The present study demonstrates that agonist-mediated activation of α2A adrenergic receptors (α 2A AR) is voltage-dependent. By resolving the kinetics of conformational changes of α 2A AR at defined membrane potentials, we show that negative membrane potentials in the physiological range promote agonist-mediated activation of α 2A AR. We discovered that the conformational change of α 2A AR by voltage is independent from receptor-G protein docking and regulates receptor signaling, including β-arrestin binding, activation of G proteins, and G protein-activated inwardly rectifying K + currents. Comparison of the dynamics of voltage-dependence of clonidine-vs. norepinephrine-activated receptors uncovers interesting mechanistic insights. For norepinephrine, the time course of voltage-dependent deactivation reflected the deactivation kinetics of the receptor after agonist withdrawal and was strongly attenuated at saturating concentrations. In contrast, clonidine-activated α 2A AR were switched by voltage even under fully saturating concentrations, and the kinetics of this switch was notably faster than dissociation of clonidine from α 2A AR, indicating voltage-dependent regulation of the efficacy. We conclude that adrenergic receptors exhibit a unique, agonist-dependent mechanism of voltage-sensitivity that modulates downstream receptor signaling.A drenergic receptors represent a clinically important group of G protein-coupled receptors (GPCRs). This large family of ligand-gated signaling molecules is involved in many physiological and pathophysiological processes, which represent important pharmacological targets (1) and share common downstream signaling pathways (2). α-adrenergic receptor type 2A (α 2A AR) are expressed in neurons of the central nervous system, where they control the regulation of neurotransmitter release (3). Therefore, they are exposed to the electric field of the membrane and may be modulated by alterations in the membrane potential (V M ), a phenomenon that is the topic of this study. Indeed, voltage-dependent binding of neurotransmitters to their receptors, or at least voltage-dependent regulation of their downstream signal, has been described for a few other members of the GPCR family, specifically for M 1 and M 2 muscarinic receptors (4), as well as P 2 Y 1 purinergic receptors (5, 6) and metabotropic glutamate receptors (7). Voltage-dependence of GPCRs has been implicated in fine-tuning of neurotransmitter release (8, 9), in regulation of cardiac excitability (10), and in potentiation of IP 3 -dependent intracellular Ca 2+ signals (6). So far the mechanism underlying this voltage sensitivity of GPCRs is not very well understood; however, the current view is that allosteric regulation of receptor conformations by docking of the G protein seems to be required for voltage sensitivity. Notably, muscarinic M 1 and M 2 receptors display fast voltage-induced charge movements in the absence of agonists similar to the gating currents of ion channels (11,12). Recently, conformational changes directly l...

Background:The activity of several G q -and G i -coupled receptors is modulated by the membrane potential. Results: Voltage modulates catecholamine-mediated activation of G s -coupled ␤ 1 -and ␤ 2 -adrenoceptors. Conclusion: Voltage-dependence of ␤ 1 -AR is due to alterations in the efficacy of catecholamines. Significance: By modulating catecholamine efficacy on ␤ 1 -ARs, voltage can modify receptor activity on a very fast time scale.

Many different neurotransmitters and hormones control intracellular signaling by regulating the production of the second messenger cAMP. The function of the broadly expressed adenylyl cyclases (ACs) 5 and 6 is regulated by either stimulatory or inhibitory G proteins. By analyzing a well-known rebound stimulation phenomenon after withdrawal of G i protein in atrial myocytes, we discovered that AC5 and -6 are tightly regulated by the second messenger PIP 3 . By monitoring cAMP levels in real time by means of Förster resonance energy transfer (FRET)-based biosensors, we reproduced the rebound stimulation in a heterologous expression system specifically for AC5 or -6. Strikingly, this cAMP rebound stimulation was completely blocked by the PI3K inhibitor wortmannin, both in atrial myocytes and in transfected human embryonic kidney cells. Similar effects were observed by heterologous expression of the PIP 3 phosphatase and tensin homolog (PTEN). However, general kinase inhibitors or inhibitors of Akt had no effect, suggesting a PIP 3 -dependent mechanism. These findings demonstrate the existence of a novel general pathway for regulation of AC5 and -6 activity via PIP 3 that leads to pronounced alterations of cytosolic cAMP levels.-Reddy, G. R., Subramanian, H., Birk, A., Milde, M., Nikolaev, V. O., Bünemann, M. Adenylyl cyclases 5 and 6 underlie PIP 3 -dependent regulation. FASEB J. 29, 3458-3471 (2015). www.fasebj.org

G-protein coupled receptors (GPCRs) are the largest family of membrane signaling proteins. They respond to a wide-array of stimuli and contain a seven transmembrane domain that couples to heterotrimeric G-proteins. These pathways play roles in controlling second messenger levels and regulating other signaling proteins, such as ion channels and kinases. Due to their importance in many diseases, GPCRs are the most explored drug targets in biology. Despite this, the specific biochemical, physiological, and behavioral roles of many GPCRs are not well-understood and make this field ripe for the application of new tools for high-precision probing. We have developed an approach to elucidate the function of a GPCR by chemically re-engineering it to be sensitive to light. The class C glutamate-gated GPCR, mGluR2, which couples to the Gi/o pathway, was derivatized with photoswitchable ligands to generate both light-agonized (LimGluR2) and light-antagonized (''LimGluR2-block'') receptors. The bistable, azobenzene photoswitch enables activation by a light pulse to be sustained for long periods in the dark before being switched off by a longer wavelength light pulse. LimGluR2 deactivates quickly and supports multiple reproducible rounds of on/off switching with superior fidelity and speed compared to Rhodopsin. We have extended optical control to a variety of mGluRs with distinct G-protein coupling profiles. These designed receptors provide excellent tools for the dissection of the specific roles of different mGluRs in physiological functions, such as induction of synaptic plasticity, with high spatiotemporal precision. Furthermore, the high level of control afforded by tethered agonists allows for the probing of coupling of ligandbinding to receptor activation with single-subunit control. Along with single-molecule fluorescence experiments, we have used optical control via LimGluRs to probe the mechanisms of assembly and cooperative activation in the class C GPCRs.