INTERACTIONS OF A REVERSIBLE RYANOID (21-AMINO-9-ALPHA-HYDROXY-RYANODINE) WITH SINGLE SHEEP CARDIAC RYANODINE RECEPTOR CHANNELS

The binding of ryanodine to a high affinity site on the sarcoplasmic r eticulum 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 f rom its site within the time frame of a single channel experiment. In this report, we describe the interactions of a synthetic ryanoid, 21-a mino-9 alpha-hydroxy-ryanodine, with the high affinity ryanodine bindi ng site on the sheep cardiac sarcoplasmic reticulum Ca2+-release chann el. The interaction of 21-amino-9 alpha-hydroxy-ryanodine with the cha nnel induces the occurrence of a characteristic high open probability, reduced-conductance state; however, in contrast to ryanodine, the int eraction of this ryanoid with the channel is reversible under steady s tate conditions, with dwell times in the modified state lasting second s. By monitoring the reversible interaction of this ryanoid with singl e channels under voltage clamp conditions, we have established a numbe r 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 on ly from the cytosolic side of the channel and the site is available on ly when the channel is open. (c) The interaction of 21-amino-9 alpha-h ydroxy-ryanodine with its binding site is influenced strongly by trans membrane 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 transl ocation of the ryanoid into the voltage drop across the channel.