RT Journal Article
SR Electronic
T1 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.
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 1071
OP 1081
VO 45
IS 6
A1 J R Keefer
A1 J Nunnari
A1 I H Pang
A1 L E Limbird
YR 1994
UL http://molpharm.aspetjournals.org/content/45/6/1071.abstract
AB 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.