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 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.
Department of Biochemistry, Medical Faculty, Erasmus University, Rotterdam, The Netherlands.
B C
BC
Tilly
M
M
Kansen
P G
PG
van Gageldonk
N
N
van den Berghe
H
H
Galjaard
J
J
Bijman
H R
HR
de Jonge
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D003110:Colonic Neoplasms; D004848:Epithelium / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D015221:Potassium Channels / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured; D014634:Valinomycin / Q000494:pharmacology
D018118:Chloride Channels; D002712:Chlorides; D007473:Ion Channels; D008565:Membrane Proteins; D015221:Potassium Channels; D014634:Valinomycin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins