Differences in efficacy and NA⁺ sensitivity between α(2B) and α(2D) adrenergic receptors: Implications for R and R* states

The ability of Na+ ions to modulate coupling of α(2B)- and α(2D)- adrenergic receptors to G proteins was investigated in isolated membranes from transfected PC12 and NIH 3T3 fibroblast cells. The initial rate of epinephrine-stimulated [35S]GTPγS binding was higher for α(2D)-receptors (the rat homolog of the α(2A)-receptor) in both cell types, whereas both α(2B)- and α(2D)-receptor responses were higher in PC12 cell membranes. Pertussis toxin completely blocked agonist-stimulated binding. Graded increases in Na+ caused a progressive loss of basal GTP binding, indicative of its ability to reduce the level of the active R* state of the receptor. This inhibitory effect of Na+ was more pronounced in PC12/α(2B) than PC12/α(2D) membranes. Epinephrine-stimulated GTP binding in PC12/α(2B) membranes was also more sensitive to Na+ inhibition than in PC12/α(2D) membranes. In saturation [35S]GTPγS binding studies, the presence of Na+ reduced apparent GTP affinity, and its effect was greater in PC12/α(2B) membranes, consistent with a greater reduction in the active R* conformation of the receptor. The higher efficacy of epinephrine at α(2D) receptors and their lesser sensitivity to Na+ are both indicative of a more stable R* state. Together these results suggest that differences in the modulatory influence of Na+ within a family of G(i)-coupled receptors may reflect differences in the stability of the active R* state. (C) 2000 S. Karger AG, Basel.