Coupling of the muscarinic m2 receptor to G protein-activated K(+) channels via Galpha(z) and a receptor-Galpha(z) fusion protein. Fusion between the receptor and Galpha(z) eliminates catalytic (collision) coupling.
G protein-activated K(+) channel (GIRK), which is activated by the G(betagamma) subunit of heterotrimeric G proteins, and muscarinic m2 receptor (m2R) were coexpressed in Xenopus oocytes. Acetylcholine evoked a K(+) current, I(ACh), via the endogenous pertussis toxin (PTX)-sensitive G(i/o) proteins. Activation of I(ACh) was accelerated by increasing the expression of m2R, suggesting a collision coupling mechanism in which one receptor catalytically activates several G proteins. Coexpression of the alpha subunit of the PTX-insensitive G protein G(z), Galpha(z), induced a slowly activating PTX-insensitive I(ACh), whose activation kinetics were also compatible with the collision coupling mechanism. When GIRK was coexpressed with an m2R x Galpha(z) fusion protein (tandem), in which the C terminus of m2R was tethered to the N terminus of Galpha(z), part of I(ACh) was still eliminated by PTX. Thus, the m2R of the tandem activates the tethered Galpha(z) but also the nontethered G(i/o) proteins. After PTX treatment, the speed of activation of the m2R x Galpha(z)-mediated response did not depend on the expression level of m2R x Galpha(z) and was faster than when m2R and Galpha(z) were coexpressed as separate proteins. These results demonstrate that fusing the receptor and the Galpha strengthens their coupling, support the collision-coupling mechanism between m2R and the G proteins, and suggest a noncatalytic (stoichiometric) coupling between the G protein and GIRK in this model system.
Vorobiov
D
D
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv 69978, Israel.
Bera
A K
AK
Keren-Raifman
T
T
Barzilai
R
R
Dascal
N
N
D000818:Animals; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007700:Kinetics; D008845:Microinjections; D009865:Oocytes; D018408:Patch-Clamp Techniques; D037342:Pertussis Toxin; D015221:Potassium Channels / Q000378:metabolism*; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger / Q000378:metabolism; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic / Q000378:metabolism*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology; D014981:Xenopus
D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D043585:Receptor, Muscarinic M2; D011976:Receptors, Muscarinic; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins