Distinct modes of blockade in cardiac ATP-sensitive K+ channels suggest multiple targets for inhibitory drug...

Elementary K+ currents were recorded at 19 degrees C in inside-out patches from cultured neonatal rat cardiocytes to elucidate the block phenomenology in cardiac ATP-sensitive K+ channels when inhibitory drug molecules, such as the sulfonylurea glibenclamide, the phenylalkylamine verapamil or sulfonamide derivatives (HE 93 and sotalol), are interacting in an attempt to stress the hypothesis of multiple channel-associated drug targets. Similar to their adult relatives, neonatal cardiac K(ATP) channels are characterized by very individual open state kinetics, even in cytoplasmically well-controlled, cell-free conditions; at -7 mV, tau open(1) ranged from 0.7 to 4.9 msec in more than 200 patches and tau open(2) from 10 to 64 msec--an argument for a heterogeneous channel population. Nevertheless, a common response to drugs was observed. Glibenclamide and the other inhibitory molecules caused long-lasting interruptions of channel activity, after cytoplasmic application, as if drug occupancy trapped cardiac K(ATP) channels in a very stable, nonconducting configuration. The resultant NPo depression was strongest with glibenclamide (apparent IC50 13 nmol/liter) and much weaker with verapamil (apparent IC50 9 mumol/liter), HE 93 (apparent IC50 29 mumol/liter) and sotalol (apparent IC50 43 mumol/liter) and may have resulted from the occupancy of a single site with drug-specific affinity or of two sites, the high affinity glibenclamide target and a distinct nonglibenclamide, low affinity target. Changes in open state kinetics, particularly in the transition between the O1 state and the O2 state, are other manifestations of drug occupancy of the channel. Any inhibitory drug molecule reduced the likelihood of attaining the O2 state, consistent with a critical reduction of the forward rate constant governing the O1-O2 transition. But only HE 93 (10 mumol/liter) associated (with an apparent association rate constant of 2.3 x 10(6) mol-1 sec-1) to shorten significantly tau open(2) to 60.6 +/- 6% of the pre-drug value, not the expected result when the entrance in and the exit from the O2 state would be drug-unspecifically influenced. Sotalol found yet another and definitely distinctly located binding site to interfere with K+ permeation; both enantiomers associated with a rate close to 5 x 10(5) mol-1 sec-1 with the open pore thereby flicker-blocking cardiac K(ATP) channels. Clearly, these channels accommodate more than one drug-binding domain.