I. Benz et M. Kohlhardt, DISTINCT MODES OF BLOCKADE IN CARDIAC ATP-SENSITIVE K+ CHANNELS SUGGEST MULTIPLE TARGETS FOR INHIBITORY DRUG MOLECULES, The Journal of membrane biology, 142(3), 1994, pp. 309-322
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.