Epithelial chloride channels can be blocked by various inhibitors, which show considerable differences in their molecular structure. In the present patch-clamp study, we compared different blockers of one type of epithelial C1-channel with respect to their inhibitory potency. We applied the blockers to excised inside-outor outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediateconductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (ICso) for the different channel blockers were (gmol/1):
4-acetamido-4'-isothiocyanato-stilbene-2,2'disulphonic acid 100; 4,4'-diisothiocyanato-stilbene-2,Tdisulphonic acid 80; indanyloxyacetic acid 9; 4,4'-dinitrostilbene-2,2'-disulphonic acid 8; amidine 8 and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) 0.9. All compounds, when applied to the cytosolic side of the channel, induced a flicker-type block of the ICOR C1-channel at lower concentrations and a complete channel inhibition at higher concentrations. The inhibitory potency of NPPB was much higher when it was added to the external surface of the channel in outside-out-oriented membrane patches. At 1 tamol/1 the inhibition was complete. All blocker effects were fully reversible. The probe with the highest affinity (NPPB) and a closely related compound 5-nitro-2-(3-phenylethylamino)-benzoate (NPEB) were used to construct macromolecular probes by linking these blockers to aminopolyethyleneglycol (PEG) or aminoethyl-O-dextran (5 kDa). These macromolecular NPPB and NPEB derivatives inhibited the ICOR CI-channels only from the outside but had no effect on the cytosolic side. In the case of PEG-NPPB an ICso of 30 nmol/1 was determined in outside-out patches. The data indicate that the interaction site for arylaminobenzoates is accessible from the outer aspects of the C1-channel facing the Offprint requests to: R. Greger extracellular medium. Furthermore, these data show that the macromolecular probes of arylaminobenzoates have affinities to the CI-channel very similar to those of the respective parent compounds.