Neuronal GIRK Channels Research Articles

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.

Regulator of G Protein Signaling 6 (RGS6) Protein Ensures Coordination of Motor Movement by Modulating GABAB Receptor Signaling

γ-Aminobutyric acid (GABA) release from inhibitory interneurons located within the cerebellar cortex limits the extent of neuronal excitation in part through activation of metabotropic GABA(B) receptors. Stimulation of these receptors triggers a number of downstream signaling events, including activation of GIRK channels by the Gβγ dimer resulting in membrane hyperpolarization and inhibition of neurotransmitter release from presynaptic sites. Here, we identify RGS6, a member of the R7 subfamily of RGS proteins, as a key regulator of GABA(B)R signaling in cerebellum. 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 Gβ(5) and R7BP. Mice lacking RGS6 exhibit abnormal gait and ataxia characterized by impaired rotarod performance improved by treatment with a GABA(B)R antagonist. RGS6(-/-) mice administered baclofen also showed exaggerated motor coordination deficits compared with their wild-type counterparts. Isolated cerebellar neurons natively expressed RGS6, GABA(B)R, and GIRK channel subunits, and cerebellar granule neurons from RGS6(-/-) mice showed a significant delay in the deactivation kinetics of baclofen-induced GIRK channel currents. These results establish RGS6 as a key component of GABA(B)R signaling and represent the first demonstration of an essential role for modulatory actions of RGS proteins in adult cerebellum. Dysregulation of RGS6 expression in human patients could potentially contribute to loss of motor coordination and, thus, pharmacological manipulation of RGS6 levels might represent a viable means to treat patients with ataxias of cerebellar origin.

Developmental regulation of G protein‐gated inwardly‐rectifying K+(GIRK/Kir3) channel subunits in the brain

G protein-gated inwardly-rectifying K(+) (GIRK/family 3 of inwardly-rectifying K(+) ) channels are coupled to neurotransmitter action and can play important roles in modulating neuronal excitability. We investigated the temporal and spatial expression of GIRK1, GIRK2 and GIRK3 subunits in the developing and adult brain of mice and rats using biochemical, immunohistochemical and immunoelectron microscopic techniques. At all ages analysed, the overall distribution patterns of GIRK1-3 were very similar, with high expression levels in the neocortex, cerebellum, hippocampus and thalamus. Focusing on the hippocampus, histoblotting and immunohistochemistry showed that GIRK1-3 protein levels increased with age, and this was accompanied by a shift in the subcellular localization of the subunits. Early in development (postnatal day 5), GIRK subunits were predominantly localized to the endoplasmic reticulum in the pyramidal cells, but by postnatal day 60 they were mostly found along the plasma membrane. During development, GIRK1 and GIRK2 were found primarily at postsynaptic sites, whereas GIRK3 was predominantly detected at presynaptic sites. In addition, GIRK1 and GIRK2 expression on the spine plasma membrane showed identical proximal-to-distal gradients that differed from GIRK3 distribution. Furthermore, although GIRK1 was never found within the postsynaptic density (PSD), the level of GIRK2 in the PSD progressively increased and GIRK3 did not change in the PSD during development. Together, these findings shed new light on the developmental regulation and subcellular diversity of neuronal GIRK channels, and support the contention that distinct subpopulations of GIRK channels exert separable influences on neuronal excitability. The ability to selectively target specific subpopulations of GIRK channels may prove effective in the treatment of disorders of excitability.

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.

GABAB receptor-mediated tonic inhibition of noradrenergic A7 neurons in the rat

Noradrenergic (NAergic) A7 neurons that project axonal terminals to the dorsal horn of the spinal cord to modulate nociceptive signaling are suggested to receive tonic inhibition from local GABAergic interneurons, which are under the regulation of descending analgesic pathways. In support of this argument, we presently report GABA(B) receptor (GABA(B)R)-mediated tonic inhibition of NAergic A7 neurons. Bath application of baclofen induced an outward current (I(Bac)) in NAergic A7 neurons that was blocked by CGP 54626, a GABA(B)R blocker. The I(Bac) was reversed at about -99 mV, displayed inward rectification, and was blocked by Ba(2+) or Tertipian-Q, showing it was mediated by G protein-activated inward-rectifying K(+) (GIRK) channels. Single-cell RT-PCR results suggested that GIRK1/3 heterotetramers might dominate functional GIRK channels in NAergic A7 neurons. Under conditions in which GABA(A) and glycine receptors were blocked, bath application of GABA inhibited the spontaneous firing of NAergic A7 neurons in a dose-dependent manner. Interestingly, CGP 54626 application not only blocked the effect of GABA but also increased the firing rate to 126.9% of the control level, showing that GABA(B)Rs were constitutively active at an ambient GABA concentration of 2.8 μM and inhibited NAergic A7 neurons. GABA(B)Rs were also found at presynaptic excitatory and inhibitory axonal terminals in the A7 area. Pharmacological activation of these GABA(B)Rs inhibited the release of neurotransmitters. No physiological role was found for GABA(B)Rs on excitatory terminals, whereas those on the inhibitory terminals were found to exert autoregulatory control of GABA release.

Mechanism underlying selective regulation of G protein-gated inwardly rectifying potassium channels by the psychostimulant-sensitive sorting nexin 27

G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases.

Targeting GIRK Channels for the Development of New Therapeutic Agents

G protein-coupled inward rectifier K+ (GIRK) channels represent novel targets for the development of new therapeutic agents. GIRK channels are activated by a large number of G protein-coupled receptors (GPCRs) and regulate the electrical activity of neurons, cardiac myocytes, and β-pancreatic cells. Abnormalities in GIRK channel function have been implicated in the patho-physiology of neuropathic pain, drug addiction, cardiac arrhythmias, and other disorders. However, the pharmacology of these channels remains largely unexplored. In this paper we describe the development of a screening assay for identifying new modulators of neuronal and cardiac GIRK channels. Pituitary (AtT20) and cardiac (HL-1) cell lines expressing GIRK channels were cultured in 96-well plates, loaded with oxonol membrane potential-sensitive dyes and measured using a fluorescent imaging plate reader. Activation of the endogenous GPCRs in the cells caused a rapid, time-dependent decrease in the fluorescent signal; indicative of K+ efflux through the GIRK channels (GPCR stimulation versus control, Z′-factor = 0.5–0.7). As expected this signal was inhibited by addition of Ba2+ and the GIRK channel toxin tertiapin-Q. To test the utility of the assay for screening GIRK channel blockers, cells were incubated for 5 min with a compound library of Na+ and K+ channel modulators. Ion transporter inhibitors such as 5-(N,N-hexamethylene)-amiloride and SCH-28080 were identified as blockers of the GIRK channel at sub-micromolar concentrations. Thus, the screening assay will be useful for expanding the limited pharmacology of the GIRK channel and in developing new agents for the treatment of GIRK channelopathies.

CRF Enhancement of GIRK Channel-Mediated Transmission in Dopamine Neurons

Dopamine neurons in the ventral midbrain contribute to learning and memory of natural and drug-related rewards. Corticotropin-releasing factor (CRF), a stress-related peptide, is thought to be involved in aspects of relapse following drug withdrawal, but the cellular actions are poorly understood. This study investigates the action of CRF on G-protein-linked inhibitory postsynaptic currents (IPSCs) mediated by GIRK (Kir3) channels in dopamine neurons. CRF enhanced the amplitude and slowed the kinetics of IPSCs following activation of D2-dopamine and GABA(B) receptors. This action was postsynaptic and dependent on the CRF(1) receptor. The enhancement induced by CRF was attenuated by repeated in vivo exposures to psychostimulants or restraint stress. The results indicate that CRF influences dopamine- and GABA-mediated inhibition in the midbrain, suggesting implications for the chronic actions of psychostimulants and stress on dopamine-mediated behaviors.

Diverse Regulation of the Neuronal G-Protein Gated K+ Channel (GIRK), GIRK1 and GIRK2 by Gα and Gβγ

G-protein activated K+ channels (GIRK, Kir3) mediate postsynaptic inhibitory effects of neurotransmitters via activation of G-protein coupled receptors, followed by activation of Gi/o proteins and direct binding of Gβγ. The main neuronal GIRK channel is composed of GIRK1 and GIRK2 heteromers. These channels express in various regions of the brain. Unlike GIRK1, GIRK2 can also form function homomeric channels predominantly expressed in the substantia nigra.In vitro protein interaction studies showed that the binding of the whole cytosolic domain of GIRK1 to Gαi3GDP or Gαi3GTP was enhanced by the presence of Gβγ. This increment was not observed with GIRK2, implying subunit specific modulated by G-proteins.Functional implication of these diversions were explored using homomeric GIRK1∗ (a pore mutant that forms functional homomers) and GIRK2 channels in Xenopus oocytes. In addition, two Gαi3 mutants were utilized to simulating the GTP/GDP bound states: “constitutively active” Gαi3Q204L (“QL”; poor GTPase) and “constitutively inactive” Gαi3G203A (“GA”) which forms a stable complex with Gβγ. GIRK2 behaved like a “classical” Gβγ effector, demonstrating very low basal activity and strong Gβγ-dependent activation, while Gα expression was without effect. GIRK1∗ exhibited large basal currents and no response to coexpressed Gβγ, whilst retaining activation by agonist. Furthermore, in excised patches GIRK1∗ homomers displayed a reverse correlation between the basal activity and the Gβγ evoked currents. Gαi3GA restored the ability of Gβγ to activate GIRK1∗, whereas Gαi3QL elicited no effect.These results suggest a specific role for GIRK1 as the scaffold for Gαiβγ within GIRK-G-protein signaling complex, while GIRK2 is the Gβγ sensitive, responsive subunit. Moreover, we suggest that GIRK1/2 may contribute to regulation of resting potential and excitability in neurons, whereas GIRK2 homomers serves as a high-gain neurotransmitter-induced inhibitory relay.

Addictive drugs modulate GIRK-channel signaling by regulating RGS proteins

Regulator of G-protein signaling (RGS) proteins are strong modulators of G-protein-mediated pathways in the nervous system. One function of RGS proteins is to accelerate the activation-deactivation kinetics of G-protein-coupled inwardly rectifying potassium (GIRK) channels. The opening of GIRK channels reduces the firing rates of neurons. Recent studies indicate that RGS proteins also modulate the coupling efficiency between gamma-aminobutyric acid type B (GABA(B)) receptors and GIRK channels in dopamine neurons of the ventral tegmental area (VTA), the initial target for addictive drugs in the brain reward pathway. Chronic drug exposure can dynamically regulate the expression levels of RGS. Functional and behavioral studies now reveal that levels of RGS2 protein, through selective association with GIRK3, critically determine whether GABA(B) agonists are excitatory or inhibitory in the VTA. The regulation of RGS protein in the reward pathway might underlie adaptation to different types of addictive drugs.

Amplitude Histogram-Based Method of Analysis of Patch Clamp Recordings that Involve Extreme Changes in Channel Activity Levels

Many ion channels show low basal activity, which is increased hundreds-fold by the relevant gating factor. A classical example is the activation G-protein-activated K(+) channels (GIRK) by Gbetagamma subunit dimer. The extent of activation (relative to basal current), R(a), is an important physiological parameter, usually readily estimated from whole cell recordings. However, calculation of R(a) often becomes non-trivial in multi-channel patches because of extreme changes in activity upon activation, from a seemingly single-channel pattern to a macroscopic one. In such cases, calculation of the net current flowing through the channels in the patch, I, before and after activation may require different methods of analysis. To address this problem, we utilized neuronal GIRK channels activated by purified Gbetagamma in excised patches of Xenopus oocytes. Channels were expressed at varying densities, from a few to several hundreds per patch. We present a simple and fast method of calculating I using amplitude histogram analysis and establish its accuracy by comparing with I calculated from event lists. This method allows the analysis of extreme changes in I in multichannel patches, which would be impossible using the standard methods of idealization and event list generation.

Behavioral characterization of mice lacking GIRK/Kir3 channel subunits

G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels mediate the postsynaptic inhibitory effects of many neurotransmitters and drugs of abuse. The lack of drugs selective for GIRK channels has hindered our ability to study their contributions to behavior. Here, we assessed the impact of GIRK subunit ablation on several behavioral endpoints. Mice were evaluated with respect to open-field motor activity and habituation, anxiety-related behavior, motor co-ordination and ataxia and operant performance. GIRK3 knockout ((-/-)) mice behaved indistinguishably from wild-type mice in this panel of tests. GIRK1(-/-) mice and GIRK2(-/-) mice, however, showed elevated motor activity and delayed habituation to an open field. GIRK2(-/-) mice, and to a lesser extent GIRK1(-/-) mice, also displayed reduced anxiety-related behavior in the elevated plus maze. Both GIRK1(-/-) mice and GIRK2(-/-) mice displayed marked resistance to the ataxic effects of the GABA(B) receptor agonist baclofen in the rotarod test. All GIRK(-/-) mice were able to learn an operant task using food as the reinforcing agent. Within-session progressive ratio scheduling, however, showed elevated lever press behavior in GIRK2(-/-) mice and, to a lesser extent, in GIRK1(-/-) mice. Phenotypic differences between mice lacking GIRK1, GIRK2 and GIRK3 correlate well with the known impact of GIRK subunit ablation on neurotransmitter-gated GIRK currents, arguing that most neuronal GIRK channels contain GIRK1 and/or GIRK2. Altogether, our data suggest that GIRK channels make important contribution to a range of behaviors and may represent points of therapeutic intervention in disorders of anxiety, spasticity and reward.

G protein gated potassium channels (GIRK/ Kir3) mediate the motor stimulatory effects of opiates

Neuronal GIRK channels mediate the postsynaptic inhibitory effect of many neurotransmitters and drugs of abuse, including opioids. GIRK subunits are expressed in dopamine and GABA neurons of the VTA, a structure implicated in opioid‐induced behaviors. We measured the impact of genetic ablation of different GIRK subunits in mice on the motor stimulatory effects of morphine. While GIRK1 knockout and GIRK2 knockout mice showed higher motor activity levels than wild‐type controls, GIRK3 knockout mice showed decreased sensitivity to morphine. We next used a stereotaxic approach to probe the relevance of GIRK signaling in the VTA to the observed responses to systemic morphine. We challenged wild‐type and knockout mice with intra‐VTA administration of the MOR selective agonist DAMGO, and measured its motor inducing effects. Our findings mirrored phenotypes seen with systemic morphine administration. Differences in opioid‐induced behaviors seen in mice lacking different GIRK subunits likely relate to the differential distribution of GIRK subunits in GABA and dopamine neurons of the VTA. Our data suggest that GIRK channels in the VTA modulate the sensitivity to opiate‐induced behaviors in a subunit‐dependent manner.

Losing RGS2 and a Taste for GHB

Depending on concentration, agonists of the γ-aminobutyric acid type B (GABA B ) receptor can either potentiate or inhibit the activity of dopaminergic neurons arising in the ventral tegmentum (a pathway involved in addiction). Labouèbe et al.-- researchers who provided a plausible mechanism for these opposing effects by showing that the coupling efficiency of GABA B receptors to G protein-activated inwardly rectifying potassium (GIRK) channels was greater in inhibitory GABAergic neurons than in dopaminergic neurons--explored regulation of GABA B -GIRK coupling. Immunoelectron microscopy of transgenic mice lacking different GIRK subunits showed that GIRK2 and GIRK3 were present in both ventral tegmental neurons in which they could detect tyrosine hydroxylase (TH, the rate-limiting enzyme for dopamine synthesis) and in neurons lacking TH, whereas GIRK1 was not found in TH-containing neurons. Both GIRK2 and GIRK3 contributed to currents evoked in dopaminergic neurons by the high-affinity GABA B agonist baclofen. Pharmacological analysis indicated that inhibition of regulator of G protein signaling proteins (RGS proteins, which act as GTPase-activating proteins) increased coupling efficiency (lowered EC 50 ) of GABA B to GIRK. Furthermore, RGS2 was preferentially expressed in dopaminergic neurons and coupling efficiency was increased in mice lacking RGS2. Fluorescence resonance energy transfer (FRET) analysis, as well as electrophysiological analysis of knockout mice, indicated that RGS2 interacted directly with GIRK3 but not GIRK2. Repeated exposure of mice to γ-hydroxybutyrate (GHB, an addictive, low-affinity GABA B receptor agonist) or morphine over several days led to an increase in coupling efficiency between GABA B receptors and GIRK in dopaminergic neurons, as well as a decrease in RGS2 mRNA in the ventral tegmentum. Intriguingly, chronic exposure to GHB caused mice to lose their preference for solutions containing it and instead to avoid them. The authors suggest that these data may provide insight into new approaches to targeting dopaminergic activity in the ventral tegmentum to treat addiction. G. Labouèbe, M. Lomazzi, H. G. Cruz, C. Creton, R. Luján, M. Li, Y. Yanagawa, K. Obata, M. Watanabe, K. Wickman, S. B. Boyer, P. A. Slesinger, C. Lüscher, RGS2 modulates coupling between GABA B receptors and GIRK channels in dopamine neurons of the ventral tegmental area. Nat. Neurosci. 10 , 1559-1568 (2007). [PubMed]

RGS2 modulates coupling between GABAB receptors and GIRK channels in dopamine neurons of the ventral tegmental area

Agonists of GABA(B) receptors exert a bi-directional effect on the activity of dopamine (DA) neurons of the ventral tegmental area, which can be explained by the fact that coupling between GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK) channels is significantly weaker in DA neurons than in GABA neurons. Thus, low concentrations of agonists preferentially inhibit GABA neurons and thereby disinhibit DA neurons. This disinhibition might confer reinforcing properties on addictive GABA(B) receptor agonists such as gamma-hydroxybutyrate (GHB) and its derivatives. Here we show that, in DA neurons of mice, the low coupling efficiency reflects the selective expression of heteromeric GIRK2/3 channels and is dynamically modulated by a member of the regulator of G protein signaling (RGS) protein family. Moreover, repetitive exposure to GHB increases the GABA(B) receptor-GIRK channel coupling efficiency through downregulation of RGS2. Finally, oral self-administration of GHB at a concentration that is normally rewarding becomes aversive after chronic exposure. On the basis of these results, we propose a mechanism that might underlie tolerance to GHB.

Molecular and Cellular Diversity of Neuronal G-Protein-Gated Potassium Channels

Neuronal G-protein-gated potassium (GIRK) channels mediate the inhibitory effects of many neurotransmitters. Although the overlapping distribution of GIRK subunits suggests that channel composition varies in the CNS, little direct evidence supports the existence of structural or functional diversity in the neuronal GIRK channel repertoire. Here we show that the GIRK channels linked to GABAB receptors differed in two neuron populations. In the substantia nigra, GIRK2 was the principal subunit, and it was found primarily in dendrites of neurons in the substantia nigra pars compacta (SNc). Baclofen evoked prominent barium-sensitive outward current in dopamine neurons of the SNc from wild-type mice, but this current was completely absent in neurons from GIRK2 knock-out mice. In the hippocampus, all three neuronal GIRK subunits were detected. The loss of GIRK1 or GIRK2 was correlated with equivalent, dramatic reductions in baclofen-evoked current in CA1 neurons. Virtually all of the barium-sensitive component of the baclofen-evoked current was eliminated with the ablation of both GIRK2 and GIRK3, indicating that channels containing GIRK3 contribute to the postsynaptic inhibitory effect of GABAB receptor activation. The impact of GIRK subunit ablation on baclofen-evoked current was consistent with observations that GIRK1, GIRK2, and GABAB receptors were enriched in lipid rafts isolated from mouse brain, whereas GIRK3 was found primarily in higher-density membrane fractions. Altogether, our data show that different GIRK channel subtypes can couple to GABAB receptors in vivo. Furthermore, subunit composition appears to specify interactions between GIRK channels and organizational elements involved in channel distribution and efficient receptor coupling.

Neuronal Kir3.1/Kir3.2a channels coupled to serotonin 1A and muscarinic m2 receptors are differentially modulated by the ‘short’ RGS3 isoform

‘Regulators of G protein signaling’ (RGS proteins) have profound effects on ion channels regulated by G protein-coupled receptor (GPCR) signaling, including the G protein-gated inwardly rectifying K + (GIRK) channels that inhibit excitability of neuronal, endocrine, and cardiac cells. Here we describe the effects of an alternatively spliced ‘short’ RGS3 isoform (RGS3s) in comparison to RGS4, on the temporal and steady-state gating properties of neuronal GIRK channels (Kir3.1/Kir3.2a) activated by either serotonin 1A (5-HT 1A) receptors or muscarinic m2 receptors expressed in Chinese hamster ovary (CHO-K1) cells. RGS3s is abundantly expressed in brain and contains a unique short N-terminus via alternative splicing that distinguishes it from other RGS3 isoforms as well as other members of the B/R4 RGS gene subfamily. Our results indicate that RGS3s and RGS4 similarly affect the temporal and steady-state gating properties of 5-HT 1A receptor-coupled Kir3.1/Kir3.2a channels, but differentially modulate muscarinic m2 receptor-coupled channels. RGS3s caused a significant ∼45% reduction in the maximal acetylcholine (ACh)-evoked GIRK current amplitude and a marked shift in the steady-state ACh dose–response relation indicative of a reduction in functionally coupled m2 receptor-GIRK channel complexes. Yet RGS3s still accelerated the m2 receptor-dependent GIRK activation, deactivation, and acute desensitization time course consistent with the RGS-enhanced GAP activity that was also observed with RGS4. Several mechanisms that may contribute to the receptor-dependent effects of RGS3s are discussed with particular attention to the role of the distinct N-terminal domain. Our findings highlight the potential impact of selective RGS-GPCR interactions on neuronal GIRK channel function that may affect the properties of inhibitory postsynaptic potentials activated by different GPCR-GIRK channel complexes.

Pertussis-toxin-sensitive G α subunits selectively bind to C-terminal domain of neuronal GIRK channels: evidence for a heterotrimeric G-protein-channel complex

Neuronal G-protein-gated inwardly rectifying potassium (Kir3; GIRK) channels are activated by G-protein-coupled receptors that selectively interact with PTX-sensitive (G αi/o) G proteins. Although the G βγ dimer is known to activate GIRK channels, the role of the G αi/o subunit remains unclear. Here, we established that G αo subunits co-immunoprecipitate with neuronal GIRK channels. In vitro binding studies led to the identification of six amino acids in the GIRK2 C-terminal domain essential for G αo binding. Further studies suggested that the G αi/oβγ heterotrimer binds to the GIRK2 C-terminal domain via G α and not G βγ. G αi/o binding-impaired GIRK2 channels exhibited reduced receptor-activated currents, but retained normal ethanol- and G βγ-activated currents. Finally, PTX-insensitive G αq or G αs subunits did not bind to the GIRK2 C-terminus. Together, these results suggest that the interaction of PTX-sensitive G αi/o subunit with the GIRK2 C-terminal domain regulates G-protein receptor coupling, and may be important for establishing specific G αi/o signaling pathways.

Measuring the Modulatory Effects of RGS Proteins on GIRK Channels

Discovery of "regulators of G-protein signaling" (RGS) as GTPase-activating proteins for heterotrimeric G proteins has provided a highly sought "missing link," reconciling past discrepancies between the in vitro GTPase activity of purified G proteins and the kinetics of physiological responses mediated by G-protein signaling in vivo. With the number of RGS genes in the mammalian genome at more than 30, associating specific RGS proteins to specific G-protein-coupled receptor (GPCR) signaling events has become a focus of RGS investigators. The ubiquitous expression of multiple RGS proteins has complicated this effort, yet the outlook has been encouraged with the identification of RGS9 as the determinant mediating rapid recovery of the transducin-dependent photoresponse. G-protein-gated inwardly rectifying potassium (GIRK) channels that mediate inhibitory synaptic transmission via GPCR activation of pertussis toxin-sensitive G proteins are similarly accelerated by RGS proteins when reconstituted in heterologous cell expression systems and fully reproduce the gating properties of native GIRK channels in neurons and cardiomyocytes. The endogenous neuronal and cardiac RGS protein(s) that accelerate GPCR-->GIRK channel-gating kinetics are currently not known. This article describes methods used to measure the receptor-dependent GIRK channel-gating parameters reconstituted in Chinese hamster ovary (CHO-K1) cells and Xenopus oocytes, as well as rat atrial myocytes and rat cerebellar granule neurons as model cells with native GPCR-->GIRK channel signaling. Applications of these methods for structure-function-based studies of RGS proteins, G proteins, and GPCRs are discussed. We also describe single cell reverse transcriptase polymerase chain reaction methods developed to profile atrial myocyte and neuronal RGS expression to identify specific RGS proteins for targeted knockdown or knockout.