Nonequilibrium activation of a G-protein-coupled receptor.

G-protein-coupled receptor activation is generally analyzed under equilibrium conditions. However, real-life receptor functions are often dependent on very short, transient stimuli that may not allow the achievement of a steady state. This is particularly true for synaptic receptors such as the α(2A)-adrenergic receptor (α(2A)-AR). Therefore, we developed a fluorescence resonance energy transfer-based technology to study nonequilibrium α(2A)-AR function in living cells. To examine the effects of increasing concentrations of the endogenous agonist norepinephrine on the speed and extent of α(2A)-AR activation with very high temporal resolution, we took advantage of a fluorophore-containing α(2A)-AR sensor. The results indicated that the efficacy of norepinephrine in eliciting receptor activation increased in a time-dependent way, reaching the maximum with a half-life of ~60 ms. The EC(50) values under nonequilibrium conditions start at ~26 μM (at 40 ms) and show a 10-fold decrease until the steady state is achieved. To analyze the ability of norepinephrine to trigger a downstream intracellular response after α(2A)-AR stimulation, we monitored the kinetics and amplitude of G(i) activation in real time by using a fluorophore-containing G(i) sensor. The results show that both the efficacy and the potency of norepinephrine in inducing G(i) activation achieve a steady state more slowly, compared with receptor activation, and that the initial EC(50) value of ~100 nM decreases in an exponential way, reaching the minimal value of ~10 nM at equilibrium. Therefore, both the efficacy and the potency of norepinephrine increase ~10-fold over a few seconds of agonist stimulation, which illustrates that receptor and G-protein signaling and signal amplification are highly time-dependent phenomena.