Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. ...However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood. Here we examined the contribution of the m2R-I(KACh) intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-I(KACh) signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-I(KACh) signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The time course of signaling via heterotrimeric G proteins is controlled through their activation by G-protein coupled receptors and deactivation through the action of GTPase accelerating proteins ...(GAPs). Here we identify RGS7 and RGS11 as the key GAPs in the mGluR6 pathway of retinal rod ON bipolar cells that set the sensitivity and time course of light-evoked responses. We showed using electroretinography and single cell recordings that the elimination of RGS7 did not influence dark-adapted light-evoked responses, but the concurrent elimination of RGS11 severely reduced their magnitude and dramatically slowed the onset of the response. In RGS7/RGS11 double-knockout mice, light-evoked responses in rod ON bipolar cells were only observed during persistent activation of rod photoreceptors that saturate rods. These observations are consistent with persistently high G-protein activity in rod ON bipolar cell dendrites caused by the absence of the dominant GAP, biasing TRPM1 channels to the closed state.
Our robust visual experience is based on the reliable transfer of information from our photoreceptor cells, the rods and cones, to higher brain centers. At the very first synapse of the visual ...system, information is split into two separate pathways, ON and OFF, which encode increments and decrements in light intensity, respectively. The importance of this segregation is borne out in the fact that receptive fields in higher visual centers maintain a separation between ON and OFF regions. In the past decade, the molecular mechanisms underlying the generation of ON signals have been identified, which are unique in their use of a G-protein signaling cascade. In this review, we consider advances in our understanding of G-protein signaling in ON-bipolar cell (BC) dendrites and how insights about signaling have emerged from visual deficits, mostly night blindness. Studies of G-protein signaling in ON-BCs reveal an intricate mechanism that permits the regulation of visual sensitivity over a wide dynamic range.
Modulation of neuronal circuits is key to information processing in the brain. The majority of neuromodulators exert their effects by activating G-protein-coupled receptors (GPCRs) that control the ...production of second messengers directly impacting cellular physiology. How numerous GPCRs integrate neuromodulatory inputs while accommodating diversity of incoming signals is poorly understood. In this study, we develop an in vivo tool and analytical suite for analyzing GPCR responses by monitoring the dynamics of a key second messenger, cyclic AMP (cAMP), with excellent quantitative and spatiotemporal resolution in various neurons. Using this imaging approach in combination with CRISPR/Cas9 editing and optogenetics, we interrogate neuromodulatory mechanisms of defined populations of neurons in an intact mesolimbic reward circuit and describe how individual inputs generate discrete second-messenger signatures in a cell- and receptor-specific fashion. This offers a resource for studying native neuronal GPCR signaling in real time.
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•CAMPER reporter mice developed to probe neuromodulation in the endogenous setting•CAMPER mice report modulation of cAMP dynamics by a variety of neurotransmitter GPCRs•Probing real-time cAMP flux with optogenetics reports signaling in intact circuits•CAMPER imaging approach reveals principles of dopamine signaling in the striatum
Muntean et al. develop an in vivo reagent to study processing of neurotransmitter GPCR signals by monitoring real-time dynamics of cAMP responses. They demonstrate application of this approach, in combination with CRISPR/Cas9 gene editing and optogenetics, to interrogate the functional organization of a striatal circuit.
The extent and temporal characteristics of G protein-coupled receptor (GPCR) signaling are shaped by the regulator of G protein signaling (RGS) proteins, which promote G protein deactivation. With ...hundreds of GPCRs and dozens of RGS proteins, compartmentalization plays a key role in establishing signaling specificity. However, the molecular details and mechanisms of this process are poorly understood. In this paper, we report that the R7 group of RGS regulators is controlled by interaction with two previously uncharacterized orphan GPCRs: GPR158 and GPR179. We show that GPR158/179 recruited RGS complexes to the plasma membrane and augmented their ability to regulate GPCR signaling. The loss of GPR179 in a mouse model of night blindness prevented targeting of RGS to the postsynaptic compartment of bipolar neurons in the retina, illuminating the role of GPR179 in night vision. We propose that the interaction of RGS proteins with orphan GPCRs promotes signaling selectivity in G protein pathways.
Mechanisms of Gβγ Release upon GPCR Activation Martemyanov, Kirill A.
Trends in biochemical sciences (Amsterdam. Regular ed.),
September 2021, 2021-09-00, 20210901, Letnik:
46, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Gβγ release is a key event in the transduction of GPCR signals. However, the molecular mechanisms of this process have been unclear. A recent report by Knight et al. provides important clues into the ...sequence of events that lead to the liberation of Gβγ upon G protein activation by GPCRs.
Mutations in LRIT3 lead to complete congenital stationary night blindness (cCSNB). The exact role of LRIT3 in ON‐bipolar cell signaling cascade remains to be elucidated. Recently, we have ...characterized a novel mouse model lacking Lrit3 no b‐wave 6, (Lrit3nob6/nob6), which displays similar abnormalities to patients with cCSNB with LRIT3 mutations. Here we compare the localization of components of the ON‐bipolar cell signaling cascade in wild‐type and Lrit3nob6/nob6 retinal sections by immunofluorescence confocal microscopy. An anti‐LRIT3 antibody was generated. Immunofluorescent staining of LRIT3 in wild‐type mice revealed a specific punctate labeling in the outer plexiform layer (OPL), which was absent in Lrit3nob6/nob6 mice. LRIT3 did not co‐localize with ribeye or calbindin but co‐localized with mGluR6. TRPM1 staining was severely decreased at the dendritic tips of all depolarizing bipolar cells in Lrit3nob6/nob6 mice. mGluR6, GPR179, RGS7, RGS11 and Gβ5 immunofluorescence was absent at the dendritic tips of cone ON‐bipolar cells in Lrit3nob6/nob6 mice, while it was present at the dendritic tips of rod bipolar cells. Furthermore, peanut agglutinin (PNA) labeling was severely reduced in the OPL in Lrit3nob6/nob6 mice. This study confirmed the localization of LRIT3 at the dendritic tips of depolarizing bipolar cells in mouse retina and demonstrated the dependence of TRPM1 localization on the presence of LRIT3. As tested components of the ON‐bipolar cell signaling cascade and PNA revealed disrupted localization, an additional function of LRIT3 in cone synapse formation is suggested. These results point to a possibly different regulation of the mGluR6 signaling cascade between rod and cone ON‐bipolar cells.
The focus of this work was to elucidate the role of LRIT3, a protein implicated in complete congenital stationary night blindness (cCSNB), by using the no b‐wave 6 cCSNB mouse model (Lrit3nob6/nob6), lacking Lrit3. We showed that LRIT3 is localized at the dendritic tips of ON‐bipolar cells in mouse retina, that LRIT3 is essential for the correct localization of TRPM1 at the dendritic tips of ON‐bipolar cells and that other components of the cascade reveal disrupted localization at the dendritic tips of cone, but not rod, ON‐bipolar cells, suggesting an additional role of LRIT3 in cone synapse formation.
Parallel visual pathways are initiated at the first retinal synapse by signaling between the rod and cone photoreceptors and two general classes of bipolar cells. For normal function, ON or ...depolarizing bipolar cells (DBCs) require the G-protein-coupled receptor, mGluR6, an intact G-protein-coupled cascade and the transient receptor potential melastatin 1 (TRPM1) cation channel. In addition, another seven transmembrane protein, GPR179, is required for DBC function and recruits the regulators of G-protein signaling (RGS) proteins, RGS7 and RGS11, to the dendritic tips of the DBCs. Here we use the Gpr179(nob5) mouse, which lacks GPR179 and has a no b-wave electroretinogram (ERG) phenotype, to demonstrate that despite the absence of both GPR179 and RGS7/RGS11, a small dark-adapted ERG b-wave remains and can be enhanced with long duration flashes. Consistent with the ERG, the mGluR6-mediated gating of TRPM1 can be evoked pharmacologically in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs if strong stimulation conditions are used. In contrast, direct gating of TRPM1 by capsaicin in RGS7(-/-)/RGS11(-/-) and WT rod BCs is similar, but severely compromised in Gpr179(nob5) rod BCs. Noise and standing current analyses indicate that the remaining channels in Gpr179(nob5) and RGS7(-/-)/RGS11(-/-) rod BCs have a very low open probability. We propose that GPR179 along with RGS7 and RGS11 controls the ability of the mGluR6 cascade to gate TRPM1. In addition to its role in localizing RGS7 and RGS11 to the dendritic tips, GPR179 via a direct interaction with the TRPM1 channel alters its ability to be gated directly by capsaicin.
A large body of work has established the prominent roles of the atrial M2R-IKACh signaling pathway, and the negative regulatory protein RGS6, in modulating critical aspects of parasympathetic ...influence on cardiac function, including pace-making, heart rate (HR) variability (HRV), and atrial arrhythmogenesis. Despite increasing evidence of its innervation of the ventricles, and the expression of M2R, IKACh channel subunits, and RGS6 in ventricle, the effects of parasympathetic modulation on ventricular electrophysiology are less clear. The main objective of our study was to investigate the contribution of M2R-IKACh signaling pathway elements in murine ventricular electrophysiology, using in-vivo ECG measurements, isolated whole-heart optical mapping and constitutive knockout mice lacking IKACh (Girk4-/-) or RGS6 (Rgs6-/-). Consistent with previous findings, mice lacking GIRK4 exhibited diminished HR and HRV responses to the cholinergic agonist carbachol (CCh), and resistance to CCh-induced arrhythmic episodes. In line with its role as a negative regulator of atrial M2R-IKACh signaling, loss of RGS6 correlated with a mild resting bradycardia, enhanced HR and HRV responses to CCh, and increased propensity for arrhythmic episodes. Interestingly, ventricles from mice lacking GIRK4 or RGS6 both exhibited increased action potential duration (APD) at baseline, and APD was prolonged by CCh across all genotypes. Similarly, CCh significantly increased the slope of APD restitution in all genotypes. There was no impact of genotype or CCh on either conduction velocity or heterogeneity. Our data suggests that altered parasympathetic signaling through the M2R-IKACh pathway can affect ventricular electrophysiological properties distinct from its influence on atrial physiology.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
In vertebrate retina, light responses generated by the rod photoreceptors are transmitted to the second-order neurons, the ON-bipolar cells (ON-BC), and this communication is indispensible for vision ...in dim light. In ON-BCs, synaptic transmission is initiated by the metabotropic glutamate receptor, mGluR6, that signals via the G-protein Go to control opening of the effector ion channel, TRPM1. A key role in this process belongs to the GTPase Activating Protein (GAP) complex that catalyzes Go inactivation upon light-induced suppression of glutamate release in rod photoreceptors, thereby driving ON-BC depolarization to changes in synaptic input. The GAP complex has a striking molecular complexity. It contains two Regulator of G-protein Signaling (RGS) proteins RGS7 and RGS11 that directly act on Go and two adaptor subunits: RGS Anchor Protein (R9AP) and the orphan receptor, GPR179. Here we examined the organizational principles of the GAP complex in ON-BCs. Biochemical experiments revealed that RGS7 binds to a conserved site in GPR179 and that RGS11 in vivo forms a complex only with R9AP. R9AP and GPR179 are further integrated via direct protein-protein interactions involving their cytoplasmic domains. Elimination of GPR179 prevents postsynaptic accumulation of R9AP. Furthermore, concurrent knock-out of both R9AP and RGS7 does not reconfigure the GAP complex and completely abolishes synaptic transmission, resulting in a novel mouse model of night blindness. Based on these results, we propose a model of hierarchical assembly and function of the GAP complex that supports ON-BCs visual signaling.
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