RESUMO
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 voltage-dependent agonist binding.
Assuntos
Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Sítio Alostérico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cisteína/metabolismo , Fluorescência , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação/genética , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Toxina Pertussis/farmacologia , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Receptor Muscarínico M2/genética , Rodaminas/metabolismo , XenopusRESUMO
Activation by agonist binding of G-protein-coupled receptors (GPCRs) controls most signal transduction processes. Although these receptors span the cell membrane, they are not considered to be voltage sensitive. Recently it was shown that both the activity of GPCRs and their affinity towards agonists are regulated by membrane potential. However, it remains unclear whether GPCRs intrinsically respond to changes in membrane potential. Here we show that two prototypical GPCRs, the m2 and m1 muscarinic receptors (m2R and m1R), display charge-movement-associated currents analogous to 'gating currents' of voltage-gated channels. The gating charge-voltage relationship of m2R correlates well with the voltage dependence of the affinity of the receptor for acetylcholine. The loop that couples m2R and m1R to their G protein has a crucial function in coupling voltage sensing to agonist-binding affinity. Our data strongly indicate that GPCRs serve as sensors for both transmembrane potential and external chemical signals.
Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Acetilcolina/metabolismo , Animais , Condutividade Elétrica , Ativação do Canal Iônico , Ligantes , Potenciais da Membrana/fisiologia , Movimento , Proteínas Mutantes/agonistas , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Oócitos/metabolismo , Ligação Proteica , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M1/genética , Receptor Muscarínico M1/metabolismo , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais , XenopusRESUMO
Ca(2+) is essential for physiological depolarization-evoked synchronous neurotransmitter release. But, whether Ca(2+) influx or another factor controls release initiation is still under debate. The time course of ACh release is controlled by a presynaptic inhibitory G protein-coupled autoreceptor (GPCR), whose agonist-binding affinity is voltage-sensitive. However, the relevance of this property for release control is not known. To resolve this question, we used pertussis toxin (PTX), which uncouples GPCR from its G(i/o) and in turn reduces the affinity of GPCR toward its agonist. We show that PTX enhances ACh and glutamate release (in mice and crayfish, respectively) and, most importantly, alters the time course of release without affecting Ca(2+) currents. These effects are not mediated by G(beta)gamma because its microinjection into the presynaptic terminal did not alter the time course of release. Also, PTX reduces the association of the GPCR with the exocytotic machinery, and this association is restored by the addition of agonist. We offer the following mechanism for control of initiation and termination of physiological depolarization-evoked transmitter release. At rest, release is under tonic block achieved by the transmitter-bound high-affinity presynaptic GPCR interacting with the exocytotic machinery. Upon depolarization, the GPCR uncouples from its G protein and consequently shifts to a low-affinity state toward the transmitter. The transmitter dissociates, the unbound GPCR detaches from the exocytotic machinery, and the tonic block is alleviated. The free machinery, together with Ca(2+) that had already entered, initiates release. Release terminates when the reverse occurs upon repolarization.
Assuntos
Sinapses/metabolismo , Transmissão Sináptica , Acetilcolina/metabolismo , Animais , Cálcio/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Camundongos , Toxina Pertussis/farmacologia , Ligação Proteica , Proteínas Qa-SNARE/metabolismo , Receptor Muscarínico M2/metabolismo , Sinapses/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Fatores de TempoRESUMO
This review discusses two theories that try to explain mechanisms of control of neurotransmitter release in fast synapses: the Ca(2+) hypothesis and the Ca(2+) voltage hypothesis. The review summarizes experimental results that are incompatible with predictions from the Ca(2+) hypothesis and concludes that Ca(2+) is involved in the control of the amount of release but not in the control of the time course of evoked release, i.e., initiation and termination of evoked release. Results summarizing direct effects of changes in membrane potential on the release machinery are then presented. These changes in membrane potential affect the affinity (for the transmitter) of presynaptic autoinhibitory G-protein coupled receptors (GPCRs). The voltage dependence of these GPCRs and their pivotal role in determining the time course of evoked release is discussed.
Assuntos
Cálcio/metabolismo , Neurotransmissores/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Sinapses/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/farmacologia , Canais de Cálcio/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Modelos BiológicosRESUMO
The effect of membrane potential on feedback inhibition of acetylcholine (ACh) release was studied using the frog neuromuscular junction. It was found that membrane potential affects the functional affinity (K(i)) of the presynaptic M2 muscarinic receptor. The K(i) for muscarine shifts from approximately 0.23 microm (at resting potential) to approximately 8 microm (at a high depolarization). Measurements of Ca2+ currents in axon terminals showed that the depolarization-mediated shift in K(i) does not stem from depolarization-dependent changes in Ca2+ influx. Pretreatments with pertussis toxin (PTX) abolished the depolarization-dependent shift in K(i); at all depolarizations K(i) was the same and higher (approximately 32 microm) than before PTX treatment. The inhibitory effect of muscarine on ACh release is produced by two independent mechanisms: a slow, PTX-sensitive process, which prevails at low to medium depolarizations and operates already at low muscarine concentrations, and a fast, PTX-insensitive and voltage-independent process, which requires higher muscarine concentrations. Neither of the two processes involves a reduction in Ca2+ influx.
Assuntos
Acetilcolina/metabolismo , Cálcio/metabolismo , Retroalimentação Fisiológica/fisiologia , Junção Neuromuscular/metabolismo , Potenciais de Ação/fisiologia , Animais , Relação Dose-Resposta a Droga , Estimulação Elétrica , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Retroalimentação Fisiológica/efeitos dos fármacos , Proteínas de Ligação ao GTP/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Junção Neuromuscular/efeitos dos fármacos , Técnicas de Patch-Clamp , Toxina Pertussis , Potássio/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Rana ridibunda , Receptor Muscarínico M2 , Receptores Muscarínicos/metabolismo , Tetrodotoxina/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
G-protein coupled receptors (GPCRs) comprise the largest protein family and mediate the vast majority of signal transduction processes in the body. Until recently GPCRs were not considered to be voltage dependent. Newly it was shown for several GPCRs that the first step in GPCR activation, the binding of agonist to the receptor, is voltage sensitive: Voltage shifts the receptor between two states that differ in their binding affinity. Here we show that this shift involves the rate constant of dissociation. We used the m2 muscarinic receptor (m2R) a prototypical GPCR and measured directly the dissociation of [(3)H]ACh from m2R expressed Xenopus oocytes. We show, for the first time, that the voltage dependent change in affinity is implemented by voltage shifting the receptor between two states that differ in their rate constant of dissociation. Furthermore, we provide evidence that suggest that the above shift is achieved by voltage regulating the coupling of the GPCR to its G protein.
Assuntos
Receptor Muscarínico M2/metabolismo , Acetilcolina/metabolismo , Animais , Cinética , Potenciais da Membrana , Ligação Proteica , XenopusRESUMO
Reliable neuronal communication depends on accurate temporal correlation between the action potential and neurotransmitter release. Although a requirement for Ca(2+) in neurotransmitter release is amply documented, recent studies have shown that voltage-sensitive G protein-coupled receptors (GPCRs) are also involved in this process. However, how slow-acting GPCRs control fast neurotransmitter release is an unsolved question. Here we examine whether the recently discovered fast depolarization-induced charge movement in the M(2)-muscarinic receptor (M(2)R) is responsible for M(2)R-mediated control of acetylcholine release. We show that inhibition of the M(2)R charge movement in Xenopus oocytes correlated well with inhibition of acetylcholine release at the mouse neuromuscular junction. Our results suggest that, in addition to Ca(2+) influx, charge movement in GPCRs is also necessary for release control.
Assuntos
Neurotransmissores/metabolismo , Receptor Muscarínico M2/metabolismo , Transdução de Sinais , Acetilcolina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Carbacol/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Trietiodeto de Galamina/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Fotólise/efeitos dos fármacos , Receptor Muscarínico M2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , XenopusRESUMO
This review describes the development of the molecular level Ca(2+)-voltage hypothesis. Theoretical considerations and feedback between theory and experiments played a key role in its development. The theory, backed by experiments, states that at fast synapses, membrane potential by means of presynaptic inhibitory autoreceptors controls initiation and termination of neurotransmitter release. A molecular kinetic scheme which depicts initiation and termination of evoked release is discussed. This scheme is able to account for both spontaneous release and evoked release. The physiological implications of this scheme are enumerated.
Assuntos
Modelos Neurológicos , Neurotransmissores/metabolismo , Animais , Cálcio/metabolismo , Retroalimentação , Matemática , Potenciais da Membrana , Camundongos , Camundongos Knockout , Receptor Muscarínico M2/fisiologiaRESUMO
G-protein-coupled receptors play a key role in signal transduction processes. Despite G-protein-coupled receptors being transmembrane proteins, the notion that they exhibit voltage sensitivity is rather novel. Here we examine whether two metabotropic glutamate receptors, mGluR3 and mGluR1a, both involved in fundamental physiological processes, exhibit, by themselves, voltage sensitivity. Measuring mGluR3-induced K(+) currents and mGluR1a-induced Ca(2+)-activated Cl(-) currents in Xenopus oocytes, we show that the apparent affinity toward glutamate decreases (mGluR3) or increases (mGluR1a) upon depolarization. Measurements of binding of [(3)H]glutamate to oocytes expressing either mGluR3 or mGluR1a corroborated the electrophysiological results. Using the chimeric Galpha subunit, we further show that the voltage sensitivity does not reside in the G-protein. To locate sites within the receptors that are involved in the voltage sensitivity, we used chimeric mGluR1a, where the intracellular loops that couple to the G-protein were replaced by those of mGluR3. The voltage sensitivity of the chimeric mGluR1a resembled that of mGluR3 and not that of the parental mGluR1a. The cumulative results indicate that the voltage sensitivity does not reside downstream to the activation of the receptors but rather in the mGluR3 and mGluR1a themselves. Furthermore, the intracellular loops play a crucial role in relaying changes in membrane potential to changes in the affinity of the receptors toward glutamate.
Assuntos
Potenciais da Membrana/fisiologia , Receptores de Glutamato Metabotrópico/fisiologia , Motivos de Aminoácidos , Animais , Proteínas de Ligação ao GTP/fisiologia , Canais Iônicos/fisiologia , Transporte de Íons/fisiologia , Ratos , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Glutamato Metabotrópico/análise , Proteínas Recombinantes de Fusão/fisiologia , Xenopus laevisRESUMO
The primacy of Ca2+ in controlling the amount of released neurotransmitter is well established. However, it is not yet clear what controls the time-course (initiation and termination) of release. Various experiments indicated that the time-course is controlled by membrane potential per se. Consequently the phenomenological Ca-Voltage-Hypothesis (CVH) was formulated. The CVH was later embodied in a molecular level mathematical model, whose key predictions were affirmed experimentally. Nonetheless, the single most important basis for the CVH, namely that depolarization per se is needed to induce physiological phasic release, was challenged by two major experimental findings. (i) Release was induced by Ca2+ alone by means of Ca2+-uncaging. (ii) There was at most a small additional effect when depolarization was applied after release was induced by Ca2+-uncaging. Point (i) was dealt with previously, but additional conclusions are drawn here. Here we concentrate on (ii) and show that the experimental results can be fully accounted for by the molecular level CVH model, with essentially the same parameters.
Assuntos
Cálcio/metabolismo , Canais Iônicos/fisiologia , Modelos Neurológicos , Neurotransmissores/metabolismo , Animais , Humanos , Potenciais da Membrana/fisiologia , Fatores de TempoRESUMO
G-protein coupled receptors are not considered to exhibit voltage sensitivity. Here, using Xenopus oocytes, we show that the M2 muscarinic receptor (m2R) is voltage-sensitive. The m2R-mediated potassium channel (GIRK) currents were used to assay the activity of m2R. We found that the apparent affinity of m2R toward acetylcholine (ACh) was reduced upon depolarization. Binding experiments of [3H]ACh to individual oocytes expressing m2R confirmed the electrophysiological findings. When the GIRK channels were activated either by overexpression of Gbetagamma subunits or by injection of GTPgammaS, the ratio between the currents measured at -60 mV and +40 mV was the same as for the basal activity of the GIRK channel. Thus, the steps downstream to agonist activation of m2R are not voltage-sensitive. We further found that, in contrast to m2R, the apparent affinity of m1R was increased upon depolarization. We also found that the voltage sensitivity of binding of [3H]ACh to oocytes expressing m2R was greatly diminished following pretreatment with pertussis toxin. The cumulative results suggest that m2R is, by itself, voltage-sensitive. Furthermore, the voltage sensitivity does not reside in the ACh binding site, rather, it most likely resides in the receptor region that couples to the G-protein.