Characteristics of apical chloride channels in human colon cells (HT29)

Abstract

Recent studies have demonstrated that active chloride secretion in mammalian colon and other epithelia, is dependent on the induction of an increase of apical chloride conductance. Since the physical characteristics of apical chloride channels in man have not been elucidated, patch clamp analysis of human colon cells (HT₂₉), in culture, was performed, after stimulation with db-cAMP 10⁻⁴ mol/l. In excised inside out patches of apical membranes two types of channels were found. The smaller and less frequent channel had a mean conductance of 15±1 pS (n=9). This type of channel showed identical I/V curves in NaCl and KCl solutions. It was inhibited by a chloride channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The more frequently observed larger conductance channel was selective for anions and was impermeable to Na⁺ and K⁺. Regarding anion selectivity, the channel was similarly permeable to Cl⁻, Br⁻, I⁻, and NO ⁻₃ , but was impermeable to gluconate. The channel was completely inhibited by the potent Cl⁻ channel blocker NPPB (10⁻⁶ mol/l). This channel exhibited rectification: The conductance was 50±4 pS at positive clamp potentials (sign referred to bath with respect to pipette interior) and 32±3 (n=33) pS at negative voltages. Moreover, the open state probability was doubled when the clamp potential was increased from −20 to +20 mV. These results demonstrate the existence of chloride channels in the apical membrane of db-cAMP treated colonic carcinoma cells.