Introduction of highly purified α2A-adrenergic receptors (α2AAR) 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 α2AAR faced the vesicle exterior. In this orientation, addition of Gi/Go G proteins yielded a 4-5-fold stimulation of agonist-dependent guanosine-5′-O-(3-[35S]thio)triphosphate binding to the G protein α subunit. These nonleaky, uniformly oriented, α2AAR-containing vesicle preparations allowed us to explore the hypothesis that the α2AAR itself, or in combination with Gi/Go 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 α2AAR by cations and amiloride analogs is not an indication that the α2AAR 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 voltagegated or receptor-operated ion channels.
|Original language||English (US)|
|Number of pages||11|
|State||Published - Jun 1994|
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