Ca²⁺‐calmodulin can activate and inactivate cardiac ryanodine receptors

Abstract

) on the gating of native sheep RyR2, reconstituted into bilayers. Suramin displaces CaM from RyR2 and we have used a gel-shift assay to provide evidence of the mechanism underlying this effect. Finally, using suramin to displace endogenous CaM from RyR2 in permeabilized cardiac cells, we have investigated the effects of 50 nmol·L -1 CaM on sarcoplasmic reticulum (SR) Ca 2+-release. Key results: Ca 2+CaM activated or inhibited single RyR2, but activation was much more likely at low (50-100 nmol·L -1 ) concentrations. Also, suramin displaced CaM from a peptide of the CaM binding domain of RyR2, indicating that, like the skeletal isoform (RyR1), suramin directly competes with CaM for its binding site on the channel. Pre-treatment of rat permeabilized ventricular myocytes with suramin to displace CaM, followed by addition of 50 nmol·L -1 CaM to the mock cytoplasmic solution caused an increase in the frequency of spontaneous Ca 2+ -release events. Application of caffeine demonstrated that 50 nmol·L -1 CaM reduced SR Ca 2+ content. Conclusions and implications: We describe for the first time how Ca 2+CaM is capable, not only of inactivating, but also of activating RyR2 channels in bilayers in a CaM kinase II-independent manner. Similarly, in cardiac cells, CaM stimulates SR Ca 2+-release and the use of caffeine suggests that this is a RyR2-mediated effect. Abbreviations: AIP, autocamtide-2-related inhibitory peptide; CaM, calmodulin; CaMKII, CaM kinase II; EC-coupling, excitation-contraction coupling; RyR2, cardiac ryanodine receptor; RyRF, RyR2 peptide (RSKKAVWHKLL-SKQRKRAVVACFRMAPLYNLP); SR, sarcoplasmic reticulum British Journal of Pharmacology IntroductionThe cardiac ryanodine receptor (RyR2; nomenclature follows Alexander et al., 2008) is the pathway for the release of intracellular Ca 2+ during excitation-contraction (EC) coupling. It also acts as a scaffolding protein localizing numerous other proteins to the dyadic cleft regions. Calmodulin (CaM) binds very tightly to RyR2, but the physiological role of this direct association is unclear (Balshaw et al., 2001;Ai et al., 2005). CaM is a Ca 2+ -sensing protein, which contains four Ca 2+ -binding sites, two on the amino and two on the carboxyl lobe. CaM interacts with numerous proteins, usually in the Ca 2+ -bound form (Ca 2+ CaM), but can also modulate certain proteins in the non-Ca 2+ -bound form (apoCaM). It has been well documented that Ca 2+ CaM can cause partial inhibition of both cardiac and skeletal (RyR1) isoforms of RyR (Fruen et al., 2000;Balshaw et al., 2001). Additionally, apoCaM is known to activate RyR1, but has little effect on RyR2 Tripathy et al., 1995;Fruen et al., 2000).The magnitude of the reported inhibition of RyR2 by Ca 2+ CaM is often exceedingly small (Yamaguchi et al., 2004;Xu and Meissner, 2004) and, interestingly, there are also rare reports suggesting that CaM may activate RyR2 (Fruen et al., 2000;Chugun et al., 2007 Xu and Meissner, 2004) and Ca 2+ -spark generation in cardiac cells (Lukyanenko and Gyorke, 1999;Ai et al., 2005;Guo ...

The effects of Suramin on Ca2+activated force and sarcoplasmic reticulum Ca2+release in skinned fast-twitch skeletal muscle fibers of the rat

“…Indeed, this was also observed by Sigalas et al. () who showed that the SR Ca 2+ ‐ATPase in permeablized cardiac cells was not affected by Suramin treatment for a similar time.…”

“…Taken together, the results indicate that the Suramin effect on the SR Ca 2+ -ATPase is fully reversible. Indeed, this was also observed by Sigalas et al (2009) who showed that the SR Ca 2+ -ATPase in permeablized cardiac cells was not affected by Suramin treatment for a similar time.…”

“…; Sigalas et al. ). If this were the case, then our results are most likely explained by an increase in the open probability when activated following the removal of CaM from the RyRs as reported in other studies (Hill et al.…”

“…It is worth noting that in this study Suramin was washed from the fibers following the 10 min treatment period and therefore, the observed effects of Suramin described in this study must result from some irreversible action of Suramin on the RyRs (in addition to the contractile myofibrillar components described earlier). Suramin is thought to compete with CaM on the RyR displacing CaM from this site (Klinger et al 1999;Papineni et al 2002;Sigalas et al 2009). If this were the case, then our results are most likely explained by an increase in the open probability when activated following the removal of CaM from the RyRs as reported in other studies (Hill et al 2004).…”

Effect of hypaconitine combined with liquiritin on the expression of calmodulin and connexin43 in rat cardiac muscle in vivo

“…In addition, CaM can inhibit the opening of the calcium release channel by binding with RyR2, and can inhibit the binding of ryanodine and RyR2 to achieve the open and close balance of calcium release channels. [13][14][15] This study indicated that the mRNA and protein expression levels of CaM were significantly decreased by hypaconitine. Although the CaM mRNA expression level of the liquiritin plus high-dose hypaconitine group had no significant difference as compared with the control, the CaM protein expression level was significantly lower than that of the control and the high-dose hypaconitine group.…”