Role of receptor kinase in short‐term desensitization of cardiac muscarinic K<sup>+</sup> channels expressed in Chinese hamster ovary cells

Shui, Z.; Khan, Iftikhar Ahmad; Tsuga, Hirofumi; Haga, Tatsuya; Boyett, Mark R.

doi:10.1111/j.1469-7793.1998.325bt.x

Cited by 21 publications

(40 citation statements)

References 35 publications

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“…CaR⌬886-EGFP may desensitize normally because the amino acid residues between 868 and 886 retard access of the proteins involved in desensitization to the relevant phosphorylation site(s). Direct interactions between G protein-coupled receptor kinases and possibly arrestin with muscarinic receptors have been functionally demonstrated (44), although the consensus binding site(s) on the receptor have not been identified. Deletion of the amino acid residues of CaR between 868 and 886 may expose potential regulatory protein interaction site(s), leading to more rapid and complete phosphorylation and desensitization of CaR.…”

Section: Discussionmentioning

confidence: 99%

A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor

Gama

Breitwieser

1998

Journal of Biological Chemistry

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Calcium sensing receptors are part of a growing G protein-coupled receptor family, which includes metabotropic glutamate, ␥-aminoisobutyric acid, and pheromone receptors. The distinctive structural features of this family include large extracellular domains that bind agonist and large intracellular, carboxyl-terminal domains of as yet undefined function(s). We have explored the contribution(s) of the carboxyl terminus of the human calcium sensing receptor (CaR) by assessing extracellular Ca 2؉ -mediated changes in intracellular Ca 2؉ in individual HEK-293 cells transfected with CaR clones. In-frame fusion of EGFP to the carboxyl terminus of CaR had no effect on either the dose response for extracellular Ca 2؉ activation or CaR desensitization. Carboxyl-terminal truncations, fused in-frame with EGFP (CaR⌬1024-EGFP, CaR⌬908-EGFP, CaR⌬886-EGFP, and CaR⌬868-EGFP), were assessed for alterations in Ca 2؉ -dependent activation or desensitization. Significant effects on the dose-response relation for extracellular Ca 2؉ were observed only for the CaR⌬868 truncation, which exhibited a decreased affinity for extracellular Ca 2؉ and a decrease in the apparent cooperativity for Ca 2؉ -dependent activation. The alterations in extracellular Ca 2؉ affinity and cooperativity observed with CaR⌬868 were recapitulated by a point mutation, T876D, in the full-length CaR-EGFP background. All truncations with wild type dose-response relations exhibited desensitization time courses that were comparable to the full-length CaR, whereas the CaR⌬868 receptor desensitized completely after two exposures to 10 mM Ca 2؉ . Interestingly, the CaR point mutation T876D exhibited desensitization comparable to wild type CaR, suggesting that this mutation specifically modifies CaR cooperativity. In conclusion, these studies suggest that amino acid residues between 868 and 886 are critical to the apparent cooperativity of Ca 2؉

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Section: Discussionmentioning

confidence: 99%

A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor

Gama

Breitwieser

1998

Journal of Biological Chemistry

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“…Previous work to define the mechanisms of GIRK desensitization suggested desensitization was resolved into fast and slow phases, where the fast phase is explained by I KACh channel dephosphorylation 33 and RGS protein GAP activity 34 and the slow phase involves G protein receptor kinase activity. 35,36 Because RGS4 has not been implicated in the regulation of kinases or phosphatases in the cell, the loss of rapid phase desensitization in RGS4-deficient SAN myocytes likely indicates the loss of a GAP-dependent desensitization mechanism. It has been shown that RGS4 forms stable protein-protein interactions with both M 2 R 14 and GIRK3 15 channels, and, thus, it is proposed to be a component of an integrated kinetic scaffolding complex that promotes efficient coordinated regulation of both G protein and GIRK activation.…”

Section: Discussionmentioning

confidence: 99%

RGS4 Regulates Parasympathetic Signaling and Heart Rate Control in the Sinoatrial Node

Cifelli

Rose

Zhang

et al. 2008

Circulation Research

114

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Abstract-Heart rate is controlled by the opposing activities of sympathetic and parasympathetic inputs to pacemaker myocytes in the sinoatrial node (SAN). Parasympathetic activity on nodal myocytes is mediated by acetylcholinedependent stimulation of M 2 muscarinic receptors and activation of G␣ i/o signaling. Although regulators of G protein signaling (RGS) proteins are potent inhibitors of G␣ i/o signaling in many tissues, the RGS protein(s) that regulate parasympathetic tone in the SAN are unknown. Our results demonstrate that RGS4 mRNA levels are higher in the SAN compared to right atrium. Conscious freely moving RGS4-null mice showed increased bradycardic responses to parasympathetic agonists compared to wild-type animals. Moreover, anesthetized RGS4-null mice had lower baseline heart rates and greater heart rate increases following atropine administration. Retrograde-perfused hearts from RGS4-null mice showed enhanced negative chronotropic responses to carbachol, whereas SAN myocytes showed greater sensitivity to carbachol-mediated reduction in the action potential firing rate. Finally, RGS4-null SAN cells showed decreased levels of G protein-coupled inward rectifying potassium (GIRK) channel desensitization and altered modulation of acetylcholine-sensitive potassium current (I KACh ) kinetics following carbachol stimulation. Taken together, our studies establish that RGS4 plays an important role in regulating sinus rhythm by inhibiting parasympathetic signaling and I KACh activity. (Circ Res. 2008;103:527-535.)Key Words: RGS proteins Ⅲ sinoatrial node Ⅲ parasympathetic Ⅲ GIRK channels H eart rate (HR) regulation by the autonomic nervous system is integrated by specialized autorhythmic (pacemaker) cells located within the sinoatrial node (SAN). Sympathetic neurotransmitters work via G s -coupled ␤-adrenergic receptors to increase adenylyl cyclase activity, intracellular cAMP concentration and protein kinase A activity. As a result, cAMP-regulated effectors such as hyperpolarizationactivated cyclic nucleotide-gated cation (HCN) channels, delayed rectifier, and voltage-gated Ca 2ϩ channels are enlisted by sympathetic activity to increase pacemaker cell firing rate. 1,2 By contrast, vagal parasympathetic activity decreases HR via G␣ i/o -coupled cholinergic M 2 muscarinic receptors (M 2 Rs). Several effects, mediated by both G␣ i/o and G␤␥ subunits, may contribute to this reduction in HR. G␤␥ heterodimers directly activate G protein-coupled inward rectifying potassium (GIRK) channels, resulting in membrane hyperpolarization. By contrast, G␣ i/o can both modulate phosphodiesterase activity and inhibit adenylyl cyclase activity, reduce both intracellular cAMP levels and protein kinase A activity, leading to decreased depolarizing currents carried by HCN and L-type calcium channels. [2][3][4][5] Because dysregulation of parasympathetic activity occurs in heart failure, sick sinus syndrome, and selected cardiac arrhythmias, 6 it is of clinical interest to identify key molecular regulators of parasympathetic signa...

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“…For GIRK current desensitization, two phases can be resolved. The slower one takes several minutes and probably is mediated by G-protein receptor kinases (GRKs, also known as ␤ARKs) (5,6). As for the acute desensitization that occurs within a minute, dephosphorylation of K ACh channels in atrial myocytes has been implicated because it is augmented by cytoplasmic ATP (4).…”

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confidence: 99%

Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K ⁺ channels

Chuang

Jan

1998

Proc. Natl. Acad. Sci. U.S.A.

116

109

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The G-protein gated inward rectifier K ؉ channel (GIRK) is activated in vivo by the G␤␥ subunits liberated upon G i -coupled receptor activation. We have recapitulated the acute desensitization of receptor-activated GIRK currents in heterologous systems and shown that it is a membrane-delimited process. Its kinetics depends on the guanine nucleotide species available and could be accounted for by the nucleotide exchange and hydrolysis cycle of G proteins. Indeed, acute desensitization is abolished by nonhydrolyzable GTP analogues. Whereas regulators of G-protein signaling (RGS) proteins by their GTPase-activating protein activities are regarded as negative regulators, a positive regulatory function of RGS4 is uncovered in our study; the opposing effects allow RGS4 to potentiate acute desensitization without compromising GIRK activation.Stimulation of G i -coupled receptors in the heart and the brain activates the G-protein gated inward rectifier K ϩ channels (GIRK) (1, 2), leading to hyperpolarization and reduction of membrane excitability. Although the receptor-activated GIRK currents vary according to the receptor subtypes stimulated and the cell types examined, they generally are biphasic, with a rapid activation followed by a slower desensitization (3, 4).Desensitization of receptor-mediated responses provides the basis for cellular adaptation to external inputs. Moreover, different receptors that activate the same signaling pathway can generate distinct temporal signals because of the differences in their desensitization kinetics. For GIRK current desensitization, two phases can be resolved. The slower one takes several minutes and probably is mediated by G-protein receptor kinases (GRKs, also known as ␤ARKs) (5, 6). As for the acute desensitization that occurs within a minute, dephosphorylation of K ACh channels in atrial myocytes has been implicated because it is augmented by cytoplasmic ATP (4). Given that ATP can serve as a substrate for various ATPases and kinases, however, the basis for the acute desensitization remains an open question.We approached this problem by defining a minimal system for acute desensitization, by using excised inside-out membrane patches exposed to cytoplasmic solutions of known composition. Like the receptor-mediated activation of GIRK current (7), acute desensitization of receptor-induced GIRK current is also a membrane-delimited process (4). Moreover, guanine nucleotides have profound effects on the GIRK current desensitization, which persists in the absence of ATP. The regulators of G-protein signaling (RGS) proteins, which speed up the activation-deactivation kinetics of GIRK currents (8, 9), also accelerate the desensitization kinetics. In addition to RGS4's ability to promote GTP hydrolysis of G␣ subunits, we found that RGS4 coexpression increased the G-protein pool available for GIRK activation, thereby allowing acceleration of current kinetics without compromising current amplitudes. Rates of GIRK current activation and desensitization under various experiment...

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Role of receptor kinase in short‐term desensitization of cardiac muscarinic K⁺ channels expressed in Chinese hamster ovary cells

Cited by 21 publications

References 35 publications

A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor

A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor

RGS4 Regulates Parasympathetic Signaling and Heart Rate Control in the Sinoatrial Node

Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K ⁺ channels

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Role of receptor kinase in short‐term desensitization of cardiac muscarinic K+ channels expressed in Chinese hamster ovary cells

Cited by 21 publications

References 35 publications

A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor

A Carboxyl-terminal Domain Controls the Cooperativity for Extracellular Ca2+ Activation of the Human Calcium Sensing Receptor

RGS4 Regulates Parasympathetic Signaling and Heart Rate Control in the Sinoatrial Node

Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K + channels

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Role of receptor kinase in short‐term desensitization of cardiac muscarinic K⁺ channels expressed in Chinese hamster ovary cells

Evidence that the nucleotide exchange and hydrolysis cycle of G proteins causes acute desensitization of G-protein gated inward rectifier K ⁺ channels