ML297 was recently identified as a potent and selective small molecule agonist of G-protein-gated inwardly rectifying K + (GIRK/Kir3) channels. Here, we show ML297 selectively activates recombinant neuronal GIRK channels containing the GIRK1 subunit in a manner that requires phosphatidylinositol-4,5-bisphosphate (PIP 2 ), but is otherwise distinct from receptor-induced, G-protein-dependent channel activation. Two amino acids unique to the pore helix (F137) and second membrane-spanning (D173) domain of GIRK1 were identified as necessary and sufficient for the selective activation of GIRK channels by ML297. Further investigation into the behavioral effects of ML297 revealed that in addition to its known antiseizure efficacy, ML297 decreases anxiety-related behavior without sedative or addictive liabilities. Importantly, the anxiolytic effect of ML297 was lost in mice lacking GIRK1. Thus, activation of GIRK1-containing channels by ML297 or derivatives may represent a new approach to the treatment of seizure and/or anxiety disorders.electrophysiology | structure-activity relationship S ignal transduction involving inhibitory (G i/o ) G proteins titrates the excitability of neurons, cardiac myocytes, and endocrine cells, influencing behavior, cardiac output, and energy homeostasis (1). G-protein-gated inwardly rectifying potassium (K + ) (GIRK/Kir3) channels are a common effector for G i/odependent signaling pathways in the heart and nervous system (2, 3). Polymorphisms and mutations in human GIRK channels have been linked to arrhythmias, hyperaldosteronism (and associated hypertension), schizophrenia, sensitivity to analgesics, and alcohol dependence (1).GIRK channels are activated by binding of the G protein Gβγ subunit (1-3). Gβγ binding strengthens channel affinity for phosphatidylinositol-4,5-bisphosphate (PIP 2 ), a necessary cofactor for channel gating (4, 5). GIRK channels are also activated in a G-protein-independent manner by ethanol (6, 7), volatile anesthetics (8, 9), and naringin (10). Many psychoactive and clinically relevant compounds with other primary molecular targets inhibit GIRK channels, albeit at relatively high doses (1, 11). The lack of selective GIRK channel modulators, and in particular, drugs that discriminate among GIRK channel subtypes, has hampered investigation into their physiological relevance and therapeutic potential.GIRK channels are homo-and heterotetramers formed by GIRK1, GIRK2, GIRK3, and GIRK4 subunits (2, 3). GIRK subunits exhibit overlapping but distinct cellular expression patterns, potentially yielding multiple channel subtypes (1). Although it cannot form functional homomers (12), GIRK1 is an integral subunit of the cardiac GIRK channel and most neuronal GIRK channels (13,14). GIRK1 confers robust basal and receptordependent activity to GIRK heteromers, attributable in part to unique residues in the pore and second transmembrane domain (15-17). The intracellular C-terminal domain also contributes to the potentiating influence of GIRK1 on channel activity, likely due to th...

Rationale:The parasympathetic reduction in heart rate involves the sequential activation of m2 muscarinic cholinergic receptors (m 2 Rs), pertussis toxin-sensitive (Gi/o) heterotrimeric G proteins, and the atrial potassium channel I KACh . Molecular mechanisms regulating this critical signal transduction pathway are not fully understood.Objective: To determine whether the G protein signaling regulator Rgs6/G␤5 modulates m 2 R-I KACh signaling and cardiac physiology. Methods and Results: Cardiac expression of Rgs6, and its interaction with G␤5, was demonstrated by immunoblotting and immunoprecipitation. Rgs6؊/؊ mice were generated by gene targeting, and the cardiac effects of Rgs6 ablation were analyzed by whole-cell recordings in isolated cardiomyocytes and ECG telemetry. Loss of Rgs6 yielded profound delays in m 2 R-I KACh deactivation kinetics in both neonatal atrial myocytes and adult sinoatrial nodal cells. Rgs6 ؊/؊ mice exhibited mild resting bradycardia and altered heart rate responses to pharmacological manipulations that were consistent with enhanced m 2 R-I KACh signaling. Conclusions:The cardiac Rgs6/G␤5 complex modulates the timing of parasympathetic influence on atrial myocytes and heart rate in mice. (Circ Res. 2010;107:1350-1354.) Key Words: G protein Ⅲ muscarinic Ⅲ knockout Ⅲ cardiac Ⅲ GIRK Ⅲ regulators of G protein signaling C ardiac output is shaped to a great extent by sympathetic and parasympathetic influences. Parasympathetic input tempers heart rate (HR), counteracts the proarrhythmic effects of sympathetic activation, and is mediated by acetylcholine (ACh). 1 ACh is released from postganglionic parasympathetic neurons and binds to m 2 muscarinic receptors (m 2 Rs) on pacemaker cells and atrial myocytes, triggering activation of pertussis toxin-sensitive (Gi/o) heterotrimeric G proteins. 2 Once activated, G proteins dissociate into G␣-GTP and G␤␥ subunits, leading to modulation of adenylyl cyclase and multiple ion channels. Central among these reactions is the binding of G␤␥ to the atrial potassium channel I KACh , a heterotetramer composed of G protein-gated inwardly rectifying K ϩ (Girk)1 and Girk4 channel subunits. 3 Binding of G␤␥ to I KACh enhances its gating, which leads to cell hyperpolarization and, ultimately, decreased HR. 4 The duration of G protein signaling is controlled by members of the RGS (regulator of G protein signaling) family. 5 RGS proteins stimulate inactivation of G␣-GTP, facilitating its reassembly with G␤␥. RGS proteins play a critical role in shaping bradycardic effects of m 2 R receptor activation. 6 -8 Indeed, eliminating RGS influence by expressing G␣ subunits insensitive to RGS action results in a substantial enhancement of I KACh regulation by m 2 R signaling, via both G␣ o and G␣ i2 pathways. 7,8 Although more than 30 RGS proteins have been identified, the involvement of specific RGS proteins in the regulation of parasympathetic input is not fully understood. Here, we report an unexpected role of the Rgs6/G␤5 complex, previously thought to be neuron-specif...

In the hippocampus, signalling through G protein-coupled receptors is modulated by Regulators of G protein Signalling (Rgs) proteins, which act to stimulate the rate of GTP hydrolysis, and consequently, G protein inactivation. The R7-Rgs subfamily selectively deactivates the Gi/o-class of Gα subunits that mediate the action of several GPCRs. Here, we used co-immunoprecipitation, electrophysiology and immunoelectron microscopy techniques to investigate the formation of macromolecular complexes and spatial relationship of Rgs7/Gβ5 complexes and its prototypical signalling partners, the GABAB receptor and Girk channel. Co-expression of recombinant GABAB receptors and Girk channels in combination with co-immunoprecipitation experiments established that the Rgs7/Gβ5 forms complexes with GABAB receptors or Girk channels. Using electrophysiological experiments, we found that GABAB-Girk current deactivation kinetics was markedly faster in cells co-expressing Rgs7/Gβ5. At the electron microscopic level, immunolabelling for Rgs7 and Gβ5 proteins was found primarily in the dendritic layers of the hippocampus and showed similar distribution patterns. Immunoreactivity was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of pyramidal cells and, to a lesser extent, to that of presynaptic terminals. Quantitative analysis of immunogold particles for Rgs7 and Gβ5 revealed an enrichment of the two proteins around excitatory synapses on dendritic spines, virtually identical to that of Girk2 and GABAB1. These data support the existence of macromolecular complexes composed of GABAB receptor-G protein-Rgs7-Girk channels, in which Rgs7 and Gβ5 proteins may preferentially modulate GABAB receptor signalling through the deactivation of Girk channels on dendritic spines. In contrast, Rgs7 and Girk2 were associated but mainly segregated from GABAB1 in dendritic shafts, where Rgs7/Gβ5 signalling complexes might modulate Girk-dependent signalling via a different metabotropic receptor(s).

HIV-associated neurocognitive disorders (HAND) afflict about half of HIV-infected patients. HIV-infected cells shed viral proteins, such as the transactivator of transcription (Tat), which can cause neurotoxicity by over activation of NMDA receptors (NMDARs). Here, we show that Tat causes a time-dependent, biphasic change in NMDA-evoked increases in intracellular Ca2+ concentration ([Ca2+]i). NMDA-evoked responses were potentiated following 2 h exposure to Tat (50 ng/mL). Tat-induced potentiation of NMDA-evoked increases in [Ca2+]i peaked by 8 h and then adapted by gradually reversing to baseline by 24 h and eventually dropping below control by 48 h. Tat-induced potentiation of NMDA-evoked responses was blocked by inhibition of lipoprotein receptors (LRP) or Src tyrosine kinase. Potentiation was unaffected by inhibition of nitric oxide synthase (NOS). However, NOS activity was required for adaptation. Adaptation was also prevented by inhibition of soluble guanylate cyclase (sGC) and cGMP-dependent protein kinase (PKG). Together, these findings indicate that Tat potentiates NMDA-evoked increases in [Ca2+]i via LRP-dependent activation of Src and that this potentiation adapts via activation of the NOS/sGC/PKG pathway. Adaptation may protect neurons from excessive Ca2+ influx and could reveal targets for the treatment of HAND.

Background: RGS4 and RGS6 are regulator of G protein signaling (RGS) proteins, and both have been proposed to modulate parasympathetic regulation of heart rate (HR). Results: RGS6 ablation enhances parasympathetic influence on the heart; RGS4 ablation does not. Conclusion: RGS6 is the primary RGS modulator of parasympathetic influence on the heart. Significance: Understanding the parasympathetic regulation of HR will improve treatment of arrhythmias.

G protein-gated inwardly rectifying K + (Girk/K IR 3) channels mediate the inhibitory effect of many neurotransmitters on excitable cells. Girk channels are tetramers consisting of various combinations of four mammalian Girk subunits (Girk1 to -4). Although Girk1 is unable to form functional homomeric channels, its presence in cardiac and neuronal channel complexes correlates with robust channel activity. This study sought to better understand the potentiating influence of Girk1, using the GABA B receptor and Girk1/Girk2 heteromer as a model system. Girk1 did not increase the protein levels or alter the trafficking of Girk2-containing channels to the cell surface in transfected cells or hippocampal neurons, indicating that its potentiating influence involves enhancement of channel activity. Structural elements in both the distal carboxyl-terminal domain and channel core were identified as key determinants of robust channel activity. In the distal carboxyl-terminal domain, residue Q404 was identified as a key determinant of receptor-induced channel activity. In the Girk1 core, three unique residues in the pore (P) loop (F137, A142, Y150) were identified as a collective potentiating influence on both receptor-dependent and receptorindependent channel activity, exerting their influence, at least in part, by enhancing mean open time and single-channel conductance. Interestingly, the potentiating influence of the Girk1 P-loop is tempered by residue F162 in the second membrane-spanning domain. Thus, discontinuous and sometime opposing elements in Girk1 underlie the Girk1-dependent potentiation of receptor-dependent and receptor-independent heteromeric channel activity.ion channel | electrophysiology | mutagenesis | baclofen