Introduction of purified alpha 2A-adrenergic receptors into uniformly oriented, unilamellar, phospholipid vesicles: productive coupling to G proteins but lack of receptor-dependent ion transport.
Introduction of highly purified alpha 2A-adrenergic receptors (alpha 2AAR) into lipid vesicles resulted in vesicle preparations that were unilamellar in structure, nonleaky to monovalent cations, and uniformly oriented such that the cytoplasmic domains of the alpha 2AAR faced the vesicle exterior. In this orientation, addition of Gi/G(o) G proteins yielded a 4-5-fold stimulation of agonist-dependent guanosine-5'-O-(3-[35S]thio)triphosphate binding to the G protein alpha subunit. These nonleaky, uniformly oriented, alpha 2AAR-containing vesicle preparations allowed us to explore the hypothesis that the alpha 2AAR itself, or in combination with Gi/G(o) proteins, is able to effect ion translocation. Measurements of 22Na+ uptake, 22Na+ efflux, and H+ movement revealed no detectable agonist-stimulated, receptor-dependent, ion translocation, even in the presence of G proteins, suggesting that allosteric regulation of alpha 2AAR by cations and amiloride analogs is not an indication that the alpha 2AAR itself is an ion transporter. Nonetheless, the methodology developed in the present studies for preparation of nonleaky vesicles containing receptor and G proteins should be well suited for evaluating the stoichiometry and selectivity of receptor-G protein interactions and, in particular, G protein specificity in mediating receptor-dependent regulation of voltage-gated or receptor-operated ion channels.
Keefer
J R
JR
Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232-6600.
Nunnari
J
J
Pang
I H
IH
Limbird
L E
LE
D000818:Animals; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006859:Hydrogen / Q000378:metabolism; D015640:Ion Channel Gating; D007473:Ion Channels / Q000502:physiology; D017136:Ion Transport; D010743:Phospholipids / Q000378:metabolism*; D018341:Receptors, Adrenergic, alpha-2 / Q000302:isolation & purification / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013552:Swine
D007473:Ion Channels; D010743:Phospholipids; D018341:Receptors, Adrenergic, alpha-2; D006859:Hydrogen; D012964:Sodium; D019204:GTP-Binding Proteins