Regulation of chloride channels in the human colon carcinoma cell line HT29.cl19A

Chloride (Cl-) channels are important in the regulation of salt and water transport in secretory epithelial cells. A disturbed Cl- secretion is the most consistent characteristic in the genetic disease cystic fibrosis. An outwardly rectifying Cl- channel (OR) with a conductance of 25-50 pS had been proposed to play a major role in Cl- secretion. Activation by Ca2+ and the protein kinases (PK) A and C (at less than 10 nM Ca2+) as well as inhibition by PKC (at 1 μM Ca2+) has been reported. In the present study, we have identified and characterized the OR in HT29.cl19A human colon carcinoma cells. The OR displayed a conductance of 31±4 pS (n=25). Its open probability in 10 nM Ca2+ was voltage-dependent in 50% of the patches, starting from 0.2 at -70 mV to 0.8 at 70 mV. The spontaneous activation in excised inside-out patches at -60 mV was Ca2+-dependent and decreased from 29% in 1 mM Ca2+ to 2% in 10 nM Ca2+. Active OR were found in (a) 25% of patches exposed to 10 nM Ca2+, ATP and cAMP only, (b) 42% of the patches exposed to 10 nM Ca2+, ATP and the catalytic subunit of PKA (CAK) and (c) 67% of the patches exposed to 1 mM Ca2+, ATP plus CAK. Inhibition of voltage-activated channels by addition of PKC in 1 μM or 1 mM Ca2+ was not observed. Attempts to activate the OR in cell-attached patches by increasing cAMP levels under different experimental conditions were unsuccessful. Our data suggest that the OR may not be as important in Cl- secretion as has been thought.

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