Structural determinants at the M2 muscarinic receptor modulate the RGS4-GIRK response to pilocarpine by impairment of the receptor voltage sensitivity.

Membrane potential controls the response of the M2 muscarinic receptor to its ligands. Membrane hyperpolarization increases response to the full agonist acetylcholine (ACh) while decreasing response to the partial agonist pilocarpine. We previously have demonstrated that the regulator of G-protein signaling (RGS) 4 protein discriminates between the voltage-dependent responses of ACh and pilocarpine; however, the underlying mechanism remains unclear. Here we show that RGS4 is involved in the voltage-dependent behavior of the M2 muscarinic receptor-mediated signaling in response to pilocarpine. Additionally we revealed structural determinants on the M2 muscarinic receptor underlying the voltage-dependent response. By electrophysiological recording in Xenopus oocytes expressing M2 muscarinic receptor and G-protein-gated inwardly rectifying K+ channels, we quantified voltage-dependent desensitization of pilocarpine-induced current in the presence or absence of RGS4. Hyperpolarization-induced desensitization of the current required for RGS4, also depended on pilocarpine concentration. Mutations of charged residues in the aspartic acid-arginine-tyrosine motif of the M2 muscarinic receptor, but not intracellular loop 3, significantly impaired the voltage-dependence of RGS4 function. Thus, our results demonstrated that voltage-dependence of RGS4 modulation is derived from the M2 muscarinic receptor. These results provide novel insights into how membrane potential impacts G-protein signaling by modulating GPCR communication with downstream effectors

RGS4 regulates partial agonism of the M2 muscarinic receptor-activated K+ currents.

Partial agonists are used clinically to avoid overstimulation of receptor-mediated signalling, as they produce a submaximal response even at 100% receptor occupancy. The submaximal efficacy of partial agonists is due to conformational change of the agonist-receptor complex, which reduces effector activation. In addition to signalling activators, several regulators help control intracellular signal transductions. However, it remains unclear whether these signalling regulators contribute to partial agonism. Here we show that regulator of G-protein signalling (RGS) 4 is a determinant for partial agonism of the M2 muscarinic receptor (M2R). In rat atrial myocytes, pilocarpine evoked smaller G-protein-gated K(+) inwardly rectifying (KG) currents than those evoked by ACh. In a Xenopus oocyte expression system, pilocarpine acted as a partial agonist in the presence of RGS4 as it did in atrial myocytes, while it acted like a full agonist in the absence of RGS4. Functional couplings within the agonist-receptor complex/G-protein/RGS4 system controlled the efficacy of pilocarpine relative to ACh. The pilocarpine-M2R complex suppressed G-protein-mediated activation of KG currents via RGS4. Our results demonstrate that partial agonism of M2R is regulated by the RGS4-mediated inhibition of G-protein signalling. This finding helps us to understand the molecular components and mechanism underlying the partial agonism of M2R-mediated physiological responses

HD Physiology Project-Japanese efforts to promote multilevel integrative systems biology and physiome research.

The HD Physiology Project is a Japanese research consortium that aimed to develop methods and a computational platform in which physiological and pathological information can be described in high-level definitions across multiple scales of time and size. During the 5 years of this project, an appropriate software platform for multilevel functional simulation was developed and a whole-heart model including pharmacokinetics for the assessment of the proarrhythmic risk of drugs was developed. In this article, we outline the description and scientific strategy of this project and present the achievements and influence on multilevel integrative systems biology and physiome research

Interactions of Cations with the Cytoplasmic Pores of Inward Rectifier K^+ Channels in the Closed State

This research was originally published in Journal of Biological Chemistry. Atsushi Inanobe, Atsushi Nakagawa, and Yoshihisa Kurachi. Interactions of Cations with the Cytoplasmic Pores of Inward Rectifier K^+ Channels in the Closed State. Journal of Biological Chemistry. 2011; 286, 41801-41811. © the American Society for Biochemistry and Molecular Biology

Conformational changes underlying pore dilation in the cytoplasmic domain of mammalian inward rectifier K^+ channels

Inanobe A, Nakagawa A, Kurachi Y (2013) Conformational Changes Underlying Pore Dilation in the Cytoplasmic Domain of Mammalian Inward Rectifier K^+ Channels. PLOS ONE 8(11): e79844. https://doi.org/10.1371/journal.pone.007984

Facilitation of I Kr current by some hERG channel blockers suppresses early afterdepolarizations.

Drug-induced block of the cardiac rapid delayed rectifying potassium current (I Kr), carried by the human ether-a-go-go-related gene (hERG) channel, is the most common cause of acquired long QT syndrome. Indeed, some, but not all, drugs that block hERG channels cause fatal cardiac arrhythmias. However, there is no clear method to distinguish between drugs that cause deadly arrhythmias and those that are clinically safe. Here we propose a mechanism that could explain why certain clinically used hERG blockers are less proarrhythmic than others. We demonstrate that several drugs that block hERG channels, but have favorable cardiac safety profiles, also evoke another effect; they facilitate the hERG current amplitude in response to low-voltage depolarization. To investigate how hERG facilitation impacts cardiac safety, we develop computational models of I Kr block with and without this facilitation. We constrain the models using data from voltage clamp recordings of hERG block and facilitation by nifekalant, a safe class III antiarrhythmic agent. Human ventricular action potential simulations demonstrate the ability of nifekalant to suppress ectopic excitations, with or without facilitation. Without facilitation, excessive I Kr block evokes early afterdepolarizations, which cause lethal arrhythmias. When facilitation is introduced, early afterdepolarizations are prevented at the same degree of block. Facilitation appears to prevent early afterdepolarizations by increasing I Kr during the repolarization phase of action potentials. We empirically test this prediction in isolated rabbit ventricular myocytes and find that action potential prolongation with nifekalant is less likely to induce early afterdepolarization than action potential prolongation with dofetilide, a hERG channel blocker that does not induce facilitation. Our data suggest that hERG channel blockers that induce facilitation increase the repolarization reserve of cardiac myocytes, rendering them less likely to trigger lethal ventricular arrhythmias

A structural determinant for the control of PIP_2 sensitivity in G protein-gated inward rectifier K^+ channels

Inward rectifier K^+ (Kir) channels are activated by phosphatidylinositol-( 4,5)-bisphosphate (PIP_2), but G protein-gated Kir (K_G) channels further require either G protein βγ subunits (Gβγ) or intracellular Na^+ for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of K_G channel subunit Kir3.2 obtained in the presence and the absence of Na^+. The Na^+ -free Kir3.2, but not the Na^+ -plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on theNterminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na^+ -dependent activation, lowered PIP_2 sensitivity. The conservation of these residues within the K_G channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP_2 sensitivity.This research was originally published in Journal of Biological Chemistry. Atsushi Inanobe, Atsushi Nakagawa, Takanori Matsuura and Yoshihisa Kurachi. A structural determinant for the control of PIP2 sensitivity in G protein-gated inward rectifier K^+ channels. Journal of Biological Chemistry. 2010; 285, 38517-38523. © the American Society for Biochemistry and Molecular Biology

Somatostatin induces hyperpolarization in pancreatic islet α cells by activating a G protein-gated K+ channel

AbstractSomatostatin inhibits glucagon-secretion from pancreatic α cells but its underlying mechanism is unknown. In mouse α cells, we found that somatostatin induced prominent hyperpolarization by activating a K+ channel, which was unaffected by tolbutamide but prevented by pre-treating the cells with pertussis toxin. The K+ channel was activated by intracellular GTP (with somatostatin), GTPγS or Gβγ subunits. It was thus identified as a G protein-gated K+ (KG) channel. RT-PCR and immunohistochemical analyses suggested the KG channel to be composed of Kir3.2c and Kir3.4. This study identified a novel ionic mechanism involved in somatostatin-inhibition of glucagon-secretion from pancreatic α cells

Molecular cloning and functional expression of a novel brain-specific inward rectifier potassium channel

AbstractWe have cloned a novel brain-specific inward rectifier K+ channel from a mouse brain cDNA library and designated it MB-IRK3. The mouse brain cDNA library was screened using a fragment of the mouse macrophage inward rectifier K+ channel (IRK1) cDNA as a probe. The amino acid sequence of MB-IRK3 shares 61% and 64% identity to MB-IRK1 and RB-IRK2, respectively.Xenopus oocytes injected with cRNA derived from this clone expressed a potassium current which showed inward-rectifying channel characteristics similar to MB-IRK1 and RB-IRK2 currents, but distinct from ROMK1 or GIRK1 current. However, the single channel conductance of MB-IRK3 was ∼ 10 pS with 140 mM extracellular K+, which was distinct from that of MB-IRK1 (20 pS). MB-IRK3 mRNA expressed specifically in the forebrain, which clearly differed from MB-IRK1 and RB-IRK2 mRNAs. These results indicate that members of the IRK family with distinct electrophysiological properties express differentially and may play heterogenous functional roles in brain functions