TY - JOUR
T1 - Introduction of purified α(2A)-adrenergic receptors into uniformly oriented, unilamellar, phospholipid vesicles
T2 - Productive coupling to G proteins but lack of receptor-dependent ion transport
AU - Keefer, J. R.
AU - Nunnari, J.
AU - Pang, I. H.
AU - Limbird, L. E.
PY - 1994
Y1 - 1994
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0028174519&partnerID=8YFLogxK
M3 - Article
C2 - 7517495
AN - SCOPUS:0028174519
SN - 0026-895X
VL - 45
SP - 1071
EP - 1081
JO - Molecular Pharmacology
JF - Molecular Pharmacology
IS - 6
ER -