Ca2+Channel β3Subunit Enhances Voltage-Dependent Relief of G-Protein Inhibition Induced by Muscarinic Receptor Activation and Gβγ

Cannabinoid CB1receptor (viaGs) and dopamine D2receptor (viaGi/o) antagonistically modulate goldfish cone membrane currents. As ON bipolar cells have CB1and D1receptors, but not D2receptors, we focused on whether CB1receptor agonist and dopamine interact to modulate voltage-dependent outward membrane K+currentsIK(V)of the ON mixed rod/cone (Mb) bipolar cells. Whole-cell currents were recorded from Mb bipolar cells in goldfish retinal slices. Mb bipolar cells were identified by intracellular filling with Lucifer yellow. The bath solution was calcium-free and contained 1 mM cobalt to block indirect calcium-dependent effects. Dopamine (10 μM) consistently increasedIK(V)by a factor of 1.57 ± 0.12 (S.E.M.,n= 15). A CB receptor agonist, WIN 55212-2 (0.25–1 μM), had no effect, but 4 μM WIN 55212-2 suppressedIK(V)by 60%. IfIK(V)was first increased by 10 μM dopamine, application of WIN 55212-2 (0.25–1 μM) reversibly blocked the effect of dopamine even though these concentrations of WIN 55212-2 had no effect of their own. If WIN 55212-2 was applied first and dopamine (10 μM) was added to the WIN-containing solution, 0.1 μM WIN 55212-2 blocked the effect of dopamine. All effects of WIN 55212-2 were blocked by coapplication of SR 141716A (CB1antagonist) and pretreatment with pertussis toxin (blocker of Gi/o) indicating actionviaCB1receptor activation of G protein Gi/o. Coactivation of CB1and D1receptors on Mb bipolar cells produces reciprocal effects onIK(V). The CB1-evoked suppression ofIK(V)is mediated by G protein Gi/o, whereas the D1-evoked enhancement is mediated by G protein Gs. As dopamine is a retinal “light” signal, these data support our notion that endocannabinoids function as a “dark” signal, interacting with dopamine to set retinal sensitivity.

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G protein-coupled receptor kinase 5 regulates airway responses induced by muscarinic receptor activation

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00255.2003 ◽

2004 ◽

Vol 286 (2) ◽

pp. L312-L319 ◽

Cited By ~ 44

Author(s):

J. K. L. Walker ◽

R. R. Gainetdinov ◽

D. S. Feldman ◽

P. K. McFawn ◽

M. G. Caron ◽

...

Keyword(s):

Smooth Muscle ◽

G Protein ◽

Muscarinic Receptor ◽

Airway Smooth Muscle ◽

Muscarinic Receptors ◽

Muscle Relaxation ◽

Receptor Activation ◽

Smooth Muscle Relaxation ◽

Airway Responses ◽

G Protein Coupled

G protein-coupled receptors (GPCRs) transduce extracellular signals into intracellular events. The waning responsiveness of GPCRs in the face of persistent agonist stimulation, or desensitization, is a necessary event that ensures physiological homeostasis. GPCR kinases (GRKs) are important regulators of GPCR desensitization. GRK5, one member of the GRK family, desensitizes central M2 muscarinic receptors in mice. We questioned whether GRK5 might also be an important regulator of peripheral muscarinic receptor responsiveness in the cardiopulmonary system. Specifically, we wanted to determine the role of GRK5 in regulating muscarinic receptor-mediated control of airway smooth muscle tone or regulation of cholinergic-induced bradycardia. Tracheal pressure, heart rate, and tracheal smooth muscle tension were measured in mice having a targeted deletion of the GRK5 gene ( GRK5- /-) and littermate wild-type (WT) control mice. Both in vivo and in vitro results showed that the airway contractile response to a muscarinic receptor agonist was not different between GRK5- /- and WT mice. However, the relaxation component of bilateral vagal stimulation and the airway smooth muscle relaxation resulting from β2-adrenergic receptor activation were diminished in GRK5- /- mice. These data suggest that M2 muscarinic receptor-mediated opposition of airway smooth muscle relaxation is regulated by GRK5 and is, therefore, excessive in GRK5- /- mice. In addition, this study shows that GRK5 regulates pulmonary responses in a tissue- and receptor-specific manner but does not regulate peripheral cardiac muscarinic receptors. GRK5 regulation of airway responses may have implications in obstructive airway diseases such as asthma or chronic obstructive pulmonary disease.

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Voltage-dependent facilitation of cardiac L-type Ca channels expressed in HEK-293 cells requires β-subunit

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.278.1.h126 ◽

2000 ◽

Vol 278 (1) ◽

pp. H126-H136 ◽

Cited By ~ 15

Author(s):

Timothy J. Kamp ◽

Hai Hu ◽

Eduardo Marban

Keyword(s):

G Protein ◽

Somatostatin Receptors ◽

Ca Channel ◽

Β Subunit ◽

Ca Channels ◽

Hek 293 ◽

Hek 293 Cells ◽

Protein Inhibition ◽

293 Cells ◽

Voltage Dependent

The activity of native L-type Ca channels can be facilitated by strong depolarizations. The cardiac Ca channel α1C-subunit was transiently expressed in human embryonic kidney (HEK-293) cells, but these channels did not exhibit voltage-dependent facilitation. Coexpression of the Ca channel β1a- or β2a-subunit with the α1C-subunit enabled voltage-dependent facilitation in 40% of cells tested. The onset of facilitation in α1C + β1a-expressing HEK-293 cells was rapid after a depolarization to +100 mV (τ = 7.0 ms). The kinetic features of the facilitated currents were comparable to those observed for voltage-dependent relief of G protein inhibition demonstrated for many neuronal Ca channels; however, intracellular dialysis with guanosine 5′- O-(2-thiodiphosphate) and guanosine 5′- O-(3-thiotriphosphate) in the patch pipette had no effect on facilitation. Stimulation of G protein-coupled receptors, either endogenous (somatostatin receptors) or coexpressed (adenosine A1receptors), did not affect voltage-dependent facilitation. These results indicate that the cardiac Ca channel α1C-subunit can exhibit voltage-dependent facilitation in HEK-293 cells only when coexpressed with an auxiliary β-subunit and that this facilitation is independent of G protein pathways.

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The coupling of the M2 muscarinic receptor to its G protein is voltage dependent

PLoS ONE ◽

10.1371/journal.pone.0224367 ◽

2019 ◽

Vol 14 (10) ◽

pp. e0224367

Author(s):

Yair Ben-Chaim ◽

Chava Broide ◽

Hanna Parnas

Keyword(s):

G Protein ◽

Muscarinic Receptor ◽

M2 Muscarinic Receptor ◽

Voltage Dependent

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Scanning mutagenesis of the I-II loop of the Cav2.2 calcium channel identifies residues Arginine 376 and Valine 416 as molecular determinants of voltage dependent G protein inhibition

Molecular Brain ◽

10.1186/1756-6606-3-6 ◽

2010 ◽

Vol 3 (1) ◽

pp. 6 ◽

Cited By ~ 10

Author(s):

Hugo W Tedford ◽

Alexandra E Kisilevsky ◽

Lucienne B Vieira ◽

Diego Varela ◽

Lina Chen ◽

...

Keyword(s):

Calcium Channel ◽

G Protein ◽

Protein Inhibition ◽

Molecular Determinants ◽

Voltage Dependent

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Regulation of G-protein-coupled inward rectifying K+ channel expression by muscarinic receptor activation

Life Sciences ◽

10.1016/s0024-3205(99)90506-4 ◽

1999 ◽

Vol 64 (6-7) ◽

pp. 575

Author(s):

R.S. Chmelar ◽

S.L. Thomas ◽

N.M. Nathanson

Keyword(s):

G Protein ◽

Muscarinic Receptor ◽

Receptor Activation ◽

K Channel ◽

Channel Expression ◽

G Protein Coupled

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Differential Occurrence of Reluctant Openings in G-Protein–Inhibited N- and P/Q-Type Calcium Channels

The Journal of General Physiology ◽

10.1085/jgp.115.2.175 ◽

2000 ◽

Vol 115 (2) ◽

pp. 175-192 ◽

Cited By ~ 49

Author(s):

Henry M. Colecraft ◽

Parag G. Patil ◽

David T. Yue

Keyword(s):

Calcium Channels ◽

G Protein ◽

Action Potentials ◽

Qualitative Difference ◽

Hek 293 ◽

Protein Inhibition ◽

Voltage Dependent ◽

Dependent Inhibition ◽

Time Courses ◽

Prepulse Facilitation

Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel “slope” analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the “willing–reluctant” model, in which binding of G-protein βγ subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca2+ entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission.

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Differential regulation of cholecystokinin- and muscarinic-receptor-mediated phosphoinositide turnover in Flow 9000 cells

Biochemical Journal ◽

10.1042/bj2510625 ◽

1988 ◽

Vol 251 (3) ◽

pp. 625-630 ◽

Cited By ~ 8

Author(s):

W W Y Lo ◽

J Hughes

Keyword(s):

G Protein ◽

Muscarinic Receptor ◽

Feedback Inhibition ◽

Inositol Phosphate ◽

Binding Capacity ◽

Second Messenger ◽

Receptor Activation ◽

Maximal Response ◽

Muscarinic Agonist ◽

Stimulation Of

We have explored the hypothesis that the apparent greater efficiency of cholecystokinin (CCK-8) receptor-second messenger coupling compared with that of muscarinic receptor in Flow 9000 cells is due to differential feedback inhibitory control mechanisms. Pretreatment of Flow 9000 cells with the tumour-promoting protein kinase C (PKC)-activating agent 12-O-tetradecanoylphorbol 13-acetate (TPA) produced a time- and dose-dependent inhibition of CCK-8 and acetylcholine (ACh) stimulation of inositol phosphate production. The inhibition by TPA of ACh-induced PI (phosphoinositide) response involved reduction of the maximal response, but no change in the concentration of ACh required to evoke a half-maximal response. In contrast, TPA inhibition of CCK-8 responses could be overcome by increasing the CCK-8 concentrations. Flow 9000 cells pretreated with TPA exhibited a 52-68% reduction in [3H]quinuclidinyl benzilate ([3H]QNB) binding capacity, whereas [125I]CCK-8 binding was unchanged. In saponin-permeabilized Flow 9000 cells, TPA pretreatment had no effect on guanosine 5′-[gamma-thio]triphosphate (GTP[S])-induced inositol phosphate formation, indicating that G-protein linkage to phosphoinositidase C (PIC) was not affected. However, TPA significantly inhibited the potentiating effect of GTP[S] on CCK-8 and ACh activation of PI response, suggesting that the coupling between the receptors and the G-protein was impaired. The PKC-activator 1-oleoyl-2-acetylglycerol (OAG), a diacylglycerol analogue, also significantly reduced CCK-8 and ACh stimulation of inositol phosphate accumulation in these cells. Our results are consistent with the hypothesis that muscarinic activation of PI hydrolysis is subjected to rapid feedback inhibition via the 1,2-diacylglycerol-PKC pathway. CCK-receptor activation of PI turnover is modulated to a lesser extent, and this may partially explain apparent differences in the efficiency of receptor-second messenger coupling. It is proposed that TPA acting through PKC exerts its inhibitory action on muscarinic-agonist-mediated PI response mainly at the receptor level, whereas the inhibitory effect on CCK-8 response is at a site close to the receptor-G-protein coupling step.

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