The Metabotropic Glutamate G-protein-coupled Receptors mGluR3 and mGluR1a Are Voltage-sensitive

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...

Section: Discussionsupporting

confidence: 82%

Section: Discussionmentioning

confidence: 76%

mentioning

confidence: 99%

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Voltage regulates adrenergic receptor function

Rinne

Birk

Bünemann

2013

“…It was recently reported that mGluR activation was voltage sensitive (32,33). We tested this possibility with the hope that depolarization-induced shift of the dose-response would enable kinetic analysis with perfect time resolution.…”

Section: Discussionmentioning

confidence: 99%

Optical measurement of mGluR1 conformational changes reveals fast activation, slow deactivation, and sensitization

Marcaggi

Mutoh

Dimitrov

et al. 2009

Metabotropic glutamate receptor (mGluR) activation has been extensively studied under steady-state conditions. However, at central synapses, mGluRs are exposed to brief submillisecond glutamate transients and may not reach steady-state. The lack of information on the kinetics of mGluR activation impairs accurate predictions of their operation during synaptic transmission. Here, we report experiments designed to investigate mGluR kinetics in real-time. We inserted either CFP or YFP into the second intracellular loop of mGluR1␤. When these constructs were coexpressed in PC12 cells, glutamate application induced a conformational change that could be monitored, using fluorescence resonance energy transfer (FRET), with an EC50 of 7.5 M. The FRET response was mimicked by the agonist DHPG, abolished by the competitive antagonist MCPG, and partially inhibited by mGluR1-selective allosteric modulators. These results suggest that the FRET response reports active conformations of mGluR1 dimers. The solution exchange at the cell membrane was optimized for voltageclamped cells by recording the current induced by co-application of 30 mM potassium. When glutamate was applied at increasing concentrations up to 2 mM, the activation time course decreased to a minimum of approximately 10 ms, whereas the deactivation time course remained constant (ϳ50 ms). During long-lasting applications, no desensitization was observed. In contrast, we observed a robust sensitization of the FRET response that developed over approximately 400 ms. Activation, deactivation, and sensitization time courses and amplitudes were used to derive a kinetic scheme and rate constants, from which we inferred the EC50 and frequency dependence of mGluR1 activation under non-steady-state conditions, as occurs during synaptic transmission.he amino acid L-glutamate, the main neurotransmitter in the CNS, is referred to as a fast neurotransmitter because it mediates fast excitatory neurotransmission by activating ionotropic glutamate receptors (i.e., NMDA, AMPA, and kainate receptors). It also targets metabotropic glutamate receptors (mGluRs, 8 types) that belong to the class C G protein-coupled receptor (GPCR) subfamily. As opposed to ionotropic glutamate receptors, mGluRs mediate slower effects of glutamate, downstream of G protein activation (1). In addition, mGluRs have often been considered to play a role as extrasynaptic receptors because they are not located within the synaptic cleft (2), and their affinity for glutamate may be high enough (3) to detect glutamate concentration in extrasynaptic locations. Their extrasynaptic localization appears consistent with the slow effects of their activation because glutamate transients are expected to be less sudden at extra-and perisynaptic sites than opposite to the release site. However, even at extrasynaptic locations, glutamate transients evoked by synaptically released glutamate occur within milliseconds, as shown by recording of glutamate transporter-mediated currents in glial cells that wrap around synapses (4, 5...

“…Although GPCRs span the cell membrane, they were not considered to be affected by voltage. Recently, it was shown that depolarization affects the activity of various GPCRs belonging to classes A and C (1)(2)(3)(4)(5)(6)(7)(8)(9).…”

mentioning

confidence: 99%

Depolarization induces a conformational change in the binding site region of the M ₂ muscarinic receptor

Dekel

Priest

Parnas

et al. 2011

Self Cite

G protein-coupled receptors play a central role in signal transduction and were only known to be activated by agonists. Recently it has been shown that membrane potential also affects the activity of G protein-coupled receptors. For the M 2 muscarinic receptor, it was further shown that depolarization induces charge movement. A tight correlation was found between the voltage-dependence of the charge movement and the voltage-dependence of the agonist binding. Here we examine whether depolarization-induced charge movement causes a conformational change in the M 2 receptor that may be responsible for the voltage-dependence of agonist binding. Using site-directed fluorescence labeling we show a voltage-dependent fluorescence signal, reflecting a conformational change, which correlates with the voltage-dependent charge movement. We further show that selected mutations in the orthosteric site abolish the fluorescence signal and concomitantly, the voltage-dependence of the agonist binding. Surprisingly, mutations in the allosteric site also abolished the voltage-dependence of agonist binding but did not reduce the fluorescence signal. Finally, we show that treatments, which reduced the charge movement or hindered the coupling between the charge movement and the voltage-dependent binding, also reduced the fluorescence signal. Our results demonstrate that depolarization-induced conformational changes in the orthosteric binding site underlie the voltage-dependence of agonist binding. Our results are also unique in suggesting that the allosteric site is also involved in controlling the voltagedependent agonist binding.acetylcholine | electrochromic | fluorescence quenching | gating G protein-coupled receptors (GPCRs) are involved in many signal transduction processes within cells. Although GPCRs span the cell membrane, they were not considered to be affected by voltage. Recently, it was shown that depolarization affects the activity of various GPCRs belonging to classes A and C (1-9).Depolarization could affect GPCRs' activity by modifying agonist efficacy and/or its binding. Effect of voltage on efficacy was suggested, but was not directly demonstrated, to explain the ability of depolarization to generate a P2Y1 receptor response in the presence of a competitive antagonist and no agonist (5). However, for the M 2 muscarinic receptor (M 2 R) it was directly shown that voltage affects binding. Specifically, voltage-dependent [ 3 H]ACh binding to M 2 R was seen in synaptosomes (10) and in synaptoneurosomes (11). In the above studies (10, 11), it was shown that the M 2 R exhibits two agonist-binding affinity states. These authors suggested that depolarization shifts the high affinity state into a low affinity state.A relevant question is whether measurements of M 2 R-induced G protein-activated inwardly rectifying K + (GIRK) currents can faithfully reflect the effect of voltage on binding. To answer this question, measurements of GIRK currents and of radio-labeled agonist ([ 3 H]ACh) binding were conducted at different membra...