title: Regulation of high-conductance anion channels by G proteins and 5-HT1A receptors in CHO cells.
abstract: This study addresses the mechanisms responsible for regulation of high-conductance anion channels by GTP binding proteins in Chinese hamster ovary (CHO) cells. Single-channel currents were measured in inside-out membrane patches using patch-clamp techniques. Anion-selective channels with a unitary conductance of 381 +/- 8 pS activated spontaneously in 48% of excised patches. In patches with no spontaneous channel activity, addition of GppNHp, a nonhydrolyzable analogue of GTP, activated channels in 8 of 12 studies, and in patches with spontaneous channel activity, GppNHp increased open probability in 4 of 4 experiments. In contrast, GDP beta S, a nonhydrolyzable GDP analogue, inhibited both spontaneous and GppNHp-induced channel activity. In patches without spontaneous channel activity, addition of cholera toxin activated channels in five of eight studies. Interestingly, pertussis toxin had a similar effect, activating channels in five of seven previously quiescent patches. To further evaluate the possible role of inhibitory G proteins in channel regulation, activity was measured in cell-attached patches in cells transfected with the serotonin 5-HT1A receptor, which is coupled to effector mechanisms through a pertussis toxin-sensitive G protein. Stimulation of 5-HT1A-transfected cells with the receptor agonist (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin caused a transient decrease in open probability in either standard or high-potassium solutions. In aggregate, these findings suggest that both cholera and pertussis toxin-sensitive G proteins contribute to regulation of high-conductance anion channels in CHO cells.
authors.affiliation: Division of Gastroenterology, Duke University Medical Center, Durham, North Carolina.
authors.forename: A W
authors.initials: AW
authors.lastname: Mangel
authors.forename: J R
authors.initials: JR
authors.lastname: Raymond
authors.forename: J G
authors.initials: JG
authors.lastname: Fitz
mesh_terms: D017371:8-Hydroxy-2-(di-n-propylamino)tetralin / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D016466:CHO Cells / Q000187:drug effects / Q000502:physiology*; D002118:Calcium / Q000494:pharmacology; D002772:Cholera Toxin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004553:Electric Conductivity; D004594:Electrophysiology; D019204:GTP-Binding Proteins / Q000502:physiology*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D007473:Ion Channels / Q000502:physiology*; D009243:NAD / Q000494:pharmacology; D037342:Pertussis Toxin; D011985:Receptors, Serotonin / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D007473:Ion Channels; D011985:Receptors, Serotonin; D010566:Virulence Factors, Bordetella; D009243:NAD; D006165:Guanylyl Imidodiphosphate; D017371:8-Hydroxy-2-(di-n-propylamino)tetralin; D000255:Adenosine Triphosphate; D002772:Cholera Toxin; D000242:Cyclic AMP; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D002118:Calcium; D004229:Dithiothreitol