In the presence of high cytosolic Ca2+, calmodulin (CaM) reduces the open probability (Po) of ryanodine receptor channels (RyR) and it has been suggested that CaM binds to the same sites as suramin (Klinger et al. 1999). However, it is not understood how suramin and CaM can act at common sites as suramin has been shown only to increase the Po of RyR (Sitsapesan & Williams, 1996). We have therefore investigated the mechanisms by which CaM and suramin regulate the gating of cardiac ryanodine receptors. Sarcoplasmic reticulum (SR) membrane vesicles were isolated from sheep hearts obtained from an abattoir and were used for [3H] ryanodine binding studies or were incorporated into planar lipid bilayers for single-channel studies as previously described (Kermode et al. 1998).
In the presence of 100 µM cytosolic free Ca2+, CaM dose-dependently inhibited [3H] ryanodine binding to isolated SR (35 ± 3 % reduction at 5 µM CaM (S.E.M.; n = 4)), whereas the effect of suramin was more complex. Nanomolar and millimolar suramin inhibited binding (25 ± 9 and 58 ± 4 % reduction at 100 nM and 2 mM suramin, respectively), while micromolar suramin stimulated binding (23 ± 4.5 % increase with 100 µM suramin (S.E.M.; n = 4)). This trend was reflected in the single-channel experiments and life-time analysis provided an explanation for the mechanisms by which the changes in Po and thus [3H] ryanodine binding occur. Nanomolar [suramin] increased the mean closed life-time duration and to a lesser extent mean open time, whereas higher [suramin] led to further increases in open times but a reduction in closed times. In a typical channel, the mean closed time was 6.55, 24.19 and 2.79 ms for control, 500 nM and 50 µM suramin, respectively, whereas the mean open time was increased from 1.13 ms (control) to 2.65 ms (500 nM suramin) and 4656 ms (50 µM suramin). In contrast, CaM caused only a reduction in Po resulting from an increase in the mean closed time with no effect on open life-time duration.
The triphasic nature of the suramin dose-response suggests that suramin binds to at least three sites on RyR2. CaM, however, causes only a lengthening of closed times, leading to a decrease in Po. These results suggest that CaM either does not bind to all the suramin binding sites or may bind to some sites but have no effect on channel gating.This work was supported by the BHF.
- Kermode, H., Chan, W.M., Williams, A.J. & Sitsapesan, R. (1998). FEBS Lett. 431, 59-62.
Klinger, M., Freissmuth, M., Nickel, P., Stèbler-Schwarzbart, M., Kassack, M., Suko, J. & Hohenegger, M. (1999). Mol. Pharmacol. 55, 462-472.
Sitsapesan, R. & Williams, A.J. (1996). J. Membr. Biol. 153, 93-103.