Differences in efficacy and Na(+) sensitivity between alpha(2B) and alpha(2D) adrenergic receptors: implications for R and R* states

The ability of Na(+) ions to modulate coupling of alpha(2B)- and alpha(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 [(35)S]GTPgammaS binding was higher for alpha(2D)-receptors (the rat homolog of the alpha(2A)-receptor) in both cell types, whereas both alpha(2B)- and alpha(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/alpha(2B) than PC12/alpha(2D) membranes. Epinephrine-stimulated GTP binding in PC12/alpha(2B) membranes was also more sensitive to Na(+) inhibition than in PC12/alpha(2D) membranes. In saturation [(35)S]GTPgammaS binding studies, the presence of Na(+) reduced apparent GTP affinity, and its effect was greater in PC12/ alpha(2B) membranes, consistent with a greater reduction in the active R* conformation of the receptor. The higher efficacy of epinephrine at alpha(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.