G-proteins mediate intestinal chloride channel activation

The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carc...

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Bibliographic Details
Published in:	The Journal of biological chemistry 1991-02, Vol.266 (4), p.2036-2040
Main Authors:	TILLY, B. C, KANSEN, M, VAN GAGELDONK, P. G. M, VAN DEN BERGHE, N, GALJAARD, H, BIJMAN, J, DE JONGE, H. R
Format:	Article
Language:	eng
Subjects:	Animals Biological and medical sciences Cell Membrane - metabolism Cell physiology chloride Chloride Channels Chlorides - metabolism Colonic Neoplasms Epithelium - metabolism Fundamental and applied biological sciences. Psychology GTP-Binding Proteins - metabolism guanine nucleotide-binding protein Guanosine 5'-O-(3-Thiotriphosphate) - metabolism Guanosine 5'-O-(3-Thiotriphosphate) - pharmacology Humans Intestinal Mucosa - metabolism Ion Channels - metabolism Kinetics Membrane and intracellular transports Membrane Proteins - metabolism Molecular and cellular biology Potassium Channels - metabolism Rats Tumor Cells, Cultured Valinomycin - pharmacology
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Summary:	The localization of several GTP-binding regulatory proteins in teh apical membrane of intestinal epithelial cells has prompted us to investigate a possible role for G-proteins as modulators of apical Cl- channels. In membrane vesicles isolated from rat small intestine or human HT29-cl.19A colon carcinoma cells, the entrapment of guanosine 5'-O-(3-thiophosphate (GTP gamma S) led to a large increase in Cl- conductance, as evidenced by an increased 125I- uptake and faster SPQ quenching. The enhancement was observed in the presence, but not in the absence of the K+ ionophore valinomycin, indicating that the increased Cl- permeability is not secondary to the opening of K+ channels. The effect of GTP gamma S was counteracted by guanosine 5'-O-(2-thiophosphate (GDP beta S) and appeared to be independent of cytosolic messengers, including ATP, cAMP, and Ca2+, suggesting that protein phosphorylation and/or phospholipase C activation is not involved. Patch clamp analysis of apical membrane patches of HT29-cl.19A colonocytes revealed a GTP gamma S-activated, inwardly rectifying, anion-selective channel with a unitary conductance of 20 +/- 4 pS. No spontaneous channel openings were observed in the absence of GTP gamma S, while the open time probability (Po) increases dramatically to 0.81 +/- 0.09 upon addition with GTP gamma S. Since the electrophysiological characteristics and regulatory properties of this channel are markedly different from those of the more widely studied cAMP/protein kinase A-operated channel, we propose the existence of a separate Cl(-)-selective ion channel in the apical border of intestinal epithelial cells. Our results suggest an alternative regulatory pathway in transepithelial salt transport and a possible site for anomalous channel regulation as observed in cystic fibrosis patients.
ISSN:	0021-9258 1083-351X