas the apparent Ca2+ sensitivity of the RYR1 isoform was enhanced in the presence of CaM, the RYR2 displayed the opposite response (RYR2 Ca2+ EC50 increased 7-10-fold in the presence of 5 microM wild-type CaM). CaM inhibition of the RYR2 was nonetheless abolished by each of four mutations targeting individual CaM Ca2+ sites. Furthermore, a mutant CaM deficient in Ca2+ binding at all four Ca2+ sites significantly activated the RYR2 and acted as a competitive inhibitor of RYR2 regulation by wild-type Ca2+CaM. We conclude that Ca2+ binding to CaM determines the effect of CaM on both RYR1 and RYR2 channels and that isoform differences in CaM regulation reflect the differential tuning of Ca2+ binding sites on CaM when bound to the different RYRs. These results thus suggest a novel mechanism by which CaM may contribute to functional diversity among the RYR isoforms.

Mapping the Ryanodine Receptor FK506-binding Protein Subunit Using Fluorescence Resonance Energy Transfer

“…Materials-Sarcoplasmic reticulum (SR) membrane vesicles were isolated from pig longissimus dorsi and pig ventricular tissue by differential ultracentrifugation of homogenized muscle (23). Cysteine-reactive fluorescent dyes were purchased from Invitrogen.…”

FRET-based mapping of calmodulin bound to the RyR1 Ca 2+ release channel

“…Skeletal muscle SR membrane vesicles were isolated from pig longissimus dorsi muscle by differential ultracentrifugation of homogenized muscle (5,18). Samples enriched in RyR1 were obtained by sucrose gradient fractionation of CHAPS-solubilized SR (23).…”

“…A single-cysteine FKBP (T14C, C22A, C76I FKBP12.6) was derived from the human FKBP12.6 cDNA by site-directed mutagenesis (QuikChange kit; Stratagene) and expressed in Escherichia coli BL21(DE3)pLysS, and the FKBP was purified as described previously (24,25). Single-cysteine CaMs with substitutions within either the N lobe (T34C), central linker (K75C), or C lobe (T110C) were expressed and purified as described previously (5,15). A singlecysteine Ca 2ϩ -insensitive CaM was synthesized by introducing the T34C substitution into a CaM 1234 mutant (E-to-A substitutions at positions 31, 67, 104, and 140) (5).…”

Role of abnormal sarcoplasmic reticulum function in atrial fibrillation

“…RyR2 are tetramers comprised of four RyR2 subunits, each of which associates with several regulatory subunits. Calmodulin (CaM) is an accessory protein that binds to and regulates RyR2 gating [24]. The FK506-binding protein (FKBP12.6, also known as calstabin2) stabilizes the RyR2 closed conformational state [20,25], and facilitates coupled gating between connected RyR2 channels [26].…”