Multiple actions of pinacidil on adenosine triphosphate-sensitive potassium channels in guinea-pig ventricular myocytes

Abstract

1. The patch-clamp method was used to study the effects of pinacidil on the adenosine 5'-triphosphate (ATP)-sensitive K+ channel current in guinea-pig ventricular myocytes. 2. In the inside-out configuration of the patch membranes, the channel activity revealed a nearly fully open state in the absence of ATP, whereas application of ATP (0.1-5 mM) markedly suppressed the channel opening. Addition of pinacidil (0.02-1.0 mM) antagonized the inhibitory action of ATP and induced channel opening without marked change in conductance. An increase in ATP concentration depressed the maximal effect of pinacidil. Consequently, the dose-response relationship of ATP inhibition was shifted to the right, but the shift approached a limiting value as pinacidil concentration was increased. The results indicate that the antagonism between pinacidil and ATP is not competitive. 3. The dose-response curve for activation of the channel by pinacidil examined at -50 mV showed a sigmoidal shape but at +50 mV it had a convex shape, revealing asymmetry in the activating effects of pinacidil at these two voltages. 4. In the absence of ATP, pinacidil produced a voltage-dependent block at positive voltages by decreasing the mean open time and increasing the mean closed time, whereas no such effects were observed at negative voltages. The concentration-block relation at a given voltage was fitted to a first-order Hill saturation function. The Kd (dissociation constant) decreased with depolarization from 2.2 mM at +20 mV to 0.15 mM at + 80 mV. 5. The kinetics of block and unblock by pinacidil were shown to be slow, and were expressed by a first-order transition model. The blocking and unblocking rate constants were voltage dependent. 6. The slow block of single-channel current showed an exponential decay in the ensemble current. The time constant of the decay was voltage dependent, reaching a maximal value at around +50 mV. 7. In the absence of ATP, the channel activity gradually decreased and eventually stopped within 12-20 min, a process known as run-down of channel activity. Calcium accelerated this run-down process. Application of pinacidil partially reactivated the channel. Such channel reactivation by pinacidil during the course of run-down depended upon the conditions of the patch and the time course of the run-down. Pretreatment of the channel with ATP markedly strengthened the reactivation effect of pinacidil. 8. These results indicate that there are multiple sites or processes for interaction of pinacidil with the ATP-sensitive K+ channel.