On the mechanism of G protein beta gamma subunit activation of the muscarinic K+ channel in guinea pig atrial cell membrane. Comparison with the ATP-sensitive K+ channel

• Published in: The Journal of general physiology Vol. 99; no. 6; pp. 961 - 983
• Main Authors: Ito, H, Tung, R T, Sugimoto, T, Kobayashi, I, Takahashi, K, Katada, T, Ui, M, Kurachi, Y
• Format: Journal Article
• Language: English
• Published: United States The Rockefeller University Press 01.06.1992
• Subjects: Adenosine Triphosphate - pharmacology; Animals; Atrial Function; Cell Membrane - drug effects; Cell Membrane - physiology; Cell Membrane - ultrastructure; Dose-Response Relationship, Drug; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - physiology; Guanosine Triphosphate; Guinea Pigs; Heart Atria - cytology; Heart Atria - ultrastructure; Heart Ventricles - cytology; Heart Ventricles - ultrastructure; Parasympathomimetics - pharmacology; Pertussis Toxin; Potassium Channels - drug effects; Potassium Channels - physiology; Time Factors; Trypsin - pharmacology; Ventricular Function; Virulence Factors, Bordetella - pharmacology
• ISSN: 0022-1295; 1540-7748
• DOI: 10.1085/jgp.99.6.961

The mechanism of G protein beta gamma subunit (G beta gamma)-induced activation of the muscarinic K+ channel (KACh) in the guinea pig atrial cell membrane was examined using the inside-out patch clamp technique. G beta gamma and GTP-gamma S-bound alpha subunits (G alpha *'s) of pertussis toxin (PT)-sensitive G proteins were purified from bovine brain. Either in the presence or absence of Mg2+, G beta gamma activated the KACh channel in a concentration-dependent fashion. 10 nM G beta gamma almost fully activated the channel in 132 of 134 patches (98.5%). The G beta gamma-induced maximal channel activity was equivalent to or sometimes larger than the GTP-gamma S-induced one. Half-maximal activation occurred at approximately 6 nM G beta gamma. Detergent (CHAPS) and boiled G beta gamma preparation could not activate the KACh channel. G beta gamma suspended by Lubrol PX instead of CHAPS also activated the channel. Even when G beta gamma was pretreated in Mg(2+)-free EDTA internal solution containing GDP analogues (24-48 h) to inactivate possibly contaminating G i alpha *'s, the G beta gamma activated the channel. Furthermore, G beta gamma preincubated with excessive GDP-bound G o alpha did not activate the channel. These results indicate that G beta gamma itself, but neither the detergent CHAPS nor contaminating G i alpha *, activates the KACh channel. Three different kinds of G i alpha * at 10 pM-10 nM could weakly activate the KACh channel. However, they were effective only in 40 of 124 patches (32.2%) and their maximal channel activation was approximately 20% of that induced by GTP-gamma S or G beta gamma. Thus, G i alpha * activation of the KACh channel may not be significant. On the other hand, G i alpha *'s effectively activated the ATP-sensitive K+ channel (KATP) in the ventricular cell membrane when the KATP channel was maintained phosphorylated by the internal solution containing 100 microM Mg.ATP. G beta gamma inhibited adenosine or mACh receptor-mediated, intracellular GTP-induced activation of the KATP channel. G i alpha *'s also activated the phosphorylated KATP channel in the atrial cell membrane, but did not affect the background KACh channel. G beta gamma subsequently applied to the same patch caused prominent KACh channel activation. The above results may indicate two distinct regulatory systems of cardiac K+ channels by PT-sensitive G proteins: G i alpha activation of the KATP channel and G beta gamma activation of the KACh channel.