Activation of the sheep cardiac sarcoplasmic reticulum Ca 2+ ‐release channel by analogues of sulmazole

1 The effect of sulmazole and several structurally related analogues on cardiac sarcoplasmic reticulum (SR) Ca 2+ ‐release channel gating and on [ 3 H]‐ryanodine binding to isolated SR membrane vesicles has been investigated. 2 The optical isomers, (+)‐ and (–)‐sulmazole, increased the open probability ( P o ) of single Ca 2+ ‐release channels incorporated into phospholipid bilayers held under voltage clamp by increasing the frequency and duration of open events. The respective EC 50 s were 423 μ m and 465 μ m at 10 μ m activating cytosolic Ca 2+ and the Hill coefficients for activation were approximately two, suggesting that at least two molecules of either enantiomer are required to bind for channel activation. 3 Similarly the related enantiomers, (+)‐ and (–)‐isomazole, which differ from sulmazole in the position of the pyridine nitrogen (4,5b for sulmazole; 4,5c for isomazole), were approximately as potent as each other and as potent as the isomers of sulmazole with EC 50 s of approximately 445 μ m . 4 In contrast, EMD 46512 and EMD 41000, which are sulmazole and isomazole analogues respectively, each with the methylsulphinyl oxygen removed, increased single‐channel P o with EC 50 s of 42 μ m and 40 μ m . The open and closed lifetime distributions were similar to those of the less potent analogues and the Hill coefficients were the same, suggesting that these compounds act at the sulmazole site on the Ca 2+ ‐release channel. 5 All of the compounds tested were able to increase the P o of channels in the absence of activating Ca 2+ but were less potent than in the presence of Ca 2+ . The drugs were effective only when added to the cytosolic face of the channel. None of the drugs could fully activate the channel in the absence of Ca 2+ , partly due to only one drug molecule binding in the absence of Ca 2+ , which is in contrast to the situation when activating Ca 2+ is present. This suggests a synergistic action of these drugs and Ca 2+ in Ca 2+ ‐release channel activation. 6 EMD 46512 and EMD 41000 increased [ 3 H]‐ryanodine binding to HSR vesicles with Hill coefficients of approximately two and EC 50 s of 25 μ m and 28 μ m , respectively, at 10 μ m Ca 2+ . These drugs also increased [ 3 H]‐ryanodine binding to HSR vesicles at p m Ca 2+ but with Hill slopes of only one and EC 50 s of 112 and 133 μ m for EMD 46152 and EMD 41000, respectively. In addition, maximal binding was reduced at p m Ca 2+ in comparison to 10 μ m Ca 2+ . 7 These data show that analogues of sulmazole increase the P o of the cardiac SR Ca 2+ ‐release channel and this occurs as the result of an increase in the frequency and duration of open events. They also demonstrate that the activation of the channel by these drugs is not stereoselective and therefore the configuration of the oxygen atom or methyl group attached to the sulphur atom does not affect their ability to elicit their effect. Similarly, the results show that the nitrogen in the 4, 5b or 4, 5c position of the pyridine ring does not affect Ca 2+ ‐dependent or Ca 2+ ‐independent activation of the Ca 2+ ‐release channel. However, removal of the methylsulphinyl oxygen in sulmazole and isomazole results in two drugs which display a ten fold increase in potency over their respective parent compound in the activation of the Ca 2+ ‐release channel. It is apparent that minor modifications of the sulmazole or isomazole molecules around the terminal sulphur atom dramatically affect potency but not maximal attainable effect, suggesting that the area around the sulphur atom may be critically involved in channel activation.