G protein activation inhibits amiloride-blockable highly selective sodium channels in A6 cells.
Single-channel methods were used to examine the regulation of amiloride-blockable highly selective sodium channels by guanine nucleotide-binding proteins (G proteins). A6 cells (a renal cell line derived from Xenopus laevis kidney) were cultured on permeable collagen films, and patch recordings were made from the apical membranes of confluent cells. The predominant channel in the apical membranes is a highly selective, 4-pS, amiloride-blockable sodium channel (the Na(+)-to-K+ permeability ratio is > 30). In inside-out patches, application to the cytosolic surface of guanosine-5'-O-(2-thiodiphosphate) (GDP beta S), which deactivates G proteins, increased sodium channel activity. GDP beta S produced a sevenfold increase in channel activity. In contrast, GTP and guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) decreased sodium channel activity to about one-twentieth of the untreated value. The effect of GTP (but not GTP gamma S) was reversible. In cell-attached patches, a 3- to 4-h exposure of the apical membrane to pertussis toxin (PTX) increased the mean open time of sodium channels approximately 2.7 times and the open probability approximately 1.6-fold, but pretreatment of apical membranes with cholera toxin (250 ng/ml) for 3-4 h had no effect on open probability or mean open time. These results imply that a PTX-sensitive G protein regulates amiloride-blockable highly selective sodium channels in the apical membranes of A6 cells and that the G protein in a GTP-bound, activated state inhibits sodium channel activity.
Department of Physiology, Emory University School of Medicine, Atlanta, Georgia 30322.
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A
Ohara
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H
Matsunaga
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DC
Eaton
D000584:Amiloride / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D004553:Electric Conductivity; D019204:GTP-Binding Proteins / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000031:analogs & derivatives / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D037342:Pertussis Toxin; D015222:Sodium Channels / Q000187:drug effects* / Q000502:physiology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D015222:Sodium Channels; D010566:Virulence Factors, Bordetella; C034883:guanosine 5'-O-(2-thiotriphosphate); D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000584:Amiloride; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins