Chloride conductance in HT29 cells: investigations with apical membrane vesicles and RT-PCR

Vesicles enriched in a marker enzyme for apical membranes were isolated fro m HT29 cells. These vesicles contain an anion conductance with the selectiv ity gluconate approximate to sulphate<F-<Cl-<Br-<NO3-<I-<SCN-. K+ diffusion potential-driven Cl-36(-) uptake was inhibited by 5-nitro-2-(3-phenylpropy lamino)benzoate (NPPB)>4,4'-diisothiocyanatostilbene-2,2'-disulphonate (DID S)>glibenclamide. The Cl- conductance was insensitive to Ca2+ and to extrav esicular cyclic adenosine monophosphate (cAMP). cyclic guanosine monophosph ate (cGMP) and inosine 5'-triphosphate (ITP). Using the reverse transcripti on polymerase chain reaction (RT-PCR) technique and sequencing of the ampli fied products we detected messenger ribonucleic acid (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR). the putative Cl- chann el or Cl- channel regulator pI(Cln), and the Cl- channels ClC-2, ClC-3, ClC -5 and ClC-6 in HT29 cells. The properties of the vesicles' Cl- conductance resemble those of the intermediate conductance outwardly rectifying Cl- ch annel and tentatively exclude contributions of CFTR. pI(Cln) and ClC-2. Whe ther ClC-3, ClC-5, ClC-6 are involved in Cl- conductance remains to be dete rmined.