Neuronal GIRK Channels Research Articles

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels: Molecular, cellular, and subcellular diversity.

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels are mainly expressed in excitable cells such as neurons and atrial myocytes, where they can respond to a wide variety of neurotransmitters. Four GIRK subunits have been found in mammals (GIRK1-4) and act as downstream targets for various Gαi/o-linked G protein-coupled receptors (GPCRs). Activation of GIRK channels produces a postsynaptic efflux of potassium from the cell, responsible for hyperpolarization/inhibition of the neuron. A growing body of evidence suggests that dysregulation of GIRK signalling can lead to excessive or deficient neuronal excitability, which contributes to neurological diseases and disorders. Therefore, GIRK channels are proposed as new pharmacological targets. The function of GIRK channels in neurons is not only determined by their biophysical properties but also by their cellular and subcellular localization patterns and densities on the neuronal surface. GIRK channels can be located within several subcellular compartments, where they have many different functional implications. This subcellular localization changes dynamically along the neuronal surface in response to drug intake. Ongoing research is focusing on determining the proteins that form macromolecular complexes with GIRK channels and are responsible for fast and precise signalling under physiological conditions, and how their alteration is implicated in pathological conditions. In this review, the distinct regional, cellular, and subcellular distribution of GIRK channel subunits in the brain will be discussed in view of their possible functional and pathological implications.

Spatial Memory Training Counteracts Hippocampal GIRK Channel Decrease in the Transgenic APPSw,Ind J9 Alzheimer's Disease Mouse Model.

G-protein-gated inwardly rectifying potassium (GIRK) channels are critical determinants of neuronal excitability. They have been proposed as potential targets to restore excitatory/inhibitory balance in acute amyloidosis models, where hyperexcitability is a hallmark. However, the role of GIRK signaling in transgenic mice models of Alzheimer's disease (AD) is largely unknown. Here, we study whether progressive amyloid-β (Aβ) accumulation in the hippocampus during aging alters GIRK channel expression in mutant β-amyloid precursor protein (APPSw,Ind J9) transgenic AD mice. Additionally, we examine the impact of spatial memory training in a hippocampal-dependent task, on protein expression of GIRK subunits and Regulator of G-protein signaling 7 (RGS7) in the hippocampus of APPSw,Ind J9 mice. Firstly, we found a reduction in GIRK2 expression (the main neuronal GIRK channels subunit) in the hippocampus of 6-month-old APPSw,Ind J9 mice. Moreover, we found an aging effect on GIRK2 and GIRK3 subunits in both wild type (WT) and APPSw,Ind J9 mice. Finally, when 6-month-old animals were challenged to a spatial memory training, GIRK2 expression in the APPSw,Ind J9 mice were normalized to WT levels. Together, our results support the evidence that GIRK2 could account for the excitatory/inhibitory neurotransmission imbalance found in AD models, and training in a cognitive hippocampal dependent task may have therapeutic benefits of reversing this effect and lessen early AD deficits.

Neuronal G protein-gated K+ channels.

G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels exert a critical inhibitory influence on neurons. Neuronal GIRK channels mediate the G protein-dependent, direct/postsynaptic inhibitory effect of many neurotransmitters including γ-aminobutyric acid (GABA), serotonin, dopamine, adenosine, somatostatin, and enkephalin. In addition to their complex regulation by G proteins, neuronal GIRK channel activity is sensitive to phosphatidylinositol 4,5-bisphosphate (PIP2), phosphorylation, regulator of G protein signaling (RGS) proteins, intracellular Na+ and Ca2+, and cholesterol. The application of genetic and viral manipulations in rodent models, together with recent progress in the development of GIRK channel modulators, has increased our understanding of the physiological and behavioral impact of neuronal GIRK channels. Work in rodent models has also revealed that neuronal GIRK channel activity is modified, transiently or persistently, by various stimuli including exposure drugs of abuse, changes in neuronal activity patterns, and aversive experience. A growing body of preclinical and clinical evidence suggests that dysregulation of GIRK channel activity contributes to neurological diseases and disorders. The primary goals of this review are to highlight fundamental principles of neuronal GIRK channel biology, mechanisms of GIRK channel regulation and plasticity, the nascent landscape of GIRK channel pharmacology, and the potential relevance of GIRK channels to the pathophysiology and treatment of neurological diseases and disorders.

Exploring GIRK Functionality in Human and Mouse Peripheral Neurons

G protein coupled receptors (GPCRs) comprise over 17% of all approved drug targets, constituting the largest family of proteins targeted by currently available pharmaceutical treatments, including those for acute and chronic pain conditions. An important factor in inhibitory GPCR function is the activation of G protein-gated inwardly rectifying potassium (GIRK) channels. Upon activation GIRK channels hyperpolarize the cells and function as an important regulator of neuronal excitability. Selective targeting of these inhibitory GPCRs in peripheral neurons has garnered recent attention as this may help to reduce nociceptor activity that might avoid the negative side effects associated with these receptors in the central nervous system. The expression of various proteins differs greatly in the central nervous system from the periphery and much of our current knowledge on GIRK activation, and interplay with nociceptive circuits, is from the central nervous system. Here we asked whether activation of inhibitory GPCRs in peripheral sensory neurons couple to GIRK and voltage-gated Ca2+ channels to dampen excitability in human neurons. To investigate the availability of GIRKs we used RT-PCR to study the different GIRK subtypes expression in extracted whole DRG, from both human donors and C57BL/6J mice. To test for functional coupling, we used whole-cell patch clamp recordings in mouse and human neurons to directly study GIRK channel activation following treatment with several different families of Gi-coupled GPCR agonists. This allowed us to directly examine both the electrical and molecular components that might comprise peripheral GIRK channel activation, which adds new layers to the complexity of GPCR function in different neuron types. This study revealed previously unknown information about GIRK channels in both human and mouse DRG neurons. Going forward, the use of human DRG for preclinical molecular and functional studies may improve translational success in pharmaceutical treatments for numerous targets. Grant support from Gereau, R.W.: NS042595; The Dr. Seymour and Rose T. Brown Professorship in Anesthesiology Del Rosario, J: K00NS113422 Chamessian, A: American Neuromuscular Foundation Career Development Award Slivicki, R: F32DA051160-01.

Characterization of VU0468554, a New Selective Inhibitor of Cardiac G Protein-Gated Inwardly Rectifying K+ Channels.

G protein-gated inwardly rectifying K+ (GIRK) channels are critical mediators of excitability in the heart and brain. Enhanced GIRK-channel activity has been implicated in the pathogenesis of supraventricular arrhythmias, including atrial fibrillation. The lack of selective pharmacological tools has impeded efforts to investigate the therapeutic potential of cardiac GIRK-channel interventions in arrhythmias. Here, we characterize a recently identified GIRK-channel inhibitor, VU0468554. Using whole-cell electrophysiological approaches and primary cultures of sinoatrial nodal cells and hippocampal neurons, we show that VU0468554 more effectively inhibits the cardiac GIRK channel than the neuronal GIRK channel. Concentration-response experiments suggest that VU0468554 inhibits Gβγ-activated GIRK channels in noncompetitive and potentially uncompetitive fashion. In contrast, VU0468554 competitively inhibits GIRK-channel activation by ML297, a GIRK-channel activator containing the same chemical scaffold as VU0468554. In the isolated heart model, VU0468554 partially reversed carbachol-induced bradycardia in hearts from wild-type mice but not Girk4-/- mice. Collectively, these data suggest that VU0468554 represents a promising new pharmacological tool for targeting cardiac GIRK channels with therapeutic implications for relevant cardiac arrhythmias. SIGNIFICANCE STATEMENT: Although cardiac GIRK-channel inhibition shows promise for the treatment of supraventricular arrhythmias, the absence of subtype-selective channel inhibitors has hindered exploration into this therapeutic strategy. This study utilizes whole-cell patch-clamp electrophysiology to characterize the new GIRK-channel inhibitor VU0468554 in human embryonic kidney 293T cells and primary cultures. We report that VU0468554 exhibits a favorable pharmacodynamic profile for cardiac over neuronal GIRK channels and partially reverses GIRK-mediated bradycardia in the isolated mouse heart model.

Structural Basis of GIRK2 Channel Modulation by Cholesterol and PIP <sub>2</sub>

G protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability and have been implicated in a number of neurological diseases. In addition to G proteins, GIRK channels are also regulated by membrane cholesterol, which is elevated in the brain for neurodegenerative diseases like Alzheimer’s Dementia and Parkinson’s Disease. The structural mechanism of cholesterol modulation of GIRK channels is not well understood. Here, we present cryo-electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) and PIP2. The structures reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain. Our analysis suggests that CHS stabilizes PIP2 interaction with the channel and promotes the engagement of the cytoplasmic domain onto the transmembrane region. Mutagenesis of residues in one CHS binding pocket occludes cholesterol-dependent potentiation. Elucidating the cholesterol-binding site and characterizing the structural mechanisms underlying modulation of neuronal GIRK channels could facilitate the development of novel therapeutics for treating human diseases.

High Cholesterol Diet Up-Regulates Atrial and Neuronal GIRK Channel Activity

High Cholesterol Diet Up-Regulates Atrial and Neuronal GIRK Channel Activity

Direct activation of G-protein-gated inward rectifying K+ channels promotes nonrapid eye movement sleep.

A major challenge in treating insomnia is to find effective medicines with fewer side effects. Activation of G-protein-gated inward rectifying K+ channels (GIRKs) by GABAB agonists baclofen or γ-hydroxybutyric acid (GHB) promotes nonrapid eye movement (NREM) sleep and consolidates sleep. However, baclofen has poor brain penetration, GHB possesses abuse liability, and in rodents both drugs cause spike-wave discharges (SWDs), an absence seizure activity. We tested the hypothesis that direct GIRK activation promotes sleep without inducing SWD using ML297, a potent and selective GIRK activator. Whole-cell patch-clamp recordings from hypocretin/orexin or hippocampal neurons in mouse brain slices were made to study neuronal excitability and synaptic activity; spontaneous activity, locomotion, contextual and tone-conditioned memory, and novel object recognition were assessed. Electroencephalogram/electromyogram (EEG/EMG) recordings were used to study GIRK modulation of sleep. ML297, like baclofen, caused membrane hyperpolarization, decreased input resistance, and blockade of spontaneous action potentials. Unlike baclofen, ML297 (5-10 µM) did not cause significant depression of postsynaptic excitatory and inhibitory currents (EPSCs-IPSCs), indicating preferential postsynaptic inhibition. ML297 (30 mg/kg, i.p.) inhibited wake activity and locomotion, and preferentially increased NREM sleep without altering EEG delta power, REM sleep, inducing SWDs, or impairing conditioned memory and novel object recognition. This study finds that direct activation of neuronal GIRK channels modulates postsynaptic membrane excitability and prolongs NREM sleep without changing sleep intensity, inducing SWDs, or impairing memory in rodents. These results suggest that direct GIRK activation with a selective compound may present an innovative approach for the treatment of chronic insomnia.

Deletion of GIRK2 subunit containing GIRK channels of neurons expressing dopamine transporter decrease immobility time on forced swimming in mice.

We previously reported that non-narcotic antitussives possessing inhibitory actions on G protein-coupled inwardly rectifying potassium (GIRK) channels have antidepressant-like effects in the forced swimming test in normal and adrenocoticotropic hormone (ACTH) treated rats. Furthermore, the antidepressant-like effects of the antitussives such as tipepidine were blocked by dopamine D1 receptor antagonist, and inhibitory actions on GIRK channels of dopamine neurons may be involved in the antidepressant-like effects of tipepidine. In this study, we generated GIRK2DATKO mice with Girk2/Kcnj6 conditional deletion and assessed depression-related behavior of the mice. The Cre/loxP system was used to selectively delete GIRK2 subunit containing GIRK channels in the neurons expressing dopamine transporter. First, deletion of GIRK2 subunits in the ventral tegmental area (VTA) neurons expressing dopamine transporters was confirmed by hisitochemically and electrophysiologically. In the mice, a significant decrease in the immobility time of forced swimming test was observed. Locomotor activity of the mice was not changed compared to that of GIRK2floxed mice, when tested in the open field. These results suggest that the antidepressant-like effect of antitussives such as tipepidine may be caused partly through the inhibitory actions on GIRK channels in the dopamine neurons.

N-(2-methoxyphenyl) benzenesulfonamide, a novel regulator of neuronal G protein-gated inward rectifier K+ channels

G protein-gated inward rectifier K+ (GIRK) channels are members of the super-family of proteins known as inward rectifier K+ (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin, dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K+ channel, another member of the Kir channel family, is regulated by sulfonamide drugs. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the somatostatin type-2 and -5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by somatostatin. In whole-cell patch clamp experiments, application of MPBS both shifted the somatostatin concentration-response curve (EC50 = 3.5nM [control] vs.1.0nM [MPBS]) for GIRK channel activation and increased the maximum GIRK current measured with 100nM somatostatin. However, GIRK channel activation was not observed when MPBS was applied to the cells in the absence of somatostatin. While the MPBS structural analog 4-fluoro-N-(2-methoxyphenyl) benzenesulfonamide also augmented the somatostatin-induced GIRK fluorescent signal, no increase in the signal was observed with the sulfonamides tolbutamide, sulfapyridine and celecoxib. In conclusion, MPBS represents a novel prototypic GPCR-dependent regulator of neuronal GIRK channels.

Dynamic role of the tether helix in PIP2-dependent gating of a G protein-gated potassium channel.

G protein-gated inwardly rectifying potassium (GIRK) channels control neuronal excitability in the brain and are implicated in several different neurological diseases. The anionic phospholipid phosphatidylinositol 4,5 bisphosphate (PIP2) is an essential cofactor for GIRK channel gating, but the precise mechanism by which PIP2 opens GIRK channels remains poorly understood. Previous structural studies have revealed several highly conserved, positively charged residues in the "tether helix" (C-linker) that interact with the negatively charged PIP2 However, these crystal structures of neuronal GIRK channels in complex with PIP2 provide only snapshots of PIP2's interaction with the channel and thus lack details about the gating transitions triggered by PIP2 binding. Here, our functional studies reveal that one of these conserved basic residues in GIRK2, Lys200 (6'K), supports a complex and dynamic interaction with PIP2 When Lys200 is mutated to an uncharged amino acid, it activates the channel by enhancing the interaction with PIP2 Atomistic molecular dynamic simulations of neuronal GIRK2 with the same 6' substitution reveal an open GIRK2 channel with PIP2 molecules adopting novel positions. This dynamic interaction with PIP2 may explain the intrinsic low open probability of GIRK channels and the mechanism underlying activation by G protein Gβγ subunits and ethanol.

GIRK2 splice variants and neuronal G protein-gated K+ channels: implications for channel function and behavior

Many neurotransmitters directly inhibit neurons by activating G protein-gated inwardly rectifying K+ (GIRK) channels, thereby moderating the influence of excitatory input on neuronal excitability. While most neuronal GIRK channels are formed by GIRK1 and GIRK2 subunits, distinct GIRK2 isoforms generated by alternative splicing have been identified. Here, we compared the trafficking and function of two isoforms (GIRK2a and GIRK2c) expressed individually in hippocampal pyramidal neurons lacking GIRK2. GIRK2a and GIRK2c supported comparable somato-dendritic GIRK currents in Girk2−/− pyramidal neurons, although GIRK2c achieved a more uniform subcellular distribution in pyramidal neurons and supported inhibitory postsynaptic currents in distal dendrites better than GIRK2a. While over-expression of either isoform in dorsal CA1 pyramidal neurons restored contextual fear learning in a conditional Girk2−/− mouse line, GIRK2a also enhanced cue fear learning. Collectively, these data indicate that GIRK2 isoform balance within a neuron can impact the processing of afferent inhibitory input and associated behavior.

N‐(2‐Methoxyphenyl) Benzenesulfonamide, a Novel Agonist of Neuronal G Protein‐Gated Inward Rectifier K+ Channels

G protein-gated inward rectifier K+ (GIRK) channels are members of the super-family of proteins known as inward rectifier K+ (Kir) channels and are expressed throughout the peripheral and central nervous systems. Neuronal GIRK channels are the downstream targets of a number of neuromodulators including opioids, somatostatin (SST), dopamine and cannabinoids. Previous studies have demonstrated that the ATP-sensitive K+ channel, another member of the Kir channel family, is regulated by sulfonamide drugs such as tolbutamide and diazoxide. Therefore, to determine if sulfonamides also modulate GIRK channels, we screened a library of arylsulfonamide compounds using a GIRK channel fluorescent assay that utilized pituitary AtT20 cells expressing GIRK channels along with the SST type-2 and 5 receptors. Enhancement of the GIRK channel fluorescent signal by one compound, N-(2-methoxyphenyl) benzenesulfonamide (MPBS), was dependent on the activation of the channel by SST. In order to determine the mechanism for the augmentation in the fluorescent signal, GIRK currents were recorded in the AtT20 cells using the whole-cell patch clamp technique (Figure 1). In control solution, application of a low concentration (0.5 nM) of SST produced little if any activation of the GIRK channel (Figure 1a). As expected, the subsequent addition of a high concentration (100 nM) of SST caused a robust activation of the current. In contrast, application of 0.5 nM SST caused a strong activation of the current in cells pretreated with MPBS (Figure 1b). In conclusion, MPBS represents a novel prototypic SST-dependent agonist of neuronal GIRK channels. Structural analogs of MPBS are currently being tested for GIRK channel potency, selectivity and mechanism of action. Support or Funding InformationSupported by NSF grant CBET-1606882 to KW. Figure 1Open in figure viewerPowerPoint GIRK currents measured in AtT20 cells during stimulation by somatostatin (SST). Currents were recorded during voltage steps applied from a holding potential of −40 mV to −100 mV during stimulation with either 0.5 or 100 nM SST.

Selective Ablation of GIRK Channels in Dopamine Neurons Alters Behavioral Effects of Cocaine in Mice.

The increase in dopamine (DA) neurotransmission stimulated by in vivo cocaine exposure is tempered by G protein-dependent inhibitory feedback mechanisms in DA neurons of the ventral tegmental area (VTA). G protein-gated inwardly rectifying K+ (GIRK/Kir3) channels mediate the direct inhibitory effect of GABAB receptor (GABABR) and D2 DA receptor (D2R) activation in VTA DA neurons. Here we examined the effect of the DA neuron-specific loss of GIRK channels on D2R-dependent regulation of VTA DA neuron excitability and on cocaine-induced, reward-related behaviors. Selective ablation of Girk2 in DA neurons did not alter the baseline excitability of VTA DA neurons but significantly reduced the magnitude of D2R-dependent inhibitory somatodendritic currents and blunted the impact of D2R activation on spontaneous activity and neuronal excitability. Mice lacking GIRK channels in DA neurons exhibited increased locomotor activation in response to acute cocaine administration and an altered locomotor sensitization profile, as well as increased responding for and intake of cocaine in an intravenous self-administration test. These mice, however, showed unaltered cocaine-induced conditioned place preference. Collectively, our data suggest that feedback inhibition to VTA DA neurons, mediated by GIRK channel activation, tempers the locomotor stimulatory effect of cocaine while also modulating the reinforcing effect of cocaine in an operant-based self-administration task.

G-protein–gated Inwardly Rectifying Potassium Channels Modulate Respiratory Depression by Opioids

Drugs acting on μ-opioid receptors (MORs) are widely used as analgesics but present side effects including life-threatening respiratory depression. MORs are G-protein-coupled receptors inhibiting neuronal activity through calcium channels, adenylyl cyclase, and/or G-protein-gated inwardly rectifying potassium (GIRK) channels. The pathways underlying MOR-dependent inhibition of rhythmic breathing are unknown. By using a combination of genetic, pharmacological, and physiological tools in rodents in vivo, the authors aimed to identify the role of GIRK channels in MOR-mediated inhibition of respiratory circuits. GIRK channels were expressed in the ventrolateral medulla, a neuronal population regulating rhythmic breathing, and GIRK channel activation with flupirtine reduced respiratory rate in rats (percentage of baseline rate in mean ± SD: 79.4 ± 7.4%, n = 7), wild-type mice (82.6 ± 3.8%, n = 3), but not in mice lacking the GIRK2 subunit, an integral subunit of neuronal GIRK channels (GIRK2, 101.0 ± 1.9%, n = 3). Application of the MOR agonist [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO) to the ventrolateral medulla depressed respiratory rate, an effect partially reversed by the GIRK channel blocker Tertiapin-Q (baseline: 42.1 ± 7.4 breath/min, DAMGO: 26.1 ± 13.4 breath/min, Tertiapin-Q + DAMGO: 33.9 ± 9.8 breath/min, n = 4). Importantly, DAMGO applied to the ventrolateral medulla failed to reduce rhythmic breathing in GIRK2 mice (percentage of baseline rate: 103.2 ± 12.1%, n = 4), whereas it considerably reduced rate in wild-type mice (62.5 ± 17.7% of baseline, n = 4). Respiratory rate depression by systemic injection of the opioid analgesic fentanyl was markedly reduced in GIRK2 (percentage of baseline: 12.8 ± 15.8%, n = 5) compared with wild-type mice (72.9 ± 27.3%). Overall, these results identify that GIRK channels contribute to respiratory inhibition by MOR, an essential step toward understanding respiratory depression by opioids.

G Protein-Gated K+ Channel Ablation in Forebrain Pyramidal Neurons Selectively Impairs Fear Learning

BackgroundCognitive dysfunction occurs in many debilitating conditions including Alzheimer’s disease, Down syndrome, schizophrenia, and mood disorders. The dorsal hippocampus is a critical locus of cognitive processes linked to spatial and contextual learning. G protein-gated inwardly rectifying potassium ion (GIRK/Kir3) channels, which mediate the postsynaptic inhibitory effect of many neurotransmitters, have been implicated in hippocampal-dependent cognition. Available evidence, however, derives primarily from constitutive gain-of-function models that lack cellular specificity. MethodsWe used constitutive and neuron-specific gene ablation models targeting an integral subunit of neuronal GIRK channels (GIRK2) to probe the impact of GIRK channels on associative learning and memory. ResultsConstitutive Girk2–/– mice exhibited a striking deficit in hippocampal-dependent (contextual) and hippocampal-independent (cue) fear conditioning. Mice lacking GIRK2 in gamma-aminobutyric acid neurons (GAD-Cre:Girk2flox/flox mice) exhibited a clear deficit in GIRK-dependent signaling in dorsal hippocampal gamma-aminobutyric acid neurons but no evident behavioral phenotype. Mice lacking GIRK2 in forebrain pyramidal neurons (CaMKII-Cre(+):Girk2flox/flox mice) exhibited diminished GIRK-dependent signaling in dorsal, but not ventral, hippocampal pyramidal neurons. CaMKII-Cre(+):Girk2flox/flox mice also displayed a selective impairment in contextual fear conditioning, as both cue fear and spatial learning were intact in these mice. Finally, loss of GIRK2 in forebrain pyramidal neurons correlated with enhanced long-term depression and blunted depotentiation of long-term potentiation at the Schaffer collateral/cornu ammonis 1 synapse in the dorsal hippocampus. ConclusionsOur data suggest that GIRK channels in dorsal hippocampal pyramidal neurons are necessary for normal learning involving aversive stimuli and support the contention that dysregulation of GIRK-dependent signaling may underlie cognitive dysfunction in some disorders.

Pharmacological Characterization of 5-HT1A Autoreceptor-Coupled GIRK Channels in Rat Dorsal Raphe 5-HT Neurons.

G protein-activated inwardly rectifying potassium (GIRK) channels in 5-HT neurons are assumed to be principal effectors of 5-hydroxytryptamine 1A (5-HT1A) autoreceptors, but their pharmacology, subunit composition and the role in regulation of 5-HT neuron activity have not been fully elucidated. We sought for a pharmacological tool for assessing the functional role of GIRK channels in 5-HT neurons by characterizing the effects of drugs known to block GIRK channels in the submicromolar range of concentrations. Whole-cell voltage-clamp recording in brainstem slices were used to determine concentration-response relationships for the selected GIRK channel blockers on 5-HT1A autoreceptor-activated inwardly rectifying K+ conductance in rat dorsal raphe 5-HT neurons. 5-HT1A autoreceptor-activated GIRK conductance was completely blocked by the nonselective inwardly rectifying potassium channels blocker Ba2+ (EC50 = 9.4 μM, full block with 100 μM) and by SCH23390 (EC50 = 1.95 μM, full block with 30 μM). GIRK-specific blocker tertiapin-Q blocked 5-HT1A autoreceptor-activated GIRK conductance with high potency (EC50 = 33.6 nM), but incompletely, i.e. ~16% of total conductance resulted to be tertiapin-Q-resistant. U73343 and SCH28080, reported to block GIRK channels with submicromolar EC50s, were essentially ineffective in 5-HT neurons. Our data show that inwardly rectifying K+ channels coupled to 5-HT1A autoreceptors display pharmacological properties generally expected for neuronal GIRK channels, but different from GIRK1-GIRK2 heteromers, the predominant form of brain GIRK channels. Distinct pharmacological properties of GIRK channels in 5-HT neurons should be explored for the development of new therapeutic agents for mood disorders.

GIRK Channels Modulate Opioid-Induced Motor Activity in a Cell Type- and Subunit-Dependent Manner.

G-protein-gated inwardly rectifying K(+) (GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation of Girk1 or Girk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential.

Modulation of Neuronal GIRK Channels by Cholesterol

In recent years, cholesterol emerged as a major regulator of ion channel function. The most common effect of cholesterol on ion channels is a decrease in channel activity. We have recently shown that unexpectedly cholesterol enrichment up‐regulates G‐protein gated inwardly rectifying potassium (GIRK or Kir3) currents in atrial myocytes. Here we focus on neuronal GIRK channels, and determine the effect of cholesterol on their function. Several GIRK subunits are expressed in the hippocampus: GIRK1, GIRK2a, GIRK2c and GIRK3. Among these, GIRK2 may be expressed as a homomer or as a heteromer with GIRK1 and/or GIRK3, both of which do not express as homomers in the plasma membrane. We first studied the effect of cholesterol on a pore mutant of GIRK2, GIRK2^ (GIRK2_E152D). We show that when GIRK2^ is expressed in Xenopus oocytes, its currents are up‐regulated by cholesterol. Next, using planar lipid bilayers we demonstrate that cholesterol significantly increases the open probability of the GIRK2^ channels. No apparent change was observed in the unitary current amplitude. Finally, we show that in‐vitro cholesterol enrichment of freshly isolated hippocampal neurons results in a strong increase in physiological GIRK currents. These findings point at cholesterol as a critical lipid regulator of GIRK channel activity in the brain.

Sorting nexin 27 regulation of G protein-gated inwardly rectifying K⁺ channels attenuates in vivo cocaine response.

The subcellular pathways that regulate G protein-gated inwardly rectifying potassium (GIRK or Kir3) channels are important for controlling the excitability of neurons. Sorting nexin 27 (SNX27) is a PDZ-containing protein known to bind GIRK2c/GIRK3 channels, but its function in vivo is poorly understood. Here, we investigated the role of SNX27 in regulating GIRK currents in dopamine (DA) neurons of the ventral tegmental area (VTA). Mice lacking SNX27 in DA neurons exhibited reduced GABABR-activated GIRK currents but had normal Ih currents and DA D2R-activated GIRK currents. Expression of GIRK2a, an SNX27-insensitive splice variant, restored GABABR-activated GIRK currents in SNX27-deficient DA neurons. Remarkably, mice with significantly reduced GABABR-activated GIRK currents in only DA neurons were hypersensitive to cocaine and could be restored to a normal locomotor response with GIRK2a expression. These results identify a pathway for regulating excitability of VTA DA neurons, highlighting SNX27 as a promising target for treating addiction.