Signaling by many heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) is either enhanced or attenuated by changes in plasma membrane potential. To identify structural correlates of the voltage sensitivity of GPCR signaling, we chose muscarinic acetylcholine receptors (the M1, M3, and M5 isoforms) as a model system. We combined molecular docking analysis with Förster resonance energy transfer (FRET)-based assays that monitored receptor activity under voltage clamp conditions. When human embryonic kidney (HEK) 293 cells expressing the individual receptors were stimulated with the agonist carbachol, membrane depolarization enhanced signaling by the M1 receptor but attenuated signaling by the M3 and M5 receptors. Furthermore, whether membrane depolarization enhanced or inhibited receptor signaling depended on the type of agonist. Membrane depolarization attenuated M3 receptor signaling when the receptor was bound to carbachol or acetylcholine, whereas depolarization enhanced signaling when the receptor was bound to either choline or pilocarpine. Docking calculations predicted that there were two distinct binding modes for these ligands, which were associated with the effect of depolarization on receptor function. From these calculations, we identified a residue in the M3 receptor that, when mutated, would alter the binding mode of carbachol to resemble that of pilocarpine in silico. Introduction of this mutated M3 receptor into cells confirmed that the membrane depolarization enhanced, rather than attenuated, signaling by the carbachol-bound receptor. Together, these data suggest that the directionality of the voltage sensitivity of GPCR signaling is defined by the specific binding mode of each ligand to the receptor.

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...

Proteins of the NFAT family (Nuclear Factor of Activated T-Cells) are Ca 2+ -sensitive transcription factors, which are involved in hypertrophic cardiovascular remodeling. Activation and nuclear translocation is mediated by dephosphorylation by the Ca 2+ -sensitive phosphatase calcineurin (CaN). We identified Ca 2+ signals that induced nuclear translocation of NFAT in cultured calf pulmonary artery endothelial (CPAE) cells using confocal fluorescence microscopy to measure simultaneously [Ca 2+ ] i and subcellular localization of NFAT-GFP (isoforms NFATc1 and NFATc3). The vasoactive agonists ATP (5 μM) or bradykinin (20 μM) in the presence of 2 mM extracellular Ca 2+ induced Ca 2+ release from the endoplasmic reticulum (ER) and activated capacitative Ca 2+ entry (CCE), which caused robust translocation of NFAT to the nucleus. This effect was sensitive to the CaNinhibitor Cyclosporin A (1 μM). Influx of extracellular Ca 2+ via CCE, but not ER Ca 2+ release was identified as the activating Ca 2+ source. NFAT was also activated by Ca 2+ influx induced by cell swelling, reverse mode Na/Ca exchange or ionomycin treatment. NFAT regulation was isoformspecific. Whereas activation of NFATc1-GFP by ATP resulted in persistent nuclear localization, NFATc3-GFP was only transiently imported into the nucleus, followed by rapid export back to the cytoplasm. Inhibition of nuclear kinases, which mediate export of NFAT via phosphorylation, or direct block of nuclear export (Leptomycin B) resulted in stable nuclear localization of NFATc3. These data demonstrate that extracellular Ca 2+ entry mediates NFAT activation. Furthermore, the regulation of nuclear localization of NFAT is isoform-specific and dependent on nuclear export processes.

In SHR, hypertension induces early subcellular LA myocyte Ca2+ remodelling during compensated LV hypertrophy. In basal conditions, atrial myocyte CaTs are not changed. At increased stimulation frequency, however, SHR atrial myocytes become more prone to arrhythmogenic Ca2+ alternans, suggesting a link between hypertension, atrial Ca2+ homeostasis, and development of atrial tachyarrhythmias.

Nuclear factors of activated T cells (NFATs) are Ca(2+)-sensitive transcription factors that have been implicated in hypertrophy, heart failure (HF), and arrhythmias. Cytosolic NFAT is activated by dephosphorylation by the Ca(2+)-sensitive phosphatase calcineurin, resulting in translocation to the nucleus, which is opposed by kinase activity, rephosphorylation, and nuclear export. Four different NFAT isoforms are expressed in the heart. The activation and regulation of NFAT in adult cardiac myocytes, which may depend on the NFAT isoform and cell type, are not fully understood. This study compared basal localization, import, and export of NFATc1 and NFATc3 in adult atrial and ventricular myocytes to identify isoform- and tissue-specific regulatory mechanisms of NFAT activation under physiological conditions and in HF. NFAT-green fluorescent protein fusion proteins and NFAT immunocytochemistry were used to analyze NFAT regulation in adult cat and rabbit myocytes. NFATc1 displayed basal nuclear localization in atrial and ventricular myocytes, an effect that was attenuated by reducing intracellular Ca(2+) concentration and inhibiting calcineurin, and enhanced by the inhibition of nuclear export. In contrast, NFATc3 was localized to the cytoplasm but could be driven to the nucleus by angiotensin II and endothelin-1 stimulation in atrial, but not ventricular, cells. Inhibition of nuclear export (by leptomycin B) facilitated nuclear localization in both cell types. Ventricular myocytes from HF rabbits showed increased basal nuclear localization of endogenous NFATc3 and reduced responsiveness of NFAT translocation to phenylephrine stimulation. In control myocytes, Ca(2+) overload, leading to spontaneous Ca(2+) waves, induced substantial translocation of NFATc3 to the nucleus. We conclude that the activation of NFAT in adult cardiomyocytes is isoform and tissue specific and is tightly controlled by nuclear export. NFAT is activated in myocytes from HF animals and may be secondary to Ca(2+) overload.