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Correspondence to Tsung-Yu Chen: tycchen{at}ucdavis.edu
Intracellularly applied amphiphilic molecules, such as p-chlorophenoxy acetate (CPA) and octanoate, block various pore-open mutants of CLC-0. The voltage-dependent block of a particular pore-open mutant, E166G, was found to be multiphasic. In symmetrical 140 mM Cl–, the apparent affinity of the blocker in this mutant increased with a negative membrane potential but, paradoxically, decreased when the negative membrane potential was greater than –80 mV, a phenomenon similar to the blocker "punch-through" shown in many blocker studies of cation channels. To provide further evidence of the punch-through of CPA and octanoate, we studied the dissociation rate of the blocker from the pore by measuring the time constant of relief from the block under various voltage and ionic conditions. Consistent with the voltage dependence of the effect on the steady-state current, the rate of CPA dissociation from the E166G pore reached a minimum at –80 mV in symmetrical 140 mM Cl–, and the direction of current recovery suggested that the bound CPA in the pore can dissociate into both intracellular and extracellular solutions. Moreover, the CPA dissociation depends upon the Cl– reversal potential with a minimal dissociation rate at a voltage 80 mV more negative than the Cl– reversal potential. That the shift of the CPA-dissociation rate follows the Cl– gradient across the membrane argues that these blockers can indeed punch through the channel pore. Furthermore, a minimal CPA-dissociation rate at a voltage 80 mV more negative than the Cl– reversal potential suggests that the outward blocker movement through the CLC-0 pore is more difficult than the inward movement.
© 2009 Zhang and Chen This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).
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