INHIBITION OF SLOW-WAVE REPOLARIZATION AND CA-2+-ACTIVATED K+ CHANNELS BY QUATERNARY AMMONIUM-IONS

We studied the effects of the K+ channel blocker tetrapentylammonium (TPeA) on the electrical activity of intact circular smooth muscle from canine colon. TPeA (10 and 20 muM) increased slow-wave duration and ''locked' the membrane potential around -30 mV plateau potential after several minutes of application, suggesting that K+ channels are essential for termination of colonic slow waves. Repolarization and normal slow-wave activity resumed after 20-30 min of washout. The patch-clamp technique was used to study the block of large-conductance Ca2+-activated K+ channels (BK channels) by TPeA and tetraethylammonium (TEA) in excised and cell-attached patches from isolated colonic smooth muscle cells. Channel block was characterized by a voltage-dependent dissociation constant [K(d)(V)] for the binding of TEA and TPeA to a blocking site located a fraction of the distance across the membrane field (delta). The extracellular TEA binding site had a K(d)(0) of 0.33 mM and a delta of 0.23. The extracellular TPeA binding site had a K(d)(0) of 2.2 mM but showed significantly less voltage dependence (delta = 0.02). The intracellular binding site for TEA was of low affinity [K(d)(0) = 76 mM]. Intracellular TPeA was the most potent blocker of BK channel current [K(d)(0) = 11.7 muM]. The voltage dependence of block by intracellular TPeA (delta = -0.21) was not significantly different from that of intracellular TEA (delta = -0.3). Internal TPeA (10 muM) also blocked a 70-pS K+ channel and a 23-pS K+ channel. In cell-attached patches, TPeA (10 and 100 muM) blocked BK channels within minutes after bath application, indicating that bath-applied TPeA can enter intact cells and block K+ channels by binding to the high-affinity internal site.