Regulation of I(Cl,swell) in neuroblastoma cells by G protein signaling pathways.
Guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) activated the I(Cl,swell) anion channel in N1E115 neuroblastoma cells in a swelling-independent manner. GTPgammaS-induced current was unaffected by ATP removal and broadly selective tyrosine kinase inhibitors, demonstrating that phosphorylation events do not regulate G protein-dependent channel activation. Pertussis toxin had no effect on GTPgammaS-induced current. However, cholera toxin inhibited the current approximately 70%. Exposure of cells to 8-bromoadenosine 3',5'-cyclic monophosphate did not mimic the effect of cholera toxin, and its inhibitory action was not prevented by treatment of cells with an inhibitor of adenylyl cyclase. These results demonstrate that GTPgammaS does not act through Galpha(i/o) GTPases and that Galpha(s)/Gbetagamma G proteins inhibit the channel and/or channel regulatory mechanisms through cAMP-independent mechanisms. Swelling-induced activation of I(Cl,swell) was stimulated two- to threefold by GTPgammaS and inhibited by 10 mM guanosine 5'-O-(2-thiodiphosphate). The Rho GTPase inhibitor Clostridium difficile toxin B inhibited both GTPgammaS- and swelling-induced activation of I(Cl,swell). Taken together, these findings indicate that Rho GTPase signaling pathways regulate the I(Cl,swell) channel via phosphorylation-independent mechanisms.
Estevez
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Department of Anesthesiology, Anesthesiology Research Division, Laboratories of Cellular and Molecular Physiology, Vanderbilt University Medical Center, Nashville, Tennessee 37232, USA.
Bond
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Strange
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000838:Anions / Q000378:metabolism; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D018118:Chloride Channels / Q000378:metabolism*; D002772:Cholera Toxin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology*; D019833:Genistein / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006153:Guanosine Diphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D051379:Mice; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010766:Phosphorylation; D015290:Second Messenger Systems / Q000187:drug effects / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D020032:Tyrphostins / Q000494:pharmacology; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D000838:Anions; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D018118:Chloride Channels; D004791:Enzyme Inhibitors; D013873:Thionucleotides; D020032:Tyrphostins; C031815:toxB protein, Clostridium difficile; C114975:tyrphostin A51; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C023427:guanosine 5'-O-(2-thiodiphosphate); D000255:Adenosine Triphosphate; D002772:Cholera Toxin; D019833:Genistein; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins