DISTINCT MODES OF BLOCKADE IN CARDIAC ATP-SENSITIVE K+ CHANNELS SUGGEST MULTIPLE TARGETS FOR INHIBITORY DRUG MOLECULES

Elementary K+ currents were recorded at 19 degrees C in inside-out pat ches from cultured neonatal rat cardiocytes to elucidate the block phe nomenology in cardiac ATP-sensitive K+ channels when inhibitory drug m olecules, such as the sulfonylurea glibenclamide, the phenylalkylamine verapamil or sulfonamide derivatives (HE 93 and sotalol), are interac ting in an attempt to stress the hypothesis of multiple channel-associ ated drug targets. Similar to their adult relatives, neonatal cardiac K-(ATP) channels are characterized by very individual open state kinet ics, 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 patc hes and tau(open(2)) from 10 to 64 msec-an argument for a heterogeneou s channel population. Nevertheless, a common response to drugs was obs erved. Glibenclamide and the other inhibitory molecules caused long-la sting interruptions of channel activity, after cytoplasmic application , as if drug occupancy trapped cardiac K-(ATP) channels in a very stab le, nonconducting configuration. The resultant NP0 depression was stro ngest with glibenclamide (apparent IC50 13 nmol/liter) and much weaker with verapamil (apparent IC50 9 mu mol/liter), HE 93 (apparent IC50 2 9 mu mol/liter) and sotalol (apparent IC50 43 mu mol/liter) and may ha ve resulted from the occupancy of a single site with drug-specific aff inity or of two sites, the high affinity glibenclamide target and a di stinct nonglibenclamide, low affinity target. Changes in open state ki netics, particularly in the transition between the O-1 state and the O -2 state, are other manifestations of drug occupancy of the channel. A ny inhibitory drug molecule reduced the likelihood of attaining the O- 2 state, consistent with a critical reduction of the forward rate cons tant governing the O-1-O-2 transition. But only HE 93 (10 mu mol/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 predrug value, not the expected result when the entrance in a nd the exit from the O-2 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 acc ommodate more than one drug-binding domain.