Calmodulin (CaM) has been shown to reduce the open probability (P_o) of skeletal ryanodine receptor (RyR) channels in the presence of micromolar cytosolic Ca²⁺ but increase P_o at nanomolar levels of cytosolic Ca²⁺. It has been suggested that suramin modifies RyR function by interacting with CaM binding sites on RyR (Klinger et al. 1999). In the present study we have examined the effects of calmodulin on the sheep cardiac RyR and investigated the possibility that suramin and calmodulin act via the same binding sites. Sarcoplasmic reticulum (SR) membrane vesicles were isolated as described previously (Kermode et al. 1998) from sheep hearts obtained from an abattoir and were either incorporated into artificial membranes for single-channel studies or were used for [³H]ryanodine binding or Western blotting (Kermode et al. 1998).

In the presence of 100 mM cytosolic free Ca²⁺, 50 nM CaM reduced the P_o of RyR from 0.314 ± 0.082 to 0.031 ± 0.02 (S.E.M.; n = 5). At this same [Ca²⁺], CaM dose-dependently inhibited [³H]ryanodine binding to isolated SR vesicles (35 ± 3 % reduction at 5 mM CaM (S.E.M.; n = 4)), while CaM had no effect on binding at nanomolar Ca²⁺ (n = 3). In contrast to the apparently simple inhibitory effect of CaM in the presence of 100 mM Ca²⁺, suramin exerts a triphasic effect on cardiac RyR gating. Nanomolar concentrations of suramin inhibit [³H]ryanodine binding (25 ± 9 % reduction at 100 nM); micromolar suramin stimulates binding (23 ± 5 % increase at 100 mM suramin) and millimolar suramin inhibits binding (58 ± 4 % reduction at 2 mM suramin) (S.E.M.; n = 4). Western blotting was used to investigate if the two ligands compete for the same binding sites on RyR. SR vesicles were incubated with suramin to investigate if suramin could displace endogenously bound CaM from RyR. SR vesicles were then sedimented and the pellet and supernatant were run on a 10 % acrylamide gel and blotted with anti-CaM antibodies (Upstate Biotech, Bucks). In all cases CaM remained associated with the RyR channel protein, providing no evidence that it could be competitively displaced by suramin.

Our results demonstrate that CaM reduces the P_o of cardiac RyR channels in the presence of 100 mM Ca²⁺, but although suramin can also reduce P_o at certain concentrations we find no compelling evidence to suggest that the two ligands decrease P_o by binding to the same sites on RyR.

This work was supported by the BHF.