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

 Vesicles enriched in a marker enzyme for apical membranes were isolated from HT29 cells. These vesicles contain an anion conductance with the selectivity gluconate ≈ sulphate<F–<Cl–<Br–<NO3–<I–<SCN–. K+ diffusion potential-driven 36Cl– uptake was inhibited by 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB)>4,4′-diisothiocyanatostilbene-2,2′-disulphonate (DIDS)>glibenclamide. The Cl– conductance was insensitive to Ca2+ and to extravesicular cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP) and inosine 5′-triphosphate (ITP). Using the reverse transcription polymerase chain reaction (RT-PCR) technique and sequencing of the amplified products we detected messenger ribonucleic acid (mRNA) for the cystic fibrosis transmembrane conductance regulator (CFTR), the putative Cl– channel or Cl– channel regulator pICln, 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– channel and tentatively exclude contributions of CFTR, pICln and ClC-2. Whether ClC-3, ClC-5, ClC-6 are involved in Cl– conductance remains to be determined.