The Journal of General Physiology, Vol 79, 869-891, Copyright © 1982 by The Rockefeller University Press
Bis-quaternary ammonium blockers as structural probes of the sarcoplasmic reticulum K+ channel
C Miller
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 SR
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 at approximately 0.65 for bisQ5 to bisQ8;
the effective valence abruptly increases to 1.2-1.3 for chains of nine
carbons and longer. For the compounds of nine carbons and longer, 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 per added methylene group. All of the
voltage dependence of the binding equilibrium resides in the blocking rate,
and none in the unblocking rate. The results imply that 65% of the voltage
drop within the channel occurs over a distance of 6-7A, and that the
short-chain blockers bind in a bent-over conformation with both charges
deeply inside the channel.
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