RESUMEN
Circadian rhythms synchronize to light through the retinohypothalamic tract (RHT), which is a bundle of axons coming from melanopsin retinal ganglion cells, whose synaptic terminals release glutamate to the ventral suprachiasmatic nucleus (SCN). Activation of AMPA-kainate and NMDA postsynaptic receptors elicits the increase in intracellular calcium required for triggering the signaling cascade that ends in phase shifts. During aging, there is a decline in the synchronization of circadian rhythms to light. With electrophysiological (whole-cell patch-clamp) and immunohistochemical assays, in this work, we studied pre- and postsynaptic properties between the RHT and ventral SCN neurons in young adult (P90-120) and old (P540-650) C57BL/6J mice. Incremental stimulation intensities (applied on the optic chiasm) induced much lesser AMPA-kainate postsynaptic responses in old animals, implying a lower recruitment of RHT fibers. Conversely, a higher proportion of old SCN neurons exhibited synaptic facilitation, and variance-mean analysis indicated an increase in the probability of release in RHT terminals. Moreover, both spontaneous and miniature postsynaptic events displayed larger amplitudes in neurons from aged mice, whereas analysis of the NMDA and AMPA-kainate components (evoked by RHT electrical stimulation) disclosed no difference between the two ages studied. Immunohistochemistry revealed a bigger size in the puncta of vGluT2, GluN2B, and GluN2A of elderly animals, and the number of immunopositive particles was increased, but that of PSD-95 was reduced. All these synaptic adaptations could be part of compensatory mechanisms in the glutamatergic signaling to ameliorate the loss of RHT terminals in old animals.
Asunto(s)
Envejecimiento , Ácido Glutámico , Ratones Endogámicos C57BL , Núcleo Supraquiasmático , Transmisión Sináptica , Animales , Ratones , Núcleo Supraquiasmático/fisiología , Núcleo Supraquiasmático/metabolismo , Transmisión Sináptica/fisiología , Envejecimiento/fisiología , Ácido Glutámico/metabolismo , Masculino , Potenciales Postsinápticos Excitadores/fisiología , Vías Visuales/fisiología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Técnicas de Placa-Clamp , Receptores de N-Metil-D-Aspartato/metabolismo , Homólogo 4 de la Proteína Discs Large/metabolismoRESUMEN
The master control of mammalian circadian rhythms is the suprachiasmatic nucleus (SCN), which is formed by the ventral and dorsal regions. In SCN neurons, GABA has an important function and even excitatory actions in adulthood. However, the physiological role of this neurotransmitter in the developing SCN is unknown. Here, we recorded GABAergic postsynaptic currents (in the perforated-patch configuration using gramicidin) to determine the chloride reversal potential (ECl) and also assessed the immunological expression of the Na-K-Cl cotransporter 1 (NKCC1) at early ages of the rat (postnatal days (P) 3 to 25), during the day and night, in the two SCN regions. We detected that ECl greatly varied with age and depending on the SCN region and time of day. Broadly speaking, ECl was more hyperpolarized with age, except for the oldest age studied (P20-25) in both day and night in the ventral SCN, where it was less negative. Likewise, ECl was more hyperpolarized in the dorsal SCN both during the day and at night; while ECl was more negative at night both in the ventral and the dorsal SCN. Moreover, the total NKCC1 fluorescent expression was higher during the day than at night. These results imply that NKCC1 regulates the circadian and developmental fluctuations in the [Cl-]i to fine-tune ECl, which is crucial for either excitatory or inhibitory GABAergic actions to occur in the SCN.
Asunto(s)
Cloruros , Ritmo Circadiano , Miembro 2 de la Familia de Transportadores de Soluto 12 , Núcleo Supraquiasmático , Animales , Núcleo Supraquiasmático/metabolismo , Ritmo Circadiano/fisiología , Ratas , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Masculino , Cloruros/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Ratas Wistar , Técnicas de Placa-Clamp , Envejecimiento/fisiologíaRESUMEN
The suprachiasmatic nucleus (SCN) is the most important circadian clock in mammals. The SCN synchronizes to environmental light via the retinohypothalamic tract (RHT), which is an axon cluster derived from melanopsin-expressing intrinsic photosensitive retinal ganglion cells. Investigations on the development of the nonimage-forming pathway and the RHT are scarce. Previous studies imply that light stimulation during postnatal development is not needed to make the RHT functional at adult stages. Here, we examined the effects of light deprivation (i.e., constant darkness (DD) rearing) during postnatal development on the expression in the ventral SCN of two crucial proteins for the synchronization of circadian rhythms to light: the presynaptic vesicular glutamate transporter type 2 (vGluT2) and the GluN2B subunit of the postsynaptic NMDA receptor. We found that animals submitted to DD conditions exhibited a transitory reduction in the expression of vGluT2 (at P12-19) and of GluN2B (at P7-9) that was compensated at older stages. These findings support the hypothesis that visual stimulation during early ages is not decisive for normal development of the RHT-SCN pathway.
Asunto(s)
Receptores de N-Metil-D-Aspartato , Núcleo Supraquiasmático , Proteína 2 de Transporte Vesicular de Glutamato , Animales , Ratas , Ritmo Circadiano/fisiología , Mamíferos/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Células Ganglionares de la Retina/metabolismo , Núcleo Supraquiasmático/metabolismo , Proteína 2 de Transporte Vesicular de Glutamato/metabolismoRESUMEN
The hypothalamic suprachiasmatic nucleus (SCN) is the leading circadian pacemaker in mammals, which synchronizes with environmental light through the retinohypothalamic tract (RHT). Although the SCN regulates circadian rhythms before birth, postnatal synaptic changes are needed for the RHT-SCN pathway to achieve total functional development. However, it is unknown whether visual experience affects developmental maturation. Here, we studied the effects of constant darkness (DD) rearing on the physiology (at pre- and postsynaptic levels) of glutamatergic neurotransmission between RHT and SCN during postnatal development in rats. Upon recording spontaneous and evoked excitatory postsynaptic currents (EPSCs) by electrical stimulation of RHT fibers, we found that DD animals at early postnatal ages (P3-19) exhibited different frequencies of spontaneous EPSCs and lower synaptic performance (short-term depression, release sites, and recruitment of RHT fibers) when compared with their normal light/dark (LD) counterparts. At the oldest age evaluated (P30-35), there was a synaptic response strengthening (probability of release, vesicular re-filling rate, and reduced synaptic depression) in DD rats, which functionally equaled (or surmounted) that of LD animals. Control experiments evaluating EPSCs in ventral SCN neurons of LD rats during day and night revealed no significant differences in spontaneous or evoked EPSCs by high-frequency trains in the RHT at any postnatal age. Our results suggest that DD conditions induce a compensatory mechanism in the glutamatergic signaling of the circadian system to increase the chances of synchronization to light at adult ages, and that the synaptic properties of RHT terminals during postnatal development are not critically influenced by environmental light.
Asunto(s)
Neuronas del Núcleo Supraquiasmático , Núcleo Supraquiasmático , Animales , Ritmo Circadiano , Potenciales Postsinápticos Excitadores , Luz , Ratas , Transmisión SinápticaRESUMEN
The cardiac muscarinic receptor (M2R) regulates heart rate, in part, by modulating the acetylcholine (ACh) activated K+ current IK,ACh through dissociation of G-proteins, that in turn activate KACh channels. Recently, M2Rs were noted to exhibit intrinsic voltage sensitivity, i.e. their affinity for ligands varies in a voltage dependent manner. The voltage sensitivity of M2R implies that the affinity for ACh (and thus the ACh effect) varies throughout the time course of a cardiac electrical cycle. The aim of this study was to investigate the contribution of M2R voltage sensitivity to the rate and shape of the human sinus node action potentials in physiological and pathophysiological conditions. We developed a Markovian model of the IK,ACh modulation by voltage and integrated it into a computational model of human sinus node. We performed simulations with the integrated model varying ACh concentration and voltage sensitivity. Low ACh exerted a larger effect on IK,ACh at hyperpolarized versus depolarized membrane voltages. This led to a slowing of the pacemaker rate due to an attenuated slope of phase 4 depolarization with only marginal effect on action potential duration and amplitude. We also simulated the theoretical effects of genetic variants that alter the voltage sensitivity of M2R. Modest negative shifts in voltage sensitivity, predicted to increase the affinity of the receptor for ACh, slowed the rate of phase 4 depolarization and slowed heart rate, while modest positive shifts increased heart rate. These simulations support our hypothesis that altered M2R voltage sensitivity contributes to disease and provide a novel mechanistic foundation to study clinical disorders such as atrial fibrillation and inappropriate sinus tachycardia.
Asunto(s)
Modelos Cardiovasculares , Receptor Muscarínico M2/fisiología , Nodo Sinoatrial/fisiología , Acetilcolina/metabolismo , Biología Computacional , Humanos , Cadenas de MarkovRESUMEN
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.
Asunto(s)
Potenciales de Acción , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Miocitos Cardíacos/metabolismo , Receptor Muscarínico M2/metabolismo , Acetilcolina/metabolismo , Animales , Gatos , Células Cultivadas , Femenino , Atrios Cardíacos/citología , Magnesio/metabolismo , Masculino , Miocitos Cardíacos/fisiología , Poliaminas/metabolismoRESUMEN
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, whereas inward rectifier channels conduct greater current at hyperpolarized membrane potentials. Under most circumstances, outward currents through inwardly rectifying K(+) channels are reduced at more depolarized potentials. However, the acetylcholine-gated 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 receptor properties determine whether current conducted by KACh channels inwardly or outwardly rectifies. This 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.
Asunto(s)
Canales de Potasio de Rectificación Interna/metabolismo , Canales de Potasio/metabolismo , Receptores Muscarínicos/metabolismo , Animales , Corazón/fisiología , Humanos , Activación del Canal Iónico , Sistema Nervioso Parasimpático/fisiologíaRESUMEN
Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca²âº and Mg²âº. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd³âº (10 µM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1 µM) and SKF 96365 (250 µM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100 µM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50 µM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca²âº signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.
Asunto(s)
Calcio/metabolismo , Activación del Canal Iónico/fisiología , Mecanotransducción Celular/fisiología , Canales Catiónicos TRPV/metabolismo , Amilorida/farmacología , Compuestos de Boro/farmacología , Expresión Génica , Humanos , Imidazoles/farmacología , Transporte Iónico/efectos de los fármacos , Transporte Iónico/fisiología , Células Jurkat , Leucemia de Células T/genética , Leucemia de Células T/metabolismo , Leucemia de Células T/fisiopatología , Magnesio/metabolismo , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rojo de Rutenio/farmacología , Sodio/metabolismo , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/genéticaRESUMEN
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, pilocarpine (Pilo), and bethanechol (Beth), we evaluated the agonist-specific effects of voltage by measuring the ACh-activated K(+) current (I KACh) in feline and rabbit atrial myocytes and in HEK-293 cells expressing M2R-Kir3.1/Kir3.4. The activation of I KACh by the muscarinic agonist Beth was voltage insensitive, suggesting that the voltage-induced conformational changes in M2R do not modify its affinity for this agonist. Moreover, deactivation of the Beth-evoked I KACh was voltage insensitive. By contrast, deactivation of the ACh-induced I KACh was significantly slower at -100 mV than at +50 mV, while an opposite effect was observed when I KACh was activated by Pilo. These findings are consistent with the voltage affinity pattern observed for these three agonists. Our findings suggest that independent of how voltage disturbs the receptor binding site, the voltage dependence of the signaling pathway is ultimately determined by the agonist. These observations emphasize the pharmacological potential to regulate the M2R-parasympathetic associated cardiac function and also other cellular signaling pathways by exploiting the voltage-dependent properties of GPCRs.
Asunto(s)
Acetilcolina/farmacología , Activación del Canal Iónico/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Canales de Potasio/metabolismo , Potasio/metabolismo , Receptor Muscarínico M2/metabolismo , Animales , Sitios de Unión/efectos de los fármacos , Gatos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Células HEK293 , Humanos , Masculino , Potenciales de la Membrana/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Conejos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
The human intestinal pathogen Giardia lamblia is a flagellated unicellular protozoan with pronounced medical and biological relevance. However, the basic physiology of Giardia trophozoites has been sparsely studied, especially the electrical and ionic properties of their cellular membrane which are virtually unknown. In this study, we were able to record and characterize the macroscopic ionic currents of Giardia trophozoite membrane by electrophysiological methods of the patch clamp technique. Giardia trophozoites showed a high current density (â¼600 pA/pF at -140 mV) that was activated upon hyperpolarization. This current was carried by a chloride-selective channel (I Cl-G) and it was the most important determinant of the membrane potential in Giardia trophozoites. Moreover, this conductance was able to carry other halide anions and the sequence of permeability was Br(-) > Cl(-) ≈ I(-) â« F(-). Besides the voltage-dependent inward-rectifying nature of I Cl-G, its activation and deactivation kinetics were comparable to those observed in ClC-2 channels. Extracellular pH modified the voltage-dependent properties of I Cl-G, shifting the activation curve from a V 1/2 = -79 ± 1 mV (pH 7.4) to -93 ± 2 mV (pH 8.4) and -112 ± 2 mV (pH 5.4). Furthermore, the maximal amplitude of I Cl-G measured at -100 mV showed dependence to external pH in a bell-shaped fashion reported only for ClC-2 channels. Therefore, our results suggest that I Cl-G possesses several functional properties similar to the mammalian ClC-2 channels.
Asunto(s)
Potenciales de Acción , Canales de Cloruro/metabolismo , Giardia lamblia/metabolismo , Proteínas Protozoarias/metabolismo , Trofozoítos/metabolismo , Canales de Cloruro CLC-2 , Cloruros/metabolismo , Giardia lamblia/crecimiento & desarrollo , Giardia lamblia/fisiología , Potenciales de la Membrana , Trofozoítos/fisiologíaRESUMEN
Choline (Ch) is a precursor and metabolite of the neurotransmitter acetylcholine (ACh). In canine and guinea pig atrial myocytes, Ch was shown to activate an outward K(+) current in a delayed rectifier fashion. This current has been suggested to modulate cardiac electrical activity and to play a role in atrial fibrillation pathophysiology. However, the exact nature and identity of this current has not been convincingly established. We recently described the unique ligand- and voltage-dependent properties of muscarinic activation of ACh-activated K(+) current (IKACh) and showed that, in contrast to ACh, pilocarpine induces a current with delayed rectifier-like properties with membrane depolarization. Here, we tested the hypothesis that Ch activates IKACh in feline atrial myocytes in a voltage-dependent manner similar to pilocarpine. Single-channel recordings, biophysical profiles, specific pharmacological inhibition and computational data indicate that the current activated by Ch is IKACh. Moreover, we show that membrane depolarization increases the potency and efficacy of IKACh activation by Ch and thus gives the appearance of a delayed rectifier activating K(+) current at depolarized potentials. Our findings support the emerging concept that IKACh modulation is both voltage- and ligand-specific and reinforce the importance of these properties in understanding cardiac physiology.
Asunto(s)
Potenciales de Acción , Canales de Potasio de Tipo Rectificador Tardío/metabolismo , Atrios Cardíacos/metabolismo , Miocitos Cardíacos/metabolismo , Receptor Muscarínico M2/metabolismo , Animales , Gatos , Colina/farmacología , Femenino , Atrios Cardíacos/citología , Masculino , Potenciales de la Membrana , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Pilocarpina/farmacologíaRESUMEN
Inhibitory regulation of the heart is determined by both cholinergic M2 receptors (M2R) and adenosine A1 receptors (A1R) that activate the same signaling pathway, the ACh-gated inward rectifier K+ (KACh) channels via Gi/o proteins. Previously, we have shown that the agonist-specific voltage sensitivity of M2R underlies several voltage-dependent features of IKACh, 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 A1R and how this could impact IKACh. Upon recording whole-cell currents of guinea-pig cardiomyocytes, we found that stimulation of the A1R-Gi/o-IKACh pathway with adenosine only caused a very slight voltage dependence in concentration-response relationships (~1.2-fold EC50 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, IKACh did not exhibit relaxation. Contrarily, activation of the M2R-Gi/o-IKACh 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 EC50 increase with depolarization) and in the IKACh 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 IKACh relaxation is consequence of this property.
Asunto(s)
Acetilcolina/farmacología , Agonistas del Receptor de Adenosina A1/farmacología , Adenosina/farmacología , Activación del Canal Iónico/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Canales de Potasio/metabolismo , Receptor de Adenosina A1/metabolismo , Animales , Femenino , Cobayas , Masculino , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/metabolismoRESUMEN
Normal heart rate variability is critically dependent upon the G-protein-coupled, acetylcholine (ACh)-activated inward rectifier K+ current, I(KACh). A unique feature of I(KACh) is the so-called 'relaxation' gating property that contributes to increased current at hyperpolarized membrane potentials. I(KACh) relaxation refers to a slow decrease or increase in current magnitude with depolarization or hyperpolarization, respectively. The molecular mechanism underlying this perplexing gating behaviour remains unclear. Here, we consider a novel explanation for I(KACh) relaxation based upon the recent finding that G-protein-coupled receptors (GPCRs) are intrinsically voltage sensitive and that the muscarinic agonists acetylcholine (ACh) and pilocarpine (Pilo) manifest opposite voltage-dependent I(KACh) modulation. We show that Pilo activation of I(KACh) displays relaxation characteristics opposite to that of ACh. We explain the opposite effects of ACh and Pilo using Markov models of I(KACh) that incorporate ligand-specific, voltage-dependent parameters. Based on experimental and computational findings, we propose a novel molecular mechanism to describe the enigmatic relaxation gating process: I(KACh) relaxation represents a voltage-dependent change in agonist affinity as a consequence of a voltage-dependent conformational change in the muscarinic receptor.
Asunto(s)
Acetilcolina/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Receptores Muscarínicos/metabolismo , Acetilcolina/farmacología , Animales , Venenos de Abeja/farmacología , Gatos , Técnicas In Vitro , Activación del Canal Iónico/efectos de los fármacos , Cadenas de Markov , Potenciales de la Membrana , Modelos Biológicos , Agonistas Muscarínicos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Pilocarpina/farmacología , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Conformación Proteica , Receptores Muscarínicos/químicaRESUMEN
The ability to sense transmembrane voltage is a central feature of many membrane proteins, most notably voltage-gated ion channels. Gating current measurements provide valuable information on protein conformational changes induced by voltage. The recent observation that muscarinic G-protein-coupled receptors (GPCRs) generate gating currents confirms their intrinsic capacity to sense the membrane electrical field. Here, we studied the effect of voltage on agonist activation of M2 muscarinic receptors (M2R) in atrial myocytes and how agonist binding alters M2R gating currents. Membrane depolarization decreased the potency of acetylcholine (ACh), but increased the potency and efficacy of pilocarpine (Pilo), as measured by ACh-activated K+ current, I(KACh). Voltage-induced conformational changes in M2R were modified in a ligand-selective manner: ACh reduced gating charge displacement while Pilo increased the amount of charge displaced. Thus, these ligands manifest opposite voltage-dependent I(KACh) modulation and exert opposite effects on M2R gating charge displacement. Finally, mutations in the putative ligand binding site perturbed the movement of the M2R voltage sensor. Our data suggest that changes in voltage induce conformational changes in the ligand binding site that alter the agonistreceptor interaction in a ligand-dependent manner. Voltage-dependent GPCR modulation has important implications for cellular signalling in excitable tissues. Gating current measurement allows for the tracking of subtle conformational changes in the receptor that accompany agonist binding and changes in membrane voltage.
Asunto(s)
Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Acetilcolina/farmacología , Sustitución de Aminoácidos , Animales , Sitios de Unión/genética , Gatos , Femenino , Células HEK293 , Humanos , Técnicas In Vitro , Activación del Canal Iónico , Potenciales de la Membrana , Modelos Moleculares , Agonistas Muscarínicos/farmacología , Mutagénesis Sitio-Dirigida , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Pilocarpina/farmacología , Conformación Proteica , Receptor Muscarínico M2/agonistas , Receptor Muscarínico M2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Pilocarpine is a nonspecific agonist of muscarinic receptors which was recently found to activate the M(2) receptor subtype in a voltage-dependent manner. The purpose of our study was to investigate the role of the acetylcholine (muscarinic)-activated K(+) current (I (KACh)) on the negative chronotropic effect of pilocarpine in rabbit sinoatrial node. In multicellular preparations, we studied the effect of pilocarpine on spontaneous action potentials. In isolated myocytes, using the patch clamp technique, we studied the effects of pilocarpine on I (KACh). Pilocarpine produced a decrease in spontaneous frequency, hyperpolarization of the maximum diastolic potential, and a decrease in the diastolic depolarization rate. These effects were partially antagonized by tertiapin Q. Cesium and calyculin A in the presence of tertiapin Q partially prevented the effects of pilocarpine. In isolated myocytes, pilocarpine activated the muscarinic potassium current, I (KACh) in a voltage-dependent manner. In conclusion, the negative chronotropic effects of pilocarpine on the sinatrial node could be mainly explained by activation of I (KACh).
Asunto(s)
Acetilcolina/metabolismo , Potenciales de Acción/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Pilocarpina/farmacología , Canales de Potasio/metabolismo , Potasio/metabolismo , Nodo Sinoatrial/efectos de los fármacos , Animales , Venenos de Abeja/farmacología , Cesio/metabolismo , Inhibidores Enzimáticos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Toxinas Marinas , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Oxazoles/farmacología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Conejos , Nodo Sinoatrial/fisiologíaRESUMEN
Cardiac inward rectifier potassium currents determine the resting membrane potential and contribute repolarization capacity during phase 3 repolarization. Quinacrine is a cationic amphiphilic drug. In this work, the effects of quinacrine were studied on cardiac Kir channels expressed in HEK 293 cells and on the inward rectifier potassium currents, I(K1) and I(KATP), in cardiac myocytes. We found that quinacrine differentially inhibited Kir channels, Kir6.2 â¼ Kir2.3 > Kir2.1. In addition, we found in cardiac myocytes that quinacrine inhibited I(KATP) > I(K1). We presented evidence that quinacrine displays a double action towards strong inward rectifier Kir2.x channels, i.e., direct pore block and interference in phosphatidylinositol 4,5-bisphosphate, PIP(2)-Kir channel interaction. Pore block is evident in Kir2.1 and 2.3 channels as rapid block; channel block involves residues E224 and E299 facing the cytoplasmic pore of Kir2.1. The interference of the drug with the interaction of Kir2.x and Kir6.2/SUR2A channels and PIP(2) is suggested from four sources of evidence: (1) Slow onset of current block when quinacrine is applied from either the inside or the outside of the channel. (2) Mutation of Kir2.3(I213L) and mutation of Kir6.2(C166S) increase their affinity for PIP(2) and lowers its sensitivity for quinacrine. (3) Mutations of Kir2.1(L222I and K182Q) which decreased its affinity for PIP(2) increased its sensitivity for quinacrine. (4) Co-application of quinacrine with PIP(2) lowers quinacrine-mediated current inhibition. In conclusion, our data demonstrate how an old drug provides insight into a dual a blocking mechanism of Kir carried inward rectifier channels.
Asunto(s)
Miocitos Cardíacos/fisiología , Fosfatidilinositol 4,5-Difosfato/fisiología , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Canales de Potasio de Rectificación Interna/fisiología , Células HEK293 , Humanos , Quinacrina/farmacologíaRESUMEN
The antimalarial drug mefloquine was found to inhibit the KATP channel by an unknown mechanism. Because mefloquine is a Cationic amphiphilic drug and is known to insert into lipid bilayers, we postulate that mefloquine interferes with the interaction between PIP2 and Kir channels resulting in channel inhibition. We studied the inhibitory effects of mefloquine on Kir2.1, Kir2.3, Kir2.3(I213L), and Kir6.2/SUR2A channels expressed in HEK-293 cells, and on IK1 and IKATP from feline cardiac myocytes. The order of mefloquine inhibition was Kir6.2/SUR2A ≈ Kir2.3 (IC50 ≈ 2 µM) > Kir2.1 (IC50 > 30 µM). Similar results were obtained in cardiac myocytes. The Kir2.3(I213L) mutant, which enhances the strength of interaction with PIP2 (compared to WT), was significantly less sensitive (IC50 = 9 µM). In inside-out patches, continuous application of PIP2 strikingly prevented the mefloquine inhibition. Our results support the idea that mefloquine interferes with PIP2-Kir channels interactions.
Asunto(s)
Antimaláricos/farmacología , Mefloquina/farmacología , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Animales , Antimaláricos/administración & dosificación , Gatos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Canales KATP/antagonistas & inhibidores , Mefloquina/administración & dosificación , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/administración & dosificación , Bloqueadores de los Canales de Potasio/farmacologíaRESUMEN
The suprachiasmatic nucleus (SCN) of the hypothalamus is the brain structure that controls circadian rhythms in mammals. The SCN is formed by two neuroanatomical regions: the ventral and dorsal. Gamma-aminobutyric acid (GABA) neurotransmission is important for the regulation of circadian rhythms. Excitatory GABA effects have been described in both SCN regions displaying a circadian variation. Moreover, the GABAergic system transfers photic information from the ventral to the dorsal SCN. However, there is almost no knowledge about GABA neurotransmission during the prenatal or postnatal development of the SCN. Here, we used whole-cell patch-clamp recordings to study spontaneous inhibitory postsynaptic currents (IPSCs) in the two SCN regions, at two zeitgeber times (day or night), and at four postnatal (P) ages: P3-5, P7-9, P12-15, and P20-25. The results herein show that the three analyzed parameters of the IPSCs, frequency, amplitude, and decay time, were significantly affected by the postnatal age: mostly, the IPSC frequency increased with age, principally in the ventral SCN in both day and night recordings; similarly, the amplitude of IPSCs augmented with age, especially at night, whereas the IPSC decay time was reduced (it was faster) with postnatal age, mainly during the day. Our findings first reveal that parameters of GABA neurotransmission are modified by postnatal development, implying that synaptic adjustments are required for an appropriate maturation of the GABAergic system in the SCN.
Asunto(s)
Ritmo Circadiano , Núcleo Supraquiasmático , Animales , Técnicas de Placa-Clamp , Ratas , Transmisión Sináptica , Ácido gamma-AminobutíricoRESUMEN
Inwardly rectifying potassium (Kir) channels are broadly expressed in both excitable and nonexcitable tissues, where they contribute to a wide variety of cellular functions. Numerous studies have established that rectification of Kir channels is not an inherent property of the channel protein itself, but rather reflects strong voltage dependence of channel block by intracellular cations, such as polyamines and Mg2+. Here, we identify a previously unknown mechanism of inward rectification in Kir4.1/Kir5.1 channels in the absence of these endogenous blockers. This novel intrinsic rectification originates from the voltage-dependent behavior of Kir4.1/Kir5.1, which is generated by the flux of potassium ions through the channel pore; the inward K+-flux induces the opening of the gate, whereas the outward flux is unable to maintain the gate open. This gating mechanism powered by the K+-flux is convergent with the gating of PIP2 because, at a saturating concentration, PIP2 greatly reduces the inward rectification. Our findings provide evidence of the coexistence of two rectification mechanisms in Kir4.1/Kir5.1 channels: the classical inward rectification induced by blocking cations and an intrinsic voltage-dependent mechanism generated by the K+-flux gating.
Asunto(s)
Canales de Potasio de Rectificación Interna , Iones , Potasio , Bloqueadores de los Canales de PotasioRESUMEN
Riluzole is an anticonvulsant drug also used to treat the amyotrophic lateral sclerosis and major depressive disorder. This compound has antiglutamatergic activity and is an important multichannel blocker. However, little is known about its actions on the Kv4.2 channels, the molecular correlate of the A-type K+ current (IA) and the fast transient outward current (Itof). Here, we investigated the effects of riluzole on Kv4.2 channels transiently expressed in HEK-293 cells. Riluzole inhibited Kv4.2 channels with an IC50 of 190 ± 14 µM and the effect was voltage- and frequency-independent. The activation rate of the current (at +50 mV) was not affected by the drug, nor the voltage dependence of channel activation, but the inactivation rate was accelerated by 100 and 300 µM riluzole. When Kv4.2 channels were maintained at the closed state, riluzole incubation induced a tonic current inhibition. In addition, riluzole significantly shifted the voltage dependence of inactivation to hyperpolarized potentials without affecting the recovery from inactivation. In the presence of the drug, the closed-state inactivation was significantly accelerated, and the percentage of inactivated channels was increased. Altogether, our findings indicate that riluzole inhibits Kv4.2 channels mainly affecting the closed and closed-inactivated states.