Multiple blocking mechanisms of ATP‐sensitive potassium channels of frog skeletal muscle by tetraethylammonium ions.

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Abstract
Patch-clamp methods were used to study the action of tetracethylammonium ions (TEA+) and other quaternary ammonium ions in adenosine-5''-triphosphate (ATP)-sensitive K+ channels in sarcolemmal vesicles from frog skeletal muscle. The blocking ions were applied either to ht e external or the internal surface of the membrane patch. External TEA+ caused a very fast block, so that the amplitude of single-channel current was reduced. Open-channel variance was decreased. The block was 1:1, with a dissociation constant (Kd) of 6-7 mM. We could detect no voltage dependence of Kds. External TEA+ prolonged open times in manner consistent with the channel being unable to close when blocked by TEA+. TEA+ also blocked when applied to the internal side of the membrane. This block showed two components with different kinetics and different affinities. The slow block chopped up openings into much briefer events and had a Kd of about 1.cntdot.4 mM at .sbd.3 mV. The fast block reduced the amplitude of unitary currents and was of lower affinity, with Kd around 26 mM. The slow block by internal TEA+ was markedly voltage dependent, the Kd decreasing e-fold for a 37 mV depolarization. Both the association and dissocation rates were dependent on voltage. In contrast, the fast block by internal TEA+ appeared virtually independent of voltage. The effects of internally applied tetramethylammonium (TMA+) and tetrapentylammonium (TPA+) ions were also investigated. Internal TMA+ produced a flickery block while the block by internal TPA+ was similar to that caused by TEA+, although TPA+ was about 10 times more effective. Our results suggest that the channel has three binding sites for TEA+, one of which is accessible from the outside of the membrane. Only one of the internal sites is located so as to experience a substantial fraction of the membrane voltage field.