GIRK Currents Research Articles

Repeated Stress Dysregulates κ-Opioid Receptor Signaling in the Dorsal Raphe through a p38α MAPK-Dependent Mechanism

Repeated stress releases dynorphins and causes subsequent activation of κ-opioid receptors (KORs) in limbic brain regions. The serotonergic dorsal raphe nucleus (DRN) has previously been found to be an important site of action for the dysphoric effects of dynorphin-κ-opioid receptor system activation during stress-evoked behaviors, and KOR-induced activation of p38α mitogen-activated protein kinase (MAPK) in serotonergic neurons was found to be a critical mediator of the aversive properties of stress. Yet, how dynorphins and KORs functionally regulate the excitability of serotonergic DRN neurons both in adaptive and pathological stress states is poorly understood. Here we report that acute KOR activation by the selective agonist U69,593 [(+)-(5α,7α,8β)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4.5]dec-8-yl]benzeneacetamide] inhibits serotonergic neuronal excitability within the DRN through both presynaptic inhibition of excitatory synaptic transmission and postsynaptic activation of G-protein-gated inwardly rectifying potassium channels (GIRKs) electrophysiologically recorded in brain slices. C57BL/6 mice subjected to repeated swim, stress sessions had significantly reduced KOR-mediated GIRK currents recorded in serotonergic neurons in DRN postsynaptically, without significantly affecting presynaptic KOR-mediated regulation of excitatory transmission. This effect was blocked by genetic excision of p38α MAPK selectively from serotonergic neurons. An increase in phospho-immunoreactivity suggests that this functional dysregulation may be a consequence of tyrosine phosphorylation of GIRK (K(IR)3.1) channels. These data elucidate a mechanism for stress-induced dysregulation of the excitability of neurons in the DRN and identify a functional target of stress-induced p38α MAPK activation that may underlie some of the negative effects of pathological stress exposure.

RGS proteins maintain robustness of GPCR‐GIRK coupling

ObjectiveTo elucidate the mechanism underlying sustained GPCR‐GIRK coupling under RGS actions.ResultsRegulators of G‐protein signaling (RGS) are GTPase activating proteins (GAP) that reduce response amplitudes of activated G‐protein coupled receptors (GPCRs). We discovered that, although RGS proteins drastically accelerate kinetics of GPCR‐coupled K+ currents (GIRK), they actually increased amplitudes of inhibitory neurotransmitter‐evoked GIRK currents. The RGS‐Box domain alone is sufficient for stimulation of transmitter activation of K+ currents, but its membrane association enhances the efficiency of stimulation. Moreover, RGS4 mutants with compromised GAP activity still augment GPCR‐GIRK coupling. Among the pertussis toxin sensitive G‐proteins, we found that RGS4 selectively stimulates Gαo to maintain robustness of GPCR‐GIRK coupling, by accelerating activation kinetics of Gαo.ConclusionOpposing actions of RGS proteins both stimulate and inhibit G‐proteins to modulate ultimate amplitudes of transmitter‐induced GIRK currents and to differentiate signal intensity coupled to various G‐protein isoforms.

Regulator of G Protein Signaling 6 (RGS6) ensures coordination of motor movement by modulating GABAB Receptor (GABABR) signaling

Inhibitory interneurons located within the cerebellar cortex release GABA, which limits neuronal excitation in part through activation of metabatropic GABABRs. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key modulator of GABABR signaling in cerebellum. RGS6−/− mice exhibit abnormal gait and ataxia characterized by impaired rotarod performance that can be partially rescued by administration of a GABABR antagonist. In addition, RGS6−/− mice treated with baclofen, a GABABR agonist, show exaggerated motor coordination deficits on the rotarod compared to their wild type counterparts. Stimulation of GABABRs triggers a number of signaling events downstream of G protein activation including Gβγ subunit activation of G protein‐coupled inwardly rectifying potassium (GIRK) channels. Indeed, RGS6 is enriched in the granule cell layer of the cerebellum along with neuronal GIRK channel subunits 1 and 2 where RGS6 forms a complex with known binding partners Gβ5 and R7‐binding protein (R7BP), which stabilize and promote membrane association of R7 family members, respectively. Cerebellar granule neurons isolated from RGS6−/− mice showed a significant delay in the deactivation kinetics of baclofen‐induced GIRK currents. These results establish RGS6 as a key modulator of GABABR signaling, providing the first demonstration of an essential role for RGS proteins in cerebellar function.

Methamphetamine-Evoked Depression of GABAB Receptor Signaling in GABA Neurons of the VTA

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

Reduced GABA-B/GIRK-mediated regulation of the VTA following a single exposure to cocaine

AbstractIn this paper, Arora and colleagues expand on their previous work on GIRK channels in the ventral tegmental area (VTA) presenting evidence that a single exposure to cocaine reduces inhibitory GABAergic transmission to dopamine (DA) neurons in the ventral tegmental area. Mice receiving i.p. injections of cocaine saw a short lived (1-5 days) decrease in GABAb mediated G-protein coupled inwardly-rectifying potassium (GIRK) currents in DA neurons in the VTA. This decrease parallels an NMDA-mediated increase in the frequency of glutamatergic neurotransmission. Chronic cocaine injections had no additional effects beyond those seen with single injections. Though they found no change in mRNA levels for GABAb receptors, GIRK channels, or RGS-2 (a G-protein regulator), immunoelectron microscopy indicated a decrease in levels of GIRK channels in the plasma membrane of the dendrites of VTA DA neurons. The cocaine-mediated decrease in GIRK currents was abolished in the presence of D2/3R antagonist sulpiride, but not in the presence of D1/5 antagonist SCH23390, indicating a link between D2/3 receptor activation and GIRK activity. Interestingly, the addition of quinpirole, a D2/3 agonist, elicited similar GIRK currents, though they were smaller than those mediated by GABAb receptors. Similarly, acute injections of cocaine significantly diminished quinpirole-evoked currents.

Engineering the Alcohol Pocket to Create a Chemically-Activated GIRK Channel

G-protein gated inwardly rectifying potassium (GIRK) channels are implicated in alcohol abuse. Ethanol directly activates GIRKs through interaction with a discrete alcohol-binding pocket that we identified in the cytoplasmic domain. The structural mechanism underlying ethanol-dependent activation, however, is not well understood. Here, we hypothesized that chemically modifying the alcohol pocket with alcohol-like reagents would activate the channel. To examine this, we engineered GIRK2 containing a single cytoplasmic cysteine substitution in the βD-βE domain of the alcohol pocket; S246 is located near the edge of the pocket, and L257 is located within the pocket. All Cys substitutions were introduced into GIRK2c lacking four native Cys residues (GIRK2c(cys-)) and transiently expressed in HEK293T cells. We studied the effect of 2-Hydroxyethyl MTS (MTS-HE, alcohol-like) and two hydrophobic MTS reagents- Benzyl-MTS (MTS-Bn), and 4-hydroxy benzyl-MTS (MTS-Y), on basal (Ba2+-sensitive) and alcohol-activated GIRK currents. MTS-HE (100 μM) increased basal GIRK currents for S246C (34% ± 12%, n=7) and L257C (120% ± 24%, n=7). Similarly, MTS-Bn (10 μM) modification increased basal currents by 74% ± 18% (n=7) for S246C, and by 333% ± 31% (n=4) for L257C. MTS-Y (100 μM) increased the basal currents by 102% ± 25% (n= 5) for L257C but did not affect S246C. In addition, MTS modification decreased MPD-activated current. For L257C, we confirmed that intracellular application of MTS-HE produced a concentration-dependent increase in the time course of channel activation, having a rate constant of 90.6±17.2 M−1s−1 (n=11). Taken together, these data demonstrate that MTS modification of a single amino acid in the alcohol pocket can engage channel activation, and limit the access of bulkier diols (e.g., MPD). These results reveal novel mechanisms for chemical activation of GIRKs and underscore the importance of hydrophobic interactions in alcohol-mediated activation of GIRKs.

RGS Proteins Maintain Robustness of GPCR-GIRK Coupling

Regulators of G-protein signaling (RGS) are GTPase activating proteins (GAP) that reduce response amplitudes of activated G-protein coupled receptors (GPCRs). We discovered that, although RGS proteins drastically accelerate kinetics of GPCR-coupled K+ currents (GIRK), they actually increased amplitudes of inhibitory neurotransmitter-evoked GIRK currents. The RGS-Box domain alone is sufficient for stimulation of transmitter activation of K+ currents, but its membrane association enhances the efficiency of stimulation. Moreover, RGS4 mutants with compromised GAP activity still augment GPCR-GIRK coupling. Among the pertussis toxin sensitive G-proteins, we found that RGS4 selectively stimulates G alpha-o to maintain robustness of GPCR-GIRK coupling. Opposing actions of RGS proteins thus both stimulate and inhibit G-proteins to modulate ultimate amplitudes of transmitter-induced GIRK currents and to differentiate signal intensity coupled to various G-protein isoforms.

Ras-Associated (RA) Domain of Sorting Nexin 27 (SNX27) is Critical for Regulating GIRK Channels

G-protein-gated potassium inwardly rectifying (GIRK) channels play an important role in regulating neuronal excitability in the brain. We have previously shown that trafficking and surface expression of GIRK channels are regulated by sorting nexin 27 (SNX27). SXN27 contains three domains; a phosphoinositide-binding Phox (PX) domain that targets SNX27 preferentially to early endosomes, a PDZ domain that associates directly with carboxyl-terminal tailof GIRK3 and GIRK2c subunits, and a Ras-associated RA domain that could potentially bind to small monomeric G proteins. The functional consequence of this interaction with the RA domain was unknown. Here we show for the first time that expression of a SNX27 mutant devoid of the RA domain (SNX27ΔRA) in HEK293T cells abrogates the down-regulation of baclofen-induced GIRK2c/3 currents that is typically observed with wild-type SNX27 (12.4±3.6 pA/pF (SNX27 WT group) vs. 55.8±8.2 pA/pF (SNX27ΔRA) vs. 47.1±6.7pA/pF (control group without SNX27); mean current density±SEM). Using bimolecular fluorescence complementation (BiFC) technique to follow the intracellular localization of GIRK2c/3 heterotetramers, we observed that SNX27 but not SNX27ΔRA increases the number of GIRK2c/3 heterotetramers in early endosomes. Moreover, a single point mutation within the RA domain (K305A) was sufficient to disrupt SNX27-dependent clustering of GIRK2c/3 heterotetramers, mimicking the functional effects of the RA deletion. Recently, it was shown that the RA domain of SNX27 interacts with H-Ras in vitro. We postulate that K305A disrupts RA interaction with H-Ras. In conclusion, we provide the first functional evidence that intact SNX27 RA domain and interaction with small monomeric G proteins is necessary for the regulation of GIRK channels. These new findings suggest that H-Ras signaling directly affects the magnitude of GIRK currents through regulation of SNX27.

RGS proteins in heart: brakes on the vagus

It has been nearly a century since Otto Loewi discovered that acetylcholine (ACh) release from the vagus produces bradycardia and reduced cardiac contractility. It is now known that parasympathetic control of the heart is mediated by ACh stimulation of Gi/o-coupled muscarinic M2 receptors, which directly activate G protein-coupled inwardly rectifying potassium (GIRK) channels via Gβγ resulting in membrane hyperpolarization and inhibition of action potential (AP) firing. However, expression of M2R–GIRK signaling components in heterologous systems failed to recapitulate native channel gating kinetics. The missing link was identified with the discovery of regulator of G protein signaling (RGS) proteins, which act as GTPase-activating proteins to accelerate the intrinsic GTPase activity of Gα resulting in termination of Gα- and Gβγ-mediated signaling to downstream effectors. Studies in mice expressing an RGS-insensitive Gαi2 mutant (G184S) implicated endogenous RGS proteins as key regulators of parasympathetic signaling in heart. Recently, two RGS proteins have been identified as critical regulators of M2R signaling in heart. RGS6 exhibits a uniquely robust expression in heart, especially in sinoatrial (SAN) and atrioventricular nodal regions. Mice lacking RGS6 exhibit increased bradycardia and inhibition of SAN AP firing in response to CCh as well as a loss of rapid activation and deactivation kinetics and current desensitization for ACh-induced GIRK current (IKACh). Similar findings were observed in mice lacking RGS4. Thus, dysregulation in RGS protein expression or function may contribute to pathologies involving aberrant electrical activity in cardiac pacemaker cells. Moreover, RGS6 expression was found to be up-regulated in heart under certain pathological conditions, including doxorubicin treatment, which is known to cause life-threatening cardiotoxicity and atrial fibrillation in cancer patients. On the other hand, increased vagal tone may be cardioprotective in heart failure where acetylcholinesterase inhibitors and vagal stimulation have been proposed as potential therapeutics. Together, these studies identify RGS proteins, especially RGS6, as new therapeutic targets for diseases such as sick sinus syndrome or other maladies involving abnormal autonomic control of the heart.

Quantitative study of [Tyr10]nociceptin/orphanin FQ (1-11) at NOP receptors in rat periaqueductal gray and expressed NOP receptors in HEK293 cells

AimThe nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor was reported to be functionally heterogeneous. We investigated if [Tyr10]N/OFQ(1-11), a peptide ligand reported to selectively bind to the high affinity site of 125I-[Tyr14]N/OFQ in rodent brains, can be a tool for revealing the NOP receptor heterogeneity. We have previously founded an NOP receptor subset insensitive to Ro 64-6198 and (+)-5a Compound, two non-peptide NOP agonists, in rat ventrolateral periaqueductal gray (vlPAG) neurons. Here, we examined if [Tyr10]N/OFQ(1-11) differentiated (+)-5a Compound-sensitive and -insensitive vlPAG neurons. Certain mu-opioid (MOP) receptor ligands highly competing with [Tyr10]N/OFQ(1-11) in binding studies also showed high affinity at expressed heteromeric NOP–MOP receptors. We also examined if [Tyr10]N/OFQ(1-11) distinguished heteromeric NOP–MOP receptors from homomeric NOP receptors. Main methodsThe NOP receptor activity was evaluated by G-protein coupled inwardly rectifying potassium (GIRK) currents in rat vlPAG slices, and by inhibition of cAMP accumulation in HEK293 cells expressing NOP receptors or co-expressing NOP and MOP receptors. Key findingsIn vlPAG neurons, [Tyr10]N/OFQ(1-11), like N/OFQ, induced GIRK currents through NOP receptors. It was less potent (EC50: 8.98μM) but equi-efficacious as N/OFQ. [Tyr10]N/OFQ(1-11) displayed different pharmacological profiles as (+)-5a Compound, and was effective in both (+)-5a Compound-sensitive and -insensitive neurons. In NOP-expressing HEK293 cells and NOP- and MOP-co-expressing cells, [Tyr10]N/OFQ(1-11) displayed similar concentration–response curves in decreasing cAMP accumulation. Significance[Tyr10]N/OFQ(1-11) is an NOP full agonist and less potent than N/OFQ. However, it can neither reveal the functional heterogeneity of NOP receptors in vlPAG neurons nor differentiate heteromeric NOP–MOP and homomeric NOP receptors.

Inhibition of G Protein-Activated Inwardly Rectifying K+ Channels by Different Classes of Antidepressants

Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K+ (GIRK, Kir3) channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.

N‐Acetylaspartylglutamate is not demonstrated to be a selective mGlu3 receptor agonist

<i>N</i>‐Acetylaspartylglutamate is not demonstrated to be a selective mGlu₃ receptor agonist

The role of RGS protein in agonist-dependent relaxation of GIRK currents in Xenopus oocytes

G protein coupled inward rectifier K + channels (GIRK) are activated by the G βγ subunits of G proteins upon activation of G protein coupled receptors (GPCRs). Receptor-stimulated GIRK currents are known to possess a curious property, termed “agonist-dependent relaxation,” denoting a slow current increase upon stepping the membrane voltage from positive to negative potentials. Regulators of G protein signaling (RGS) proteins have earlier been implicated in this phenomenon since RGS coexpression was required for relaxation to be observed in heterologous expression systems. However, a recent study presented contrasting evidence that GIRK current relaxation reflects voltage sensitive agonist binding to the GPCR. The present study re-examined the role of RGS protein in agonist-dependent relaxation and found that RGS coexpression is not necessary for the relaxation phenomenon. However, RGS4 speeds up relaxation kinetics, allowing the phenomenon to be observed using shorter voltage steps. These findings resolve the controversy regarding the role of RGS protein vs. GPCR voltage sensitivity in mediating agonist-dependent relaxation of GIRK currents.

Acute Cocaine Exposure Weakens GABABReceptor-Dependent G-Protein-Gated Inwardly Rectifying K+Signaling in Dopamine Neurons of the Ventral Tegmental Area

Enhanced glutamatergic neurotransmission in dopamine (DA) neurons of the ventral tegmental area (VTA), triggered by a single cocaine injection, represents an early adaptation linked to the more enduring effects of abused drugs that characterize addiction. Here, we examined the impact of in vivo cocaine exposure on metabotropic inhibitory signaling involving G-protein-gated inwardly rectifying K(+) (Girk) channels in VTA DA neurons. Somatodendritic Girk currents evoked by the GABA(B) receptor (GABA(B)R) agonist baclofen were diminished in a dose-dependent manner in mice given a single cocaine injection. This adaptation persisted for 3-4 d, was specific for DA neurons of the VTA, and occurred in parallel with an increase in spontaneous glutamatergic neurotransmission. No additional suppression of GABA(B)R-Girk signaling was observed following repeated cocaine administration. While total Girk2 and GABA(B)R1 mRNA and protein levels were unaltered by cocaine exposure in VTA DA neurons, the cocaine-induced decrease in GABA(B)R-Girk signaling correlated with a reduction in Girk2-containing channels at the plasma membrane in VTA DA neurons. Systemic pretreatment with sulpiride, but not SCH23390 (7-chloro-3-methyl-1-phenyl-1,2,4,5-tetrahydro-3-benzazepin-8-ol), prevented the cocaine-induced suppression of GABA(B)R-Girk signaling, implicating D(2/3) DA receptor activation in this adaptation. The acute cocaine-induced weakening of somatodendritic Girk signaling complements the previously demonstrated cocaine-induced strengthening of glutamatergic neurotransmission, likely contributing to enhanced output of VTA DA neurons during the early stages of addiction.

The clozapine metabolite N-desmethylclozapine displays variable activity in diverse functional assays at human dopamine D2 and serotonin 5-HT1A receptors

N-desmethylclozapine (NDMC or norclozapine) is the major active metabolite of the antipsychotic clozapine in humans. The activity of NDMC differs from clozapine at a number of neurotransmitter receptors, probably influencing the pharmacological effects of clozapine treatment. Here, we tested the properties of NDMC in comparison with clozapine at recombinant human dopamine D(2) and serotonin 5-HT(1A) receptors, using a panel of functional assays implicating diverse signalling pathways. At dopamine D(2) receptors, NDMC as well as clozapine did not display agonist activity in measures of G protein activation by [(35)S]GTPγS binding and in the sensitive Extracellular Signal-Regulated Kinase 1/2 (ERK1/2) phosphorylation assay. In contrast, there were weak partial agonist actions of NDMC (but not of clozapine) for dopamine D(2)-dependent activation of Ca(2+) liberation via coexpressed chimeric Gα(q/o) proteins and for G protein-coupled inward rectifier potassium channel (GIRK) current induction in Xenopus oocytes. Intriguingly, GIRK currents induced by NDMC via dopamine D(2) receptors showed a rapid and transient time course, strikingly different from currents recorded with other receptor agonists. At serotonin 5-HT(1A) receptors, NDMC was a more efficacious partial agonist than clozapine for [(35)S]GTPγS binding, ERK1/2 phosphorylation and GIRK activation. Respective low and moderate partial agonist properties of NDMC at dopamine D(2) and serotonin 5-HT(1A) receptors thus differentiate the metabolite from its parent drug and may contribute to the overall effects of clozapine pharmacotherapy.

Reduction in cardiac Kir3.4 channel expression causes congenital long-QT syndrome—a functional role of GIRK currents in ventricular repolarization

Reduction in cardiac Kir3.4 channel expression causes congenital long-QT syndrome—a functional role of GIRK currents in ventricular repolarization

Quantitative study of the antagonistic effect of (−)- cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1-yl]methyl]-6,7,8,9-tetrahydro-5 H-benzocyclohepten-5-ol (SB-612111) on nociceptin/orphanin FQ-mediated potassium channel activation in rat periaqueductal gray slices

Nociceptin/orphanin FQ (N/OFQ) peptide (NOP) receptor, a non-opioid branch of the opioid receptor family, shows structural similarities to traditional opioid receptors but binds opioid with very poor affinity. This receptor has been implicated in many physiological functions including pain regulation. This study quantitatively investigated the effect of (−)- cis-1-Methyl-7-[[4-(2,6-dichlorophenyl)piperidin-1 -yl]methyl]-6,7,8,9-tetrahydro-5 H-benzocyclohepten-5-ol (SB-612111), a novel non-peptide ligand of NOP receptor, on the native NOP receptors in the midbrain ventrolateral periaqueductal gray (vlPAG), a crucial region for pain regulation. SB-612111 concentration-dependently antagonized N/OFQ-induced G-protein coupled inwardly rectifying K + (GIRK) current in vlPAG neurons. The IC 50 value of SB-612111 estimated from dose–response curves is 87.7 ± 1.2 nM. SB-612111 had no intrinsic agonistic activity and did not affect the GIRK current induced by [D-Ala 2, N-Me-Phe 4, Gly 5-ol]-enkephalin, a mu-opioid receptor agonist, when tested at concentrations of up to 1 μM. It is concluded that SB-612111 is a pure, potent and selective antagonist of NOP receptors that mediate GIRK channel activation in the vlPAG neurons.

Functional Sites of Interaction Between G-Protein βγ Subunits and GIRK Channels

G-proteins transduce extracellular signals received by cell surface receptors into cellular responses. Each of the α and the βγ subunits of G-proteins propagate signaling by activating or inhibiting various effector molecules in order to modulate cellular function. The first reported Gβγ effectors were the G-protein coupled inwardly rectifying K+ (GIRK or Kir3) channels. Previous studies have shown that Gβγ plays a necessary role in stimulation of both basal as well as agonist-induced components of GIRK channel currents. Work from our lab has shown that mutation of specific residues on the channel or Gβγ may preferentially affect one or the other of these components of GIRK current, leading to our working hypothesis: distinct interactions of Gβγ and GIRK channels contribute to basal versus agonist-induced currents.Through a novel combination of existing protein-protein docking methods, we are predicting sites of interaction between Gβγ and the GIRK1 channel. Initial rigid body docking results from ZDock and ZRank were filtered using steric restraints posed by the membrane and the Gβγ prenyl moiety. Clustering analysis (ClusPro server) was employed for selection of candidate structures for further flexible side-chain refinement with RosettaDock. Residue contacts predicted to be important for protein-protein interaction are tested and functionally characterized by site directed mutagenesis and two-electrode voltage clamp recordings in oocytes. Interaction sites are assessed for their contribution to basal activity, agonist-induced activity, or both. Predicted sites of contact include several residues on the channel or Gβγ previously shown to individually affect channel function, although the residues on both proteins comprising a complete interaction site remain unknown to date. These preliminary results give us confidence that the inclusion of further experimental restraints will further elucidate the distinct residue interactions involved in the mediation of agonist-induced versus basal currents.

Autocrine signaling via A1 adenosine receptors causes downregulation of M2 receptors in adult rat atrial myocytes in vitro

G protein-activated K(+) channels composed of Kir3 (GIRK) subunits contribute to regulation of heart rate and excitability. Opening of these channels in myocytes is increased by binding of G(βγ) upon activation of muscarinic M(2) receptors (M(2)-R) or A(1) adenosine receptors (A(1)-R). It has been shown that saturating activation of A(1)-R resulted in a smaller GIRK current than activation of M(2)-R. Adenovirus-driven overexpression of the A(1)-R caused an increase in current induced by adenosine (I(K(Ado))), whereas the M(2)-R-activated current (I(K(ACh))) was reduced. Here, we sought to get deeper insight into the mechanism causing this negative crosstalk. GIRK current in cultured rat atrial myocytes was recorded in whole cell mode. Adenovirus-driven RNA interference targeting the M(2)-R resulted in a reduction in I(K(ACh)) without affecting I(K(Ado)), arguing against a competition of the two receptors for common signaling complexes. The negative effect of A(1)-R overexpression on I(K(ACh)) was reduced by the A(1)-R antagonist DPCPX and augmented by the agonist chloro-N6-cyclopentyladenosin (CCPA). In native myocytes incubation with either CCPA or the muscarinic agonist carbachol resulted in reduction in I(K(ACh)) and I(K(Ado)), suggesting common pathways of A(1)-R and M(2)-R downregulation. In the absence of agonist, inhibition of adenosine deaminase by EHNA or exposure to AMP, less to ADP, but not ATP resulted in reduction of I(K(ACh)) and I(K(Ado)). Our data indicate that atrial myocytes generate adenosine from extracellular AMP, which activates A(1)-R in an autocrine fashion. Chronic activation of A(1)-R causes parallel downregulation of both A(1)-R and M(2)-R.

Morphine- and CaMKII-Dependent Enhancement of GIRK Channel Signaling in Hippocampal Neurons

G-protein-gated inwardly rectifying potassium (GIRK) channels, which help control neuronal excitability, are important for the response to drugs of abuse. Here, we describe a novel pathway for morphine-dependent enhancement of GIRK channel signaling in hippocampal neurons. Morphine treatment for ∼20 h increased the colocalization of GIRK2 with PSD95, a dendritic spine marker. Western blot analysis and quantitative immunoelectron microscopy revealed an increase in GIRK2 protein and targeting to dendritic spines. In vivo administration of morphine also produced an upregulation of GIRK2 protein in the hippocampus. The mechanism engaged by morphine required elevated intracellular Ca(2+) and was insensitive to pertussis toxin, implicating opioid receptors that may couple to Gq G-proteins. Met-enkephalin, but not the μ-selective (DAMGO) and δ-selective (DPDPE) opioid receptor agonists, mimicked the effect of morphine, suggesting involvement of a heterodimeric opioid receptor complex. Peptide (KN-93) inhibition of CaMKII prevented the morphine-dependent change in GIRK localization, whereas expression of a constitutively activated form of CaMKII mimicked the effects of morphine. Coincident with an increase in GIRK2 surface expression, functional analyses revealed that morphine treatment increased the size of serotonin-activated GIRK currents and Ba(2+)-sensitive basal K(+) currents in neurons. These results demonstrate plasticity in neuronal GIRK signaling that may contribute to the abusive effects of morphine.