Bis-quaternary ammonium blockers as structural probes of the sarcoplasmic reticulum K+ channel.

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Abstract
A series of n-alkyl-bis-.alpha.,.omega.-trimethylammonium (bisQn) compounds was synthesized, and their ability to block K+ currents through a K+ channel from sarcoplasmic reticulum was studied. K+ channels were inserted into planar phospholipid membranes, and single-channel K+ currents were measured in the presence of the blocking cations. These bisQn compounds block K+ currents only from the side of the membrane opposite to the addition of sarcoplasmic reticulum vesicles (the trans side). The block is dependent on transmembrane voltage, and the effective valence of the block (a measure of this voltage dependence) varies with the methylene chain length. For short chains (bisQ2-bisQ5), the effective valence decreases with chain length from 1.1 to 0.65; it then remains constant and .apprx. 0.65 for bisQ5 to bisQ8; the effective valence abruptly increases to 1.2-1.3 for chains of .gtoreq. 9 carbons. For the compounds of .gtoreq. 9 carbons, the discrete nature of the block can be observed directly as flickering noise on the open channel. The kinetics of the block were studied for these long chain blockers. Both blocking and unblocking rates of the blockers vary with chain length, with the blocking rate showing the strongest variation: an increase of 2.8-fold/added methylene group. All of the voltage dependence of the binding equilibrium resides in the blocking rate and none in the unblocking rate. Apparently, 65% of the voltage drop within the channel occurs over a distance of 6-7 .ANG., and the short chain blockers bind in an extended-chain conformation, whereas the long chain blockers may bind in a bent-over conformation with both charges deeply inside the channel.