Differential Ca²⁺ sensitivity of skeletal and cardiac muscle ryanodine receptors in the presence of calmodulin

Abstract

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor Ca(2+) release channel (RyR1) in the presence of nanomolar Ca(2+) concentrations. However, the role of CaM activation in the mechanisms that control Ca(2+) release from the sarcoplasmic reticulum (SR) in skeletal muscle and in the heart remains unclear. In media that contained 100 nM Ca(2+), the rate of (45)Ca(2+) release from porcine skeletal muscle SR vesicles was increased approximately threefold in the presence of CaM (1 microM). In contrast, cardiac SR vesicle (45)Ca(2+) release was unaffected by CaM, suggesting that CaM activated the skeletal RyR1 but not the cardiac RyR2 channel isoform. The activation of RyR1 by CaM was associated with an approximately sixfold increase in the Ca(2+) sensitivity of [(3)H]ryanodine binding to skeletal muscle SR, whereas the Ca(2+) sensitivity of cardiac SR [(3)H]ryanodine binding was similar in the absence and presence of CaM. Cross-linking experiments identified both RyR1 and RyR2 as predominant CaM binding proteins in skeletal and cardiac SR, respectively, and [(35)S]CaM binding determinations further indicated comparable CaM binding to the two isoforms in the presence of micromolar Ca(2+). In nanomolar Ca(2+), however, the affinity and stoichiometry of RyR2 [(35)S]CaM binding was reduced compared with that of RyR1. Together, our results indicate that CaM activates RyR1 by increasing the Ca(2+) sensitivity of the channel, and further suggest differences in CaM's functional interactions with the RyR1 and RyR2 isoforms that may potentially contribute to differences in the Ca(2+) dependence of channel activation in skeletal and cardiac muscle.

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).…”

IP3 Receptors: Toward Understanding Their Activation

“…In summary, all IP 3 R subtypes are inhibited by Ca 2þ -CaM, but the molecular basis of this inhibition has not been established. It seems, on balance, that CaM is unlikely to be the accessory protein through which Ca 2þ universally inhibits IP 3 R. That need not preclude a role for CaM in modulating IP 3 R function (Taylor and Laude 2002), just as it does for RyR (Chen et al 1997;Fruen et al 2000;Rodney et al 2001), but we must look elsewhere for the site through which Ca 2þ inhibits IP 3 R.…”

Different Regions in Skeletal and Cardiac Muscle Ryanodine Receptors Are Involved in Transducing the Functional Effects of Calmodulin

“…Direct CaM binding inhibits all three RyR isoforms at [Ca 2ϩ ] Ͼ 1 M, whereas at [Ca 2ϩ ] Ͻ 1 M RyR1 and RyR3 are activated, but RyR2 is inhibited by CaM (6,7). 35 S-CaM binding to sarcoplasmic reticulum vesicles (8,9) and purified RyR1 and RyR2 (10) showed that the two receptor isoforms bind one molecule of CaM per RyR subunit in the absence and presence of Ca 2ϩ . CaM protection of trypsin digestion of RyR1 (11) and site-directed mutagenesis of RyR1 (12) and RyR2 (13) demonstrated that the two RyRs have a single CaM regulatory binding domain (CaMBD) (aa 3614 -3643 in RyR1) for the Ca 2ϩ -free and Ca 2ϩ -bound (Ca 2ϩ -CaM) forms of CaM.…”