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(2A)AR) 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(2A)AR 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(2A)AR- containing vesicle preparations allowed us to explore the hypothesis that the α(2A)AR 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(2A)AR by cations and amiloride analogs is not an indication that the α(2A)AR 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.