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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Disulfonic stilbene permeation and block of the anion channel from the sarcoplasmic reticulum of rabbit skeletal muscle

School of Biomedical Sciences and Hunter Medical Research Institute, Faculty of Health, The University of Newcastle, Callaghan, New South Wales, Australia

Submitted 17 June 2005 ; accepted in final form 16 January 2006

ABSTRACT

Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives [DIDS, dibenzamidostilbene-2,2'-disulfonic acid (DBDS), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)] was investigated in planar bilayers using SO as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced nonreversible channel block from both sides within 1–3 min. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The half inhibitory concentration for cis- and trans-inhibition had voltage dependencies with minima of 190 nM and 33 µM for DBDS and 3.4 and 55 µM for DNDS. Our data supports a permeant blocker mechanism, in which stilbenes block SCl channels by lodging in the permeation pathway, where they may dissociate to either side of the membrane and thus permeate the channel. The stilbenes acted as open channel blockers where the binding of a single molecule occludes the channel. DBDS and DNDS, from opposite sides of the membrane, competed for common sites on the channel. Dissociation rates exhibited biphasic voltage dependence, indicative of two dissociation processes associated with ion movement in opposite directions within the trans-membrane electric field. The kinetics of DNDS and DBDS inhibition predict that there are two stilbene sites in the channel that are separated by 14–24 Å and that the pore constriction is 10 Å in diameter.

4,4'-dinitrostilbene-2,2'-disulfonic acid; dibenzamidostilbene-2,2'-disulfonic acid; permeant blocker