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 microM 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 Ca(2+)-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 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Kansen
M
M
Department of Cell Biology and Genetics, Medical Faculty, Erasmus University Rotterdam, The Netherlands.
Bajnath
R B
RB
Groot
J A
JA
de Jonge
H R
HR
Scholte
B
B
Hoogeveen
A T
AT
Bijman
J
J
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D018118:Chloride Channels; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms / Q000503:physiopathology*; D000242:Cyclic AMP / Q000494:pharmacology; D004553:Electric Conductivity; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D008565:Membrane Proteins / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D014407:Tumor Cells, Cultured
D018118:Chloride Channels; D008565:Membrane Proteins; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D002118:Calcium