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A–C Current activated by bethanechol (digitally subtracted) in cat atrial cells. Membrane currents activated by bethanechol A 3 µM and by B 10 µM in response to voltage steps of 3 s duration from a holding potential of –40 mV between –110 and –50 mV (upper traces) and between –30 and +70 mV (lower traces). Zero current is indicated by the arrow. C Current-voltage relationship for the current measured at the end of the test pulses at 3 µM (black circles) and 10 µM (black squares) of bethanechol, mean ± SE of n=6 cells are shown
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Recently, it has been shown that G protein-coupled receptors (GPCRs) display intrinsic voltage sensitivity. We reported that the voltage sensitivity of M2 muscarinic receptor (M2R) is also ligand specific. Here, we provide additional evidence to understand the mechanism underlying the ligand-specific voltage sensitivity of the M2R. Using ACh, piloc...
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... Finally, point mutations in M3R altered the effect of voltage on the activation of the receptor measured using FRET [35]. Another observation that may be relevant to this discussion is that the voltage dependence of muscarinic receptors has been shown to be agonist-dependent as well, i.e., while depolarization may decrease the potency of some ligands toward receptors, it may have no effect or even the opposite effect on other ligands [24,33,48]. Such ligand-specific voltage dependence has been observed for several other GPCRs [34,[49][50][51][52]. Furthermore, point mutations within the receptor protein have been shown to change this ligand specificity [33,34]. ...
... Several studies conducted following the discovery of the voltage dependence of the M2R suggest that this property of the M2R is responsible for this behavior. Namely, depolarization reduces the affinity of the receptor, and thus, the potassium currents are inactivated [48,62,63]. Another well-studied example is the role of the M2R in the control of neurotransmitter release. ...
Some signaling processes mediated by G protein-coupled receptors (GPCRs) are modulated by membrane potential. In recent years, increasing evidence that GPCRs are intrinsically voltage-dependent has accumulated. A recent publication challenged the view that voltage sensors are embedded in muscarinic receptors. Herein, we briefly discuss the evidence that supports the notion that GPCRs themselves are voltage-sensitive proteins and an alternative mechanism that suggests that voltage-gated sodium channels are the voltage-sensing molecules involved in such processes.
... Voltage dependence of GPCRs was demonstrated to play a role in several physiological processes, including controlling neurotransmitter release (Parnas and Parnas, 2010;Kupchik et al., 2011) and shaping the excitability of atrial cells (Moreno-Galindo et al., 2011;Moreno-Galindo et al., 2016;Salazar-Fajardo et al., 2018). Moreover, a recent study conducted in D. melanogaster revealed that type A muscarinic receptor exhibits voltage dependence binding of ACh and that mutating residues in the third intracellular loop of the receptor abolished the voltage dependence. ...
Serotonin (5-HT) plays a central role in various brain functions via the activation of a family of receptors, most of them G protein coupled receptors (GPCRs). 5-HT1A receptor, the most abundant 5-HT receptors, was implicated in many brain dysfunctions and is a major target for drug discovery. Several genetic polymorphisms within the 5-HT1A receptor gene were identified and linked to different conditions, including anxiety and depression. Here, we used Xenopus oocytes to examine the effects of one of the functional polymorphism, Arg220Leu, on the function of the receptor. We found that the mutated receptor shows normal activation of G protein and normal 5-HT binding. On the other hand, the mutated receptor shows impaired desensitization, probably due to impairment in activation of β arrestin-dependent pathway. Furthermore, while the 5-HT1A receptor was shown to exhibit voltage dependent activation by serotonin and by buspirone, the mutated receptor was voltage-independent. Our results suggest a pronounced effect of the mutation on the function of the 5-HT1A receptor and add to our understanding of the molecular mechanism of its voltage dependence. Moreover, the findings of this study may suggest a functional explanation for the possible link between this variant and brain pathologies.
... Recently, in cat atrial myocytes we have shown that M 2 R exhibits agonist-specific voltage dependence, where the intrinsic voltage sensitivity of this receptor [7][8][9] modifies its affinity for diverse agonists in a ligand-selective manner, which is eventually reflected on the activation of the coupled K ACh channels [10]. This property can be distinguished in the deactivation kinetics of the current carried by these channels, I KACh [11]. Also, we previously proposed that this is the molecular mechanism underlying a very distinctive attribute of receptor-stimulated Kir3.x currents (including I KACh ), known as relaxation [12], which consists of a time-dependent augment or reduction of the current upon hyperpolarizing or depolarizing, respectively, the cardiomyocyte membrane with voltage steps [13,14]. ...
... The rapid delayed rectifier (I Kr ) and slow delayed rectifier currents (I Ks ) were blocked by 3 μM E-4031 and 50 μM chromanol 293B, respectively. Recordings for concentration-response (C-R) relationships and for the estimation of the I KACh deactivation kinetics (tau) were carried out at room temperature (22-24˚C) and according to previous publications [9,11]. For these two approaches, inward rectifier current (I K1 ) was greatly reduced by adding 2 μM BaCl 2 in the recording extracellular solution [27]. ...
... In previous studies, we described the agonist-specific voltage sensitivity of M 2 R, where membrane depolarization reduces, augments, or not modifies the receptor activation by several muscarinic agonists [9,11,16]. Here, we investigated whether A 1 R exhibits a voltage-dependent interaction with its physiological agonist Ado, by measuring I KACh activation in guinea pig cardiomyocytes. The effects of different Ado concentrations on I KACh at +30 and -100 mV are shown in Fig 1A. ...
Inhibitory regulation of the heart is determined by both cholinergic M 2 receptors (M 2 R) and adenosine A 1 receptors (A 1 R) that activate the same signaling pathway, the ACh-gated inward rectifier K ⁺ (K ACh ) channels via G i/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M 2 R underlies several voltage-dependent features of I KACh , including the ‘relaxation’ property, which is characterized by a gradual increase or decrease of the current when cardiomyocytes are stepped to hyperpolarized or depolarized voltages, respectively. However, it is unknown whether membrane potential also affects A 1 R and how this could impact I KACh . Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A 1 R-G i/o - I KACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC 50 increase with depolarization) that was not manifested in the relative affinity, as estimated by the current deactivation kinetics (τ = 4074 ± 214 ms at -100 mV and τ = 4331 ± 341 ms at +30 mV; P = 0.31). Moreover, I KACh did not exhibit relaxation. Contrarily, activation of the M 2 R-G i/o - I KACh pathway with acetylcholine induced the typical relaxation of the current, which correlated with the clear voltage-dependent effect observed in the concentration-response curves (~2.8-fold EC 50 increase with depolarization) and in the I KACh deactivation kinetics (τ = 1762 ± 119 ms at -100 mV and τ = 1503 ± 160 ms at +30 mV; P = 0.01). Our findings further substantiate the hypothesis of the agonist-specific voltage dependence of GPCRs and that the I KACh relaxation is consequence of this property.
... ATS2 (not KCNJ2 associated) is much rarer than ATS1 and it is caused, for example, by a mutation in the KCNJ5 gene, that encodes for the K ir 3.4 channel, a G protein coupled inward rectifying potassium channel (GIRK) (Kokunai et al. 2014). It is activated by the βγ subunit of the G i coupled M 2 receptor and contributes to the I K,Ach that shortens the action potential in the atrium and slows down the heart rate (Moreno-Galindo et al. 2016). It was suggested that mutated K ir 3.4 form heterodimers with K ir 2.1 and subsequently inhibit the normal function of the K ir 2.1 subunit (Kokunai et al. 2014). ...
The physiological heart function is controlled by a well-orchestrated interplay of different ion channels conducting Na+, Ca2+ and K+. Cardiac K+ channels are key players of cardiac repolarization counteracting depolarizating Na+ and Ca2+ currents. In contrast to Na+ and Ca2+, K+ is conducted by many different channels that differ in activation/deactivation kinetics as well as in their contribution to different phases of the action potential. Together with modulatory subunits these K+ channel α-subunits provide a wide range of repolarizing currents with specific characteristics. Moreover, due to expression differences, K+ channels strongly influence the time course of the action potentials in different heart regions. On the other hand, the variety of different K+ channels increase the number of possible disease-causing mutations. Up to now, a plethora of gain- as well as loss-of-function mutations in K+ channel forming or modulating proteins are known that cause severe congenital cardiac diseases like the long-QT-syndrome, the short-QT-syndrome, the Brugada syndrome and/or different types of atrial tachyarrhythmias. In this chapter we provide a comprehensive overview of different K+ channels in cardiac physiology and pathophysiology.
... Since this first discovery at the M2-receptor, a number of GPCRs have been investigated under this aspect (reviewed in: Vickery et al., 2016). The majority of investigated GPCRs showed voltage dependence and the effect that voltage had on GPCR activity was ligand-specific (Navarro-Polanco et al., 2011;Sahlholm et al., 2011;Rinne et al., 2013Rinne et al., , 2015Birk et al., 2015;Moreno-Galindo et al., 2016). Voltage was able to alter either affinity, efficacy or both (Rinne et al., 2013;Birk et al., 2015). ...
... Since this first discovery at the M2-receptor, a number of GPCRs have been investigated under this aspect (reviewed in: Vickery et al., 2016). The majority of investigated GPCRs showed voltage dependence and the effect that voltage had on GPCR activity was ligandspecific (Navarro-Polanco et al., 2011;Sahlholm et al., 2011;Rinne et al., 2013Rinne et al., , 2015Birk et al., 2015;Moreno-Galindo et al., 2016). Voltage was able to alter either affinity, efficacy or both (Rinne et al., 2013;Birk et al., 2015). ...
G-protein coupled receptors (GPCRs) are the largest class of transmembrane receptors and serve as signal mediators to transduce information from extracellular signals such as neurotransmitters, hormones or drugs to cellular responses. They are exposed to the strong electrical field of the plasma membrane. In the last decade voltage modulation of ligand-induced GPCR activity has been reported for several GPCRs. Using Foerster resonance energy transfer (FRET) based biosensors in patch clamp experiments, we discovered a robust voltage dependence of the thromboxane receptor (TP receptor) on the receptor level as well as on downstream signaling. TP receptor activity doubled upon depolarization from -90 mV to +60 mV in the presence of U46619, a stable analog of prostaglandin H2 (PGH2). Half-maximal potential V0.5 determined for TP receptor was -46 mV, which is within the physiological range. We identified that depolarization affected the agonist affinity for the TP receptor. Depolarization enhanced responses of several structural analogs of U46619 with modifications to a similar extent all around the molecule, indicating that voltage modulates the general conformation of TP receptor. By means of site direct mutagenesis, we identified TP receptor R2957.40 which showed alteration of voltage sensitivity of TP receptor upon mutation. Voltage sensitivity was not limited to TP receptor, as prostaglandin F receptor (FP receptor) activated with U46619 and prostaglandin E2 receptor - subtype 3 (EP3 receptor) activated with Iloprost showed a similar reaction to depolarization as TP receptor. However, IP receptor activated with Iloprost showed no detectable voltage dependence. SIGNIFICANCE STATEMENT: Prostanoids mediate many of their physiological effects via transmembrane receptors expressed in the plasma membrane of excitable cells. We found that agonist-mediated activation of prostaglandin F receptors and prostaglandin E2 receptors as well as thromboxane receptors are activated upon depolarization, whereas prostacyclin receptors are not. The voltage-induced modulation of TP receptor activity was observed on the level of receptor conformation and downstream signaling. The range of voltage dependence was restricted by R2957.40 in the agonist-binding pocket.
... It could be anticipated that the endogenous (natural) agonists of studied GPCRs should be among those effectors whose signals are amplified by membrane voltage. Indeed, in the muscarinic acetylcholine receptor M 2 , the membrane voltage potentiated the signal from the endogenous full agonist acetylcholine, but decreased the signal from the drug pilocarpine, a partial agonist (see Fig. S3, [67,68] and Table S1 for further references). Additionally, the voltage sensitivity of the M 2 receptor is altered by mutations in the orthosteric ligand-binding site, indicating a direct connection between the agonist binding and voltage effects [67]. ...
The human genome contains about 700 genes of G protein-coupled receptors (GPCRs) of class A; these seven-helical membrane proteins are the targets of almost half of all known drugs. In the middle of the helix bundle, crystal structures reveal a highly conserved sodium-binding site, which is connected with the extracellular side by a water-filled tunnel. This binding site contains a sodium ion in those GPCRs that are crystallized in their inactive conformations but does not in those GPCRs that are trapped in agonist-bound active conformations. The escape route of the sodium ion upon the inactive-to-active transition and its very direction have until now remained obscure. Here, by modeling the available experimental data, we show that the sodium gradient over the cell membrane increases the sensitivity of GPCRs if their activation is thermodynamically coupled to the sodium ion translocation into the cytoplasm but decreases it if the sodium ion retreats into the extracellular space upon receptor activation. The model quantitatively describes the available data on both activation and suppression of distinct GPCRs by membrane voltage. The model also predicts selective amplification of the signal from (endogenous) agonists if only they, but not their (partial) analogs, induce sodium translocation. Comparative structure and sequence analyses of sodium-binding GPCRs indicate a key role for the conserved leucine residue in the second transmembrane helix (Leu2.46) in coupling sodium translocation to receptor activation. Hence, class A GPCRs appear to harness the energy of the transmembrane sodium potential to increase their sensitivity and selectivity.
... The concentration-dependent rectification is not due to changes in the blocking affinity of Mg 2+ or polyamines [14], implying that there is another element involved in this property. In the current study, we investigated the role and contribution of the novel agonist-specific [26][27][28] voltage sensitivity of M 2 R [3,4] to overall I KACh inward rectification. To our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G proteincoupled receptor. ...
... The rectification profile of I KACh is determined by the agonist-specific, voltage-sensitive properties of the M 2 R K ACh channels conduct current that inwardly rectifies when activated by ACh, and yet outwardly rectifies when activated by other ligands (e.g., pilocarpine and choline). The perplexing and paradoxical behavior of K ACh channels is due to the intrinsic voltage sensitivity of M 2 R, whereby the affinity of the receptor for distinct ligands varies in both a voltage-and ligand-specific manner [4,[26][27][28]. Unlike ACh, the affinity for pilocarpine (Pilo) increases with membrane depolarization, while decreasing with hyperpolarization such that M 2 R is~5-fold more sensitive to Pilo at + 50 mV as compared to − 100 mV [25]. ...
The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60–70% of inward rectification, with M2R voltage sensitivity contributing 30–40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.
... Charge movements within membrane proteins, such as the coupled transfer of Na + ions and protons suggested by our MD simulations and pK a calculations, should be sensitive to the membrane voltage. Indeed, it has been demonstrated that GPCR signaling is modulated by membrane voltage changes (Ben-Chaim et al., 2006;Mahaut-Smith et al., 2008;Martinez-Pinna et al., 2004;Moreno-Galindo et al., 2016;Rinne et al., 2015;Vickery et al., 2016a). This applies both to the conformation of the receptors as well as their transmitted signal. ...
Playing a central role in cell signaling, G-protein-coupled receptors (GPCRs) are the largest super-family of membrane proteins and form the majority of drug targets in humans. How extracellular agonist binding triggers the activation of GPCRs and associated intracellular effector proteins remains, however, poorly understood. Structural studies have revealed that inactive class A GPCRs harbor a conserved binding site for Na + ions in the center of their transmembrane domain, accessible from the extra-cellular space. Here, we show that the opening of a conserved hydrated channel in the activated state receptors allows the Na + ion to egress from its binding site into the cytosol. Coupled with protonation changes, this ion movement occurs without significant energy barriers, and can be driven by physiological transmembrane ion and voltage gradients. We propose that Na + ion exchange with the cytosol is a key step in GPCR activation. Further, we hypothesize that this transition locks receptors in long-lived active-state conformations.
... The M2 muscarinic receptor is the one of the most intensively studied GPCRs in its voltage-dependent behaviors and molecular mechanisms 1-4, 6-12 . For example, membrane hyperpolarization increases the affinity of the M2 muscarinic receptor for its native full agonist, acetylcholine (ACh), and decreases affinity for its partial agonist pilocarpine 3,9,10 . Conversely, pilocarpine binding enhances the voltage-induced conformational change of the M2 muscarinic receptor, while ACh binding immobilizes the charge movement 4 . ...
Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K+ channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors.
... This proposal is supported by findings in D2 dopaminergic (Sahlholm et al., 2008(Sahlholm et al., , 2011, a2A adrenergic (Rinne et al., 2013), and Gq-coupled muscarinic receptors, M 1 muscarinic receptor, M 3 muscarinic receptor, and M 5 muscarinic receptor (Rinne et al., 2015). Agonist-specific voltage sensitivity was termed to denote the relationship between specific agonists and voltage (Sahlholm et al., 2008(Sahlholm et al., , 2011Navarro-Polanco et al., 2011;Moreno-Galindo et al., 2016). ...
Potassium (K+) channels are crucial for determining the shape, duration and frequency of action potential firing in excitable cells. Broadly speaking, K+ channels can be classified based on whether their macroscopic current outwardly or inwardly rectifies, whereby rectification refers to a change in conductance with voltage. Outwardly rectifying K+ channels conduct greater current at depolarized membrane potentials, while inward rectifier channels conduct greater current at hyperpolarized membrane potentials. Under most circumstances, outward current of inwardly rectifying K+ channels reduces at more depolarized potentials. However, the acetylcholine-gated inward rectifying K+ channel (KACh) conducts current that inwardly rectifies when activated by some ligands (such as acetylcholine) and yet conducts current that outwardly rectifies when activated by other ligands (for example, pilocarpine and choline). The perplexing and paradoxical behavior of KACh channels is due to the intrinsic voltage-sensitivity of the receptor that activates KACh channels, the M2 muscarinic receptor (M2R). Emerging evidence reveals that the affinity of M2R for distinct ligands varies in a voltage-dependent and ligand-specific manner. These intrinsic properties of the receptor determine whether current conducted by KACh channels inwardly or outwardly rectifies. This mini-review summarizes the most recent concepts regarding the intrinsic voltage-sensitivity of muscarinic receptors and the consequences of this intriguing behavior on cardiac physiology and pharmacology of KACh channels.
