Ca(2+)-calmodulin can activate and inactivate cardiac ryanodine receptors.
OBJECTIVE
Ca(2+)-calmodulin (Ca(2+)CaM) is widely accepted as an inhibitor of cardiac ryanodine receptors (RyR2); however, the effects of physiologically relevant CaM concentrations have not been fully investigated.


METHODS
We investigated the effects of low concentrations of Ca(2+)CaM (50-100 nmol.L(-1)) 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.


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 x 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 x L(-1) CaM reduced SR Ca(2+) content.


CONCLUSIONS
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.
Sigalas
C
C
Department of Physiology and Pharmacology and Bristol Heart Institute, University of Bristol, Bristol, UK.
Bent
S
S
Kitmitto
A
A
O'Neill
S
S
Sitsapesan
R
R
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002120:Calcium Channel Agonists / Q000378:metabolism* / Q000494:pharmacology; D002121:Calcium Channel Blockers / Q000378:metabolism* / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology / Q000502:physiology*; D002413:Cations, Divalent; D006801:Humans; D066298:In Vitro Techniques; D015640:Ion Channel Gating; D008051:Lipid Bilayers; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D019837:Ryanodine Receptor Calcium Release Channel / Q000502:physiology*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D012756:Sheep; D013498:Suramin / Q000494:pharmacology
D002120:Calcium Channel Agonists; D002121:Calcium Channel Blockers; D002147:Calmodulin; D002413:Cations, Divalent; D008051:Lipid Bilayers; D019837:Ryanodine Receptor Calcium Release Channel; D013498:Suramin; D002118:Calcium