Stilbene disulfonate blockade of colonic secretory Cl- channels in planar lipid bilayers.
We studied blockade by 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) of a secretory Cl- channel from colonic enterocyte plasma membrane vesicles incorporated into planar lipid bilayer membranes. Except for intermittent long-lived closed periods (100 ms to several min), the control channel open probability (Po) was greater than 90%. DNDS, added to the cis or vesicle-containing side, which corresponds to the outer membrane side of the channel, caused a dramatic increase in the number of current transitions from the open-to-closed state. DNDS caused a concentration-dependent decrease in Po with a maximum inhibition of 95 +/- 2.0% and a half-maximal inhibitory concentration of 3.3 +/- 1.4 microM. DNDS added to the trans side of the channel had no effect on either the single-channel conductance or kinetic behavior of the channel. Kinetic analysis revealed that DNDS blockade from the cis side could be explained by a linear, closed-open-blocked, kinetic scheme. The estimated DNDS block rate constants were kon = 3.2 X 10(7) M-1.s-1 and koff = 52 s-1, yielding an equilibrium dissociation constant (KD) of 2.1 +/- 0.38 microM, similar to the Ki for inhibition of Po. The effects of DNDS were fully reversible after perfusion of the cis compartment with DNDS-free solution. In contrast, the covalently reactive 4,4'-diisothiocyano-substituted stilbene disulfonate caused an irreversible blockade of the Cl- channel.
Bridges
R J
RJ
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Worrell
R T
RT
Frizzell
R A
RA
Benos
D J
DJ
D000818:Animals; D018118:Chloride Channels; D002712:Chlorides / Q000502:physiology*; D003106:Colon / Q000187:drug effects / Q000502:physiology*; D004553:Electric Conductivity; D004848:Epithelium / Q000502:physiology; D005260:Female; D007700:Kinetics; D008051:Lipid Bilayers / Q000378:metabolism*; D008565:Membrane Proteins / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D013267:Stilbenes / Q000494:pharmacology*
D018118:Chloride Channels; D002712:Chlorides; D008051:Lipid Bilayers; D008565:Membrane Proteins; D013267:Stilbenes; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid