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Calmodulin regulation of the sheep cardiac ryanodine receptor is complex, involving modifications to both gating and conductance

It is generally accepted that Ca2+-bound calmodulin (Ca2+CaM) reduces the open probability (Po) of the cardiac ryanodine receptor (RyR2) channel whereas non Ca2+-bound CaM (apoCaM) either does not influence channel function or slightly reduces Po (1-3). However, this impression of how CaM regulates RyR2 function is predominantly drawn from [3H]ryanodine binding studies which can only provide a very approximate assessment of changes in RyR2 gating behaviour and cannot inform about changes in single-channel conductance. We have therefore investigated how Ca2+CaM and apoCaM modulate the single-channel properties of RyR2. As previously described (1), sarcoplasmic reticulum (SR) membrane vesicles were isolated from sheep hearts obtained from an abattoir and were either incorporated into artificial membranes for single-channel experiments or were used for [3H]ryanodine binding. In the presence of 100 μM cytosolic free Ca2+, CaM produced a concentration-dependent inhibition of [3H]ryanodine binding to SR vesicles (maximum inhibition was 33.67 ± 4.34% of control, SEM, n = 8), consistent with previous work (1). In contrast, mean data from the single-channel experiments, indicated that, in the presence of 100 μM cytosolic free Ca2+, CaM did not significantly alter Po; Po was 0.32±0.06 before and 0.34±0.09 after the cytosolic addition of 1 μM CaM (SEM, n=16). Closer inspection of the data, however, revealed that CaM produced two different effects on channel gating. CaM reduced Po (from 0.379 ± 0.149 to 0.118 ± 0.09 (SEM, n = 6, P<0.05, Student’s t test)) in 37% of the experiments but increased Po (from 0.292 ± 0.056 to 0.508 ± 0.103 (SEM, n = 10, P<0.05, Student’s t test)) in 63% of experiments. Under these experimental conditions, at a holding potential of 0 mV, CaM had no effect on single-channel current amplitude. After binding Ca2+CaM to RyR2 incorporated into bilayers by incubation with 1 μM CaM and 100 μM Ca2+, we then reduced the cytosolic free [Ca2+] to a sub-activating level (<10 nM Ca2+) to convert Ca2+CaM to apoCaM. At this free [Ca2+], the Po of the sheep RyR2, without pre-incubation with Ca2+CaM, is zero. However, after pre-incubation with Ca2+CaM, Po was 0.059 ± 0.042 (SEM, n = 10). Importantly, the observed channel open events were shown to be of significantly higher current amplitude (5.163 ± 0.101 pA) than control channel events (4.555 ± 0.057 pA (SEM, n = 10, P<0.001, Student’s t test). The data suggest that apoCaM can increase the Po and conductance of RyR2 but only if it is first pre-bound to the channel as Ca2+CaM. The results of this study indicate that CaM may bind tightly to RyR2 and influence channel function in a complex manner throughout the cycle of high and low cytosolic [Ca2+] that occurs during cardiac excitation-contraction coupling. Hill AP,Kingston O & Sitsapesan R (2004).Mol Pharmacol 65,1258-1268.