title: Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia.
abstract: 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 Cl- channel with respect to their inhibitory potency. We applied the blockers to excised inside-out-or outside-out-oriented membrane patches of cultured HT29 colon carcinoma and respiratory epithelial cells (REC) containing the outwardly rectifying intermediate-conductance (ICOR) chloride channel. Four types of inhibitory compounds were tested: stilbene disulphonate derivatives, indanyloxyacetic acid, amidine, and arylaminobenzoates. The concentrations for half-maximal inhibition (IC50) for the different channel blockers were (mumol/l): 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid 100; 4,4'-diisothiocyanato-stilbene-2,2'-disulphonic 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 Cl- 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 mumol/l 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 amino-ethyl-O-dextran (5 kDa).2+ These macromolecular NPPB and NPEB derivatives inhibited the ICOR Cl- channels only from the outside but had no effect on the cytosolic side.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Physiologisches Institut, Albert-Ludwigs-Universität Freiburg, Federal Republic of Germany.
authors.forename: M
authors.initials: M
authors.lastname: Tilmann
authors.forename: K
authors.initials: K
authors.lastname: Kunzelmann
authors.forename: U
authors.initials: U
authors.lastname: Fröbe
authors.forename: I
authors.initials: I
authors.lastname: Cabantchik
authors.forename: H J
authors.initials: HJ
authors.lastname: Lang
authors.forename: H C
authors.initials: HC
authors.lastname: Englert
authors.forename: R
authors.initials: R
authors.lastname: Greger
mesh_terms: D000578:Amidines / Q000494:pharmacology; D002478:Cells, Cultured; D018118:Chloride Channels; D004553:Electric Conductivity; D004848:Epithelium / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007189:Indans / Q000494:pharmacology; D008565:Membrane Proteins / Q000187:drug effects* / Q000378:metabolism; D015335:Molecular Probes; D009579:Nitrobenzoates / Q000494:pharmacology; D013267:Stilbenes / Q000494:pharmacology
chemical_list: D000578:Amidines; D018118:Chloride Channels; D007189:Indans; D008565:Membrane Proteins; D015335:Molecular Probes; D009579:Nitrobenzoates; D013267:Stilbenes; C060056:indanoxyacetic acid 94; C058176:5-nitro-2-(3-phenylpropylamino)benzoic acid