Blockade of swelling-induced chloride channels by phenol derivatives

1 In NIH3T3 fibroblasts, the chloride channel involved in regulatory volume decrease (RVD) was identified as I-Cln a protein isolated from a cDNA library derived from Madin Darby canine kidney (MDCK) cells. I-Cln expressed in Xenopus laevis oocytes gives rise to an outwardly rectifying chloride current, sensitive to the extracellular addition of nucleotides and the known chloride channel blockers, DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid) and NPPB (5-nitro-2-(3-phenylpropylamino)benzoic acid). We set out to study whether substances structurally similar to NPPB are able to interfere with RVD. 2 RVD in NIH3T3 fibroblasts and MDCK cells is temperature-dependent. 3 RVD, the swelling-dependent chloride current and the depolarization seen after reducing extracellular osmolarity can be blocked by gossypol and NDGA (nordihydroguaiaretic acid), both structurally related to NPPB. 4 The cyclic AMP-dependent chloride current elicited in CaCo cells is less sensitive to the two substances tested while the calcium-activated chloride current in fibroblasts is insensitive. 5 The binding site for the two phenol derivatives onto I-Cln seems to be distinct but closely related to the nucleotide binding site identified as G x G x G, a glycine repeat located at the predicted outer mouth of the I-Cln channel protein.