Introduction of purified α_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 α_2A-adrenergic receptors (α_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 α_2AAR faced the vesicle exterior. In this orientation, addition of G_i/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 α subunit. These nonleaky, uniformly oriented, α_2AAR-containing vesicle preparations allowed us to explore the hypothesis that the α_2AAR itself, or in combination with G_i/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 α_2AAR by cations and amiloride analogs is not an indication that the α_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 voltagegated or receptor-operated ion channels.