The binding of ryanodine to a high affinity site on the sarcoplasmic reticulum Ca2+-release channel results in a dramatic alteration in both gating and ion handling; the channel enters a high open probability, reduced-conductance state. Once bound, ryanodine does not dissociate from its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-amino-9α-hydroxy-ryanodine, with the high affinity ryanodine binding site on the sheep cardiac sarcoplasmic reticulum Ca2+-release channel. The interaction of 21-amino-9α-hydroxy-ryanodine with the channel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the interaction of this ryanoid with the channel is reversible under steady state conditions, with dwell times in the modified state lasting seconds. By monitoring the reversible interaction of this ryanoid with single channels under voltage clamp conditions, we have established a number of novel features of the ryanoid binding reaction. (a) Modification of channel function occurs when a single molecule of ryanoid binds to the channel protein. (b) The ryanoid has access to its binding site only from the cytosolic side of the channel and the site is available only when the channel is open. (c) The interaction of 21-amino-9α-hydroxy-ryanodine with its binding site is influenced strongly by transmembrane voltage. We suggest that this voltage dependence is derived from a voltage-driven conformational alteration of the channel protein that changes the affinity of the binding site, rather than the translocation of the ryanoid into the voltage drop across the channel.

provide a useful tool in the study of multiple subtypes of P2-purinoceptors. Furthermore the results are consistent with the hypothesis that ATP interacts with both receptor subtypes, but that the action of ADP is primarily at the P2Y-purinoceptor in these endothelial cells.

In an earlier investigation, we demonstrated that the likelihood of interaction of a positively charged ryanoid, 21-amino-9α-hydroxyryanodine, with the sarcoplasmic reticulum Ca2+-release channel (ryanodine receptor, RyR) is dependent on holding potential (Tanna, B., W. Welch, L. Ruest, J.L. Sutko, and A.J. Williams. 1998. J. Gen. Physiol. 112:55–69) and suggested that voltage dependence could result from either the translocation of the charged ligand to a site within the voltage drop across the channel or a voltage-driven alteration in receptor affinity. We now report experiments that allow us to assess the validity of these alternate mechanisms. Ryanodol is a neutral ryanoid that binds to RyR and induces modification of channel function. By determining the influence of transmembrane potential on the probability of channel modification by ryanodol and the rate constants of ryanodol association and dissociation, we demonstrate that the influence of voltage is qualitatively the same for both the neutral and positively charged ryanoids. These experiments establish that most, if not all, of the modification of ryanoid interaction with RyR by transmembrane holding potential results from a voltage-driven alteration in receptor affinity.