Submitted on June 17, 2005
Accepted on January 16, 2006
Disulfonic stilbene permeation and block of the anion channel from the sarcoplasmic reticulum of rabbit skeletal muscle
Derek R Laver1* and Katherine M Bradley1
1 Biomedical Science, University of Newcastle, Calaghan, NSW, Australia; Cardiovacular group, Hunter Mdeical Research Institute, Newcastle, NSW, Australia
* To whom correspondence should be addressed. E-mail: derek.laver{at}newcastle.edu.au.
Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives (DIDS, DBDS & DNDS) was investigated in planar bilayers using SO42- as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced non-reversible channel block from both sides within 1-3 minutes. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The Ki's for cis and trans inhibition had voltage dependencies with minima of 190 nM and 33 µM for DBDS and 3.4 µM 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, compete for common sites on the channel. Dissociation rates exhibited a 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 1.4-2.4 nM and that the pore constriction is ~ 1 nM in diameter.
