COMPARISON OF -NITRO VERSUS -AMINO 4,4'-SUBSTITUENTS OF DISULFONIC STILBENES AS CHLORIDE CHANNEL BLOCKERS

We showed previously that the disulfonic stilbene DNDS (4,4'-dinitrost ilben-2,2'-disulfonic acid) was a potent blocker of outwardly rectifyi ng chloride channels (ORCC). The studies reported here were designed t o quantify the relationship between electron withdrawal by the 4,4'-su bstituents and blocker potency. Specifically we compared the blocking effects and molecular properties of the symmetrically substituted 4,4' -diaminostilben-2,2'-disulfonic acid (DADS) and the hemi-substituted 4 -amino,4'-nitrostilben-2,2'-disulfonic acid (ANDS) with those of DNDS. Blockade was studied using outwardly rectifying colonic chloride chan nels incorporated into planar lipid bilayers. DADS was 430-fold and AN DS 44-fold less potent than DNDS as blockers of ORCC. Amplitude distri bution analysis revealed that all three disulfonic stilbenes act as op en channel blockers. Furthermore, this kinetic analysis indicated that the lower potency of DADS and ANDS was due to an increase in off rate . These results support the conclusion that the 4,4'-substituents make an important contribution to blockade by stabilizing the channel-bloc ker complex. Isopotential electron contour maps illustrated the dramat ic shift in charge at the 4,4'poles of the disulfonic stilbene molecul e from electronegative in DNDS to electropositive in DADS as well as t he bipolar contour of ANDS. Thus, the greater potency of DNDS results from the symmetric electronegative regions at the 4,4'-poles of the mo lecule. We hypothesize that the channel protein has two corresponding electropositive areas at the blocker binding site.