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G protein-gated inwardly rectifying K

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.

Drug-induced neuroadaptations in the prefrontal cortex (PFC) have been implicated in drug-associated memories that motivate continued drug use. Chronic cocaine exposure increases pyramidal neuron excitability in the prelimbic subregion of the PFC (PL), an adaptation that has been attributed in part to a suppression of inhibitory signalling mediated by the GABA

Systemic administration of ML297, a selective activator of G-protein-gated inwardly rectifying K(+) (GIRK) channels, decreases innate avoidance behavior in male C57BL/6J mice. The cellular mechanisms mediating the ML297-induced suppression of avoidance behavior are unknown. Here, we show that systemic ML297 administration suppresses elevated plus maze (EPM)-induced neuronal activation in the ventral hippocampus (vHPC) and basolateral amygdala (BLA) and that ML297 activates GIRK1-containing GIRK channels in these limbic structures. While intracranial infusion of ML297 into the vHPC suppressed avoidance behavior in the EPM test, mirroring the effect of systemic ML297, intra-BLA administration of ML297 provoked the opposite effect. Using neuron-specific viral genetic and chemogenetic approaches, we found that the combined inhibition of excitatory neurons in CA3 and dentate gyrus (DG) subregions of the vHPC was sufficient to decrease innate avoidance behavior in the EPM, open-field, and light-dark tests in male C57BL/6J mice, while performance in the marble-burying test was not impacted. Furthermore, genetic ablation of GIRK channels in CA3/DG excitatory neurons precluded the suppression of avoidance behavior evoked by systemic ML297 in the EPM test. Thus, acute inhibition of excitatory neurons in the ventral CA3 and DG subregions of the vHPC is necessary for the apparent anxiolytic efficacy of systemic ML297 and is sufficient to decrease innate avoidance behavior in male C57BL/6J mice. SIGNIFICANCE STATEMENT We interrogated the cellular mechanisms underlying the apparent anxiolytic efficacy of ML297, a selective activator of G-protein-gated inwardly rectifying K(+) (GIRK) channels and promising lead compound. Intracranial infusion of ML297 into the ventral hippocampus (vHPC) and basolateral amygdala (BLA) complex exerted opposing influence on innate avoidance behavior in male C57BL/6J mice, the former recapitulating the suppression of avoidance behavior evoked by systemic ML297. Using viral genetic and chemogenetic approaches, we showed that combined inhibition of excitatory neurons in CA3 and dentate gyrus (DG) subregions of the vHPC is sufficient to decrease innate avoidance behavior in male mice and mediates the decrease in avoidance behavior evoked by systemic ML297. These findings establish a foundation for future investigations into the therapeutic potential of GIRK channel modulation in anxiety disorders.

Drug-induced neuroadaptations in the mPFC have been implicated in addictive behaviors. Repeated cocaine exposure has been shown to increase pyramidal neuron excitability in the prelimbic (PL) region of the mouse mPFC, an adaptation attributable to a suppression of G protein-gated inwardly rectifying K+(GIRK) channel activity. After establishing that this neuroadaptation is not seen in adjacent GABA neurons, we used viral GIRK channel ablation and complementary chemogenetic approaches to selectively enhance PL pyramidal neuron excitability in adult mice, to evaluate the impact of this form of plasticity on PL-dependent behaviors. GIRK channel ablation decreased somatodendritic GABABreceptor-dependent signaling and rheobase in PL pyramidal neurons. This manipulation also enhanced the motor-stimulatory effect of cocaine but did not impact baseline activity or trace fear learning. In contrast, selective chemogenetic excitation of PL pyramidal neurons, or chemogenetic inhibition of PL GABA neurons, increased baseline and cocaine-induced activity and disrupted trace fear learning. These effects were mirrored in male mice by selective excitation of PL pyramidal neurons projecting to the VTA, but not NAc or BLA. Collectively, these data show that manipulations enhancing the excitability of PL pyramidal neurons, and specifically those projecting to the VTA, recapitulate behavioral hallmarks of repeated cocaine exposure in mice.SIGNIFICANCE STATEMENTProlonged exposure to drugs of abuse triggers neuroadaptations that promote core features of addiction. Understanding these neuroadaptations and their implications may suggest interventions capable of preventing or treating addiction. While previous work showed that repeated cocaine exposure increased the excitability of pyramidal neurons in the prelimbic cortex (PL), the behavioral implications of this neuroadaptation remained unclear. Here, we used neuron-specific manipulations to evaluate the impact of increased PL pyramidal neuron excitability on PL-dependent behaviors. Acute or persistent excitation of PL pyramidal neurons potentiated cocaine-induced motor activity and disrupted trace fear conditioning, effects replicated by selective excitation of the PL projection to the VTA. Our work suggests that hyperexcitability of this projection drives key behavioral hallmarks of addiction.

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K(+) (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2(−/−), Girk3(−/−) and Girk2/3(−/−) mice. Bones from 12-week-old Girk2(−/−) mice were normal in length, but femurs and tibiae from Girk3(−/−) and Girk2/3(−/−) mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3(−/−) mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3(−/−) chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3(−/−) mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

Autism spectrum disorder (ASD) encompasses wide-ranging neuropsychiatric symptoms with unclear etiology. Although the cerebellum is a key region implicated in ASD, it remains elusive how the cerebellar circuitry is altered and whether the cerebellum can serve as a therapeutic target to rectify the phenotype of idiopathic ASD with polygenic abnormalities. Using a syndromic ASD model, e.g., Black and Tan BRachyury T+Itpr3tf/J (BTBR) mice, we revealed that increased excitability of presynaptic interneurons (INs) and decreased intrinsic excitability of postsynaptic Purkinje neurons (PNs) resulted in low PN firing rates in the cerebellum. Knowing that downregulation of Kv1.2 potassium channel in the IN nerve terminals likely augmented their excitability and GABA release, we applied a positive Kv1.2 modulator to mitigate the presynaptic over-inhibition and social impairment of BTBR mice. Selective restoration of the PN activity by a new chemogenetic approach alleviated core ASD-like behaviors of the BTBR strain. These findings highlight complex mechanisms converging onto the cerebellar dysfunction in the phenotypic model and provide effective strategies for potential therapies of ASD.

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.

Immediate early genes (IEGs) are transcribed in response to neuronal activity from sensory stimulation during multiple adaptive processes in the brain. The transcriptional profile of IEGs is indicative of the duration of neuronal activity, but its sensitivity to the strength of depolarization remains unknown. Also unknown is whether activity history of graded potential changes influence future neuronal activity. In this work with dissociated rat cortical neurons, we found that mild depolarization-mediated by elevated extracellular potassium (K+)-induces a wide array of rapid IEGs and transiently depresses transcriptional and signaling responses to a successive stimulus. This latter effect was independent of de novo transcription, translation, and signaling via calcineurin or mitogen-activated protein kinase. Furthermore, as measured by multiple electrode arrays and calcium imaging, mild depolarization acutely subdues subsequent spontaneous and bicuculline-evoked activity via calcium- and N-methyl-d-aspartate receptor-dependent mechanisms. Collectively, this work suggests that a recent history of graded potential changes acutely depress neuronal intrinsic properties and subsequent responses. Such effects may have several potential downstream implications, including reducing signal-to-noise ratio during synaptic plasticity processes.

Imbalance in prefrontal cortical (PFC) pyramidal neuron excitation:inhibition is thought to underlie symptomologies shared across stress-related disorders and neuropsychiatric disease, including dysregulation of emotion and cognitive function. G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channels mediate excitability of medial PFC pyramidal neurons, however, the functional role of these channels in mPFC-dependent regulation of affect, cognition, and cortical dynamics is unknown. We used a viral-cre approach in male and female mice harboring a “floxed” version of the kcnj3 (Girk1) gene, to disrupt GIRK1-containing channel expression in pyramidal neurons within the prelimbic cortex (PrL). In males, loss of pyramidal GIRK1-dependent signaling differentially impacted measures of affect and impaired working memory and cognitive flexibility. Unexpectedly, ablation of PrL GIRK1-dependent signaling did not impact affect or cognition in female mice. Additional studies used a model of chronic unpredictable stress (CUS) to determine the impact on PrL GIRK-dependent signaling and cognitive function. CUS exposure in male mice produced deficits in cognition that paralleled a reduction in PrL pyramidal GIRK-dependent signaling akin to viral approaches whereas CUS exposure in female mice did not alter cognitive flexibility performance. Stress-induced behavioral deficits in male mice were rescued by systemic injection of a novel, GIRK1-selective agonist, ML297. In conclusion, GIRK1-dependent signaling in male mice, but not females, is critical for maintaining optimal PrL function and behavioral control. Disruption of this inhibition may underlie stress-related dysfunction of the PrL and represent a therapeutic target for treating stress-induced deficits in affect regulation and impaired cognition that reduce quality of life.

γ-Aminobutyric acid B receptors (GABA

BackgroundImbalance in prefrontal cortical (PFC) pyramidal neuron excitation:inhibition is thought to underlie symptomologies shared across stress-related disorders and neuropsychiatric disease, including dysregulation of emotion and cognitive function. G protein-gated inwardly rectifying K+(GIRK/Kir3) channels mediate excitability of medial PFC pyramidal neurons, however the functional role of these channels in mPFC-dependent regulation of affect, cognition, and cortical dynamics is unknown.MethodsIn mice harboring a ‘floxed’ version of the kcnj3 (Girk1) gene, we used a viral-cre approach to disrupt GIRK1-containing channel expression in pyramidal neurons within the prelimbic (PL) or infralimbic (IL) cortices. Additional studies used a novel model of chronic unpredictable stress (CUS) to determine the impact on PL GIRK-dependent signaling and cognitive function.ResultsIn males, loss of pyramidal GIRK-dependent signaling in the PL, but not IL, differentially impacted measures of affect and motivation, and impaired working memory and cognitive flexibility. CUS produced similar deficits in affect and cognition that paralleled a reduction in PL pyramidal GIRK-dependent signaling akin to viral approaches. Viral- and stress-induced behavioral deficits were rescued by systemic injection of a novel, GIRK1-selective agonist, ML-297. Unexpectedly, neither ablation of PL GIRK-dependent signaling or exposure to the CUS regimen impacted affect or cognition in female mice.ConclusionsGIRK-dependent signaling in male mice, but not females, is critical for maintaining optimal PL function and behavioral control. Disruption of this inhibition may underlie stress-related dysfunction of the PL and represent a therapeutic target for treating stress-induced deficits in affect regulation and impaired cognition that reduce quality of life.

BackgroundDrug-induced neuroadaptations in the prefrontal cortex are thought to underlie impaired executive functions that reinforce addictive behaviors. Repeated cocaine exposure increased layer 5/6 pyramidal neuron excitability in the mouse prelimbic cortex (PL), an adaptation attributable to a suppression of G protein-gated inwardly rectifying K+ (GIRK/Kir3) channel activity. GIRK channel suppression in the PL of drug-naïve mice enhanced the motor-stimulatory effect of cocaine. The impact of cocaine on PL GABA neurons, key pyramidal neuron regulators, and the behavioral relevance of increased PL pyramidal neuron excitability, remain unclear.MethodsThe effect of repeated cocaine on mouse layer 5/6 PL GABA neurons was assessed using slice electrophysiology. Adaptations enhancing PL pyramidal neuron excitability were modeled in drug-naïve mice using persistent viral Cre ablation and acute chemogenetic approaches. The impact of these manipulations on PL-dependent behavior was assessed in motor activity and trace fear conditioning tests.ResultsRepeated cocaine treatment did not impact GIRK channel activity in, or excitability of, layer 5/6 PL GABA neurons. GIRK channel ablation in PL pyramidal neurons enhanced the motor-stimulatory effect of cocaine but did not impact baseline activity or fear learning. In contrast, direct or indirect chemogenetic activation of PL pyramidal neurons increased baseline and cocaine-induced motor activity and disrupted fear learning. These effects were mirrored by chemogenetic activation of PL pyramidal neurons projecting to the ventral tegmental area.ConclusionsManipulations enhancing the excitability of PL pyramidal neurons, including those projecting to the VTA, recapitulate behavioral hallmarks of repeated cocaine exposure.

Nicotine and alcohol are the most commonly abused substances worldwide, and comorbid nicotine and alcohol addiction is highly prevalent. Nicotinic acetylcholine receptors (nAChRs) containing the α6 and β3 subunits are expressed in neural reward circuits and are critical for both nicotine and alcohol reward. nAChRs are dynamically regulated by signaling molecules such as protein kinase C epsilon (PKCε), which impact transcription of α6 and β3 subunit mRNA (Chrna6 and Chrnb3, respectively). Previous work found decreased expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain of male PKCε−/− mice, who also consume less nicotine and alcohol compared to wild-type (WT) littermates. Here, we show that female PKCε−/− mice have enhanced expression of Chrna6 and Chrnb3 transcripts in the ventral midbrain, which functionally impacts nAChR-dependent behavior, as female but not male PKCε−/− mice exhibit locomotor hypersensitivity to nicotine. Female PKCε−/− mice show no differences in alcohol-induced sedation compared to WT littermates, while male PKCε−/− have enhanced sedation compared to WT mice, a phenotype that has previously been reported in α6−/− mice. Female PKCε−/− mice also show reduced depression-like behavior in response to systemic injections of varenicline compared to WT littermates, and this effect was absent in male mice. Additionally, we found that female PKCε−/− mice show altered alcohol and nicotine consumption patterns in chronic voluntary two bottle choice assays. Our data reveal a bidirectional effect of sex in the transcriptional regulation of nicotinic receptors by PKCε, and highlight the importance of studying both sexes in preclinical animal models.

Sinus node (SAN) dysfunction (SND) manifests as low heart rate (HR) and is often accompanied by atrial tachycardia or atrioventricular (AV) block. The only currently available therapy for chronic SND is the implantation of an electronic pacemaker. Because of the growing burden of SND in the population, new pharmacological therapies of chronic SND and heart block are desirable. We developed a collection of genetically modified mouse strains recapitulating human primary SND associated with different degrees of AV block. These mice were generated with genetic ablation of L-type Cav1.3 (Cav1.3−/−), T-type Cav3.1 (Cav3.1−/−), or both (Cav1.3−/−/Cav3.1−/−). We also studied mice haplo-insufficient for the Na+ channel Nav1.5 (Nav1.5+/) and mice in which the cAMP-dependent regulation of hyperpolarization-activated f-(HCN4) channels has been abolished (HCN4-CNBD). We analysed, by telemetric ECG recording, whether pharmacological inhibition of the G-protein-activated K+ current (IKACh) by the peptide tertiapin-Q could improve HR and AV conduction in these mouse strains. Tertiapin-Q significantly improved the HR of Cav1.3−/− (19%), Cav1.3−/−/Cav3.1−/− (23%) and HCN4-CNBD (14%) mice. Tertiapin-Q also improved cardiac conduction of Nav1.5+/− mice by 24%. Our data suggest that the development of pharmacological IKACh inhibitors for the management of SND and conduction disease is a viable approach.

γ-Aminobutyric acid B receptors (GABABRs) are broadly expressed throughout the central nervous system where they play an important role in regulating neuronal excitability and synaptic transmission. GABABRs are G protein-coupled receptors that mediate slow and sustained inhibitory actions via modulation of several downstream effector enzymes and ion channels. GABABRs are obligate heterodimers that associate with diverse arrays of proteins to form modular complexes that carry out distinct physiological functions. GABABR-dependent signaling is fine-tuned and regulated through a multitude of mechanisms that are relevant to physiological and pathophysiological states. This review summarizes the current knowledge on GABABR signal transduction and discusses key factors that influence the strength and sensitivity of GABABR-dependent signaling in neurons.

Fil: Anderson, Allison. University of Minnesota. Department of Pharmacology; Estados Unidos Fil: Masuho, Ikuo. The Scripps Research Institute, Department of Neuroscience; Estados Unidos Fil: Marrón Fernández de Velasco, Ezequiel. University of Minnesota. Department of Pharmacology; Estados Unidos Fil: Nakano, Atsushi. University of California. Department of Molecular, Cell, and Developmental Biology; Estados Unidos Fil: Birnbaumer, Lutz. National Institute of Environmental Health Sciences. Neurobiology Laboratory; Estados Unidos Fil: Birnbaumer, Lutz. Pontificia Universidad Católica Argentina. Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentina Fil: Martemyanov, Kirill A. The Scripps Research Institute, Department of Neuroscience; Estados Unidos Fil: Wickman, Kevin. University of Minnesota. Department of Pharmacology; Estados Unidos Abstract: How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+ (GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCRdependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαo over Gαi as a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαo or Gαi2 was consistent with these findings. Gαi2 ablation hadminimal impact onM2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαo ablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαo substrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

Background: Endurance athletes are prone to bradyarrhythmias, which in the long-term may underscore the increased incidence of pacemaker implantation reported in this population. Our previous work in rodent models has shown training-induced sinus bradycardia to be due to microRNA (miR)-mediated transcriptional remodeling of the HCN4 channel, leading to a reduction of the “funny” (If) current in the sinoatrial node (SAN). Objective: To test if genetic ablation of G-protein-gated inwardly rectifying potassium channel, also known as IKACh channels prevents sinus bradycardia induced by intensive exercise training in mice. Methods: Control wild-type (WT) and mice lacking GIRK4 (Girk4–/–), an integral subunit of IKACh were assigned to trained or sedentary groups. Mice in the trained group underwent 1-h exercise swimming twice a day for 28 days, 7 days per week. We performed electrocardiogram recordings and echocardiography in both groups at baseline, during and after the training period. At training cessation, mice were euthanized and SAN tissues were isolated for patch clamp recordings in isolated SAN cells and molecular profiling by quantitative PCR (qPCR) and western blotting. Results: At swimming cessation trained WT mice presented with a significantly lower resting HR that was reversible by acute IKACh block whereas Girk4–/– mice failed to develop a training-induced sinus bradycardia. In line with HR reduction, action potential rate, density of If, as well as of T- and L-type Ca2+ currents (ICaT and ICaL) were significantly reduced only in SAN cells obtained from WT-trained mice. If reduction in WT mice was concomitant with downregulation of HCN4 transcript and protein, attributable to increased expression of corresponding repressor microRNAs (miRs) whereas reduced ICaL in WT mice was associated with reduced Cav1.3 protein levels. Strikingly, IKACh ablation suppressed all training-induced molecular remodeling observed in WT mice. Conclusion: Genetic ablation of cardiac IKACh in mice prevents exercise-induced sinus bradycardia by suppressing training induced remodeling of inward currents If, ICaT and ICaL due in part to the prevention of miR-mediated transcriptional remodeling of HCN4 and likely post transcriptional remodeling of Cav1.3. Strategies targeting cardiac IKACh may therefore represent an alternative to pacemaker implantation for bradyarrhythmias seen in some veteran athletes.

The G protein-gated inwardly-rectifying potassium channels (GIRK, Kir3) are a family of inward-rectifying potassium channels, and there is significant evidence supporting the roles of GIRKs in a number of physiological processes and as potential targets for numerous indications. Previously reported urea containing molecules as GIRK1/2 preferring activators have had significant pharmacokinetic (PK) liabilities. Here we report a novel series of 1H-pyrazolo-5- yl-2-phenylacetamides in an effort to improve upon the PK properties. This series of compounds display nanomolar potency as GIRK1/2 activators with improved brain distribution (rodent Kp > 0.6).

Cocaine and other drugs of abuse trigger long-lasting adaptations in excitatory and inhibitory neurotransmission in the mesocorticolimbic system, and this plasticity has been implicated in several key facets of drug addiction. For example, glutamatergic neurotransmission mediated by AMPA receptors (AMPAR) is strengthened in medium spiny neurons (MSNs) in the NAc core and shell during withdrawal following repeated in vivo cocaine administration. Repeated cocaine administration also suppresses inhibitory signaling mediated by G protein-gated inwardly rectifying K+ (GIRK) channels in pyramidal neurons of the prelimbic cortex, an important source of glutamatergic input to the NAc core that has been implicated in cocaine-seeking and behavioral sensitization. Here, we tested the hypothesis that suppression of GIRK channel activity in forebrain pyramidal neurons can promote plasticity of glutamatergic signaling in MSNs. Using novel conditional knockout mouse lines, we report that GIRK channel ablation in forebrain pyramidal neurons is sufficient to enhance AMPAR-dependent neurotransmission in D1R-expressing MSNs in the NAc core, while also increasing motor-stimulatory responses to cocaine administration. A similar increase in AMPAR-dependent signaling was seen in both D1R- and D2R-expressing MSNs in the NAc core during withdrawal from repeated cocaine administration in normal mice. Collectively, these data are consistent with the premise that the cocaine-induced suppression of GIRK-dependent signaling in glutamatergic inputs to the NAc core contributes to some of the electrophysiological and behavioral hallmarks associated with repeated cocaine administration.

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.

Background Cognitive 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. Methods We 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. Results Constitutive 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: Girk2 flox/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(+): Girk2 flox/flox mice) exhibited diminished GIRK-dependent signaling in dorsal, but not ventral, hippocampal pyramidal neurons. CaMKII-Cre(+): Girk2 flox/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. Conclusions Our 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.

Late adolescence and young adulthood, corresponding to the high school and college years, are vulnerable periods for increased alcohol and nicotine use. The dramatic increase in the prevalence of electronic cigarette use is particularly concerning in these age groups. Late adolescents and young adults are more likely to engage in cycles of binge drug consumption, and alcohol and nicotine are frequently used together. However, there are few data examining the combination of alcohol and nicotine in binge models in animal models. In this study, our objectives were to determine how voluntary nicotine consumption beginning in late adolescence influenced subsequent binge alcohol consumption in young adulthood, how a combination of alcohol and nicotine binge consumption differed from alcohol-only binge consumption, and whether nicotine would be consumed when presented in a binge procedure. Male C57BL/6J mice voluntarily consumed unsweetened alcohol and nicotine in continuous-access bottle-choice procedures in combination with cycles of drinking-in-the-dark. Our results show that experience with voluntary nicotine consumption in late adolescence did not affect subsequent binge alcohol consumption in early adulthood. However, mice that consumed nicotine in adolescence showed an initial decrease in alcohol preference, and consequently increase in nicotine preference, on the first session of combined ethanol and nicotine binge consumption in adulthood compared with mice that drank only water during late adolescence. Lastly, we found that mice readily consumed unsweetened nicotine when presented in a binge procedure, and the level of consumption exceeded the nicotine consumption observed in the combination alcohol and nicotine binge. Our data show that expansion of the patterns of alcohol and nicotine co-consumption in a mouse models is possible, which will enable us to dissect relevant molecular targets underlying these consumption patterns and better inform drug development efforts.

Background and purpose G protein-gated inwardly rectifying K+ (Kir 3) channels moderate the activity of excitable cells and have been implicated in neurological disorders and cardiac arrhythmias. Most neuronal Kir 3 channels consist of Kir 3.1 and Kir 3.2 subtypes, while cardiac Kir 3 channels consist of Kir 3.1 and Kir 3.4 subtypes. Previously, we identified a family of urea-containing Kir 3 channel activators, but these molecules exhibit suboptimal pharmacokinetic properties and modest selectivity for Kir 3.1/3.2 relative to Kir 3.1/3.4 channels. Here, we characterize a non-urea activator, VU0810464, which displays nanomolar potency as a Kir 3.1/3.2 activator, improved selectivity for neuronal Kir 3 channels, and improved brain penetration. Experimental approach We used whole-cell electrophysiology to measure the efficacy and potency of VU0810464 in neurons and the selectivity of VU0810464 for neuronal and cardiac Kir 3 channel subtypes. We tested VU0810464 in vivo in stress-induced hyperthermia and elevated plus maze paradigms. Parallel studies with ML297, the prototypical activator of Kir 3.1-containing Kir 3 channels, were performed to permit direct comparisons. Key results VU0810464 and ML297 exhibited comparable efficacy and potency as neuronal Kir 3 channel activators, but VU0810464 was more selective for neuronal Kir 3 channels. VU0810464, like ML297, reduced stress-induced hyperthermia in a Kir 3-dependent manner in mice. ML297, but not VU0810464, decreased anxiety-related behaviour as assessed with the elevated plus maze test. Conclusion and implications VU0810464 represents a new class of Kir 3 channel activator with enhanced selectivity for Kir 3.1/3.2 channels. VU0810464 may be useful for examining Kir 3.1/3.2 channel contributions to complex behaviours and for probing the potential of Kir 3 channel-dependent manipulations to treat neurological disorders.

RationaleAlcohol and nicotine addiction are prevalent conditions that co-occur. Despite the prevalence of co-use, factors that influence the suppression and enhancement of concurrent alcohol and nicotine intake are largely unknown.ObjectivesOur goals were to assess how nicotine abstinence and availability influenced concurrent alcohol consumption, and to determine the impact of quinine adulteration of alcohol on aversion resistant alcohol consumption and concurrent nicotine consumption.MethodsMale and female C57BL/6J mice voluntarily consumed unsweetened alcohol, nicotine and water in a chronic 3-bottle choice procedure. In Experiment 1, nicotine access was removed for 1 week and re-introduced the following week, while the alcohol and water bottles remained available at all times. In Experiment 2, quinine (100-1000 μM) was added to the 20% alcohol bottle, while the nicotine and water bottles remained unaltered.ResultsIn Experiment 1, we found that alcohol consumption and preference were unaffected by the presence or absence of nicotine access in both male and female mice. In Experiment 2a, we found that quinine temporarily suppressed alcohol intake and enhanced concurrent nicotine, but not water, preference in both male and female mice. In Experiment 2b, chronic quinine suppression of alcohol intake increased nicotine consumption and preference in female mice without affecting water preference, whereas it increased water and nicotine preference in male mice.ConclusionsQuinine suppression of alcohol consumption enhanced the preference for concurrent nicotine preference in male and female mice, suggesting that mice compensate for the quinine adulteration of alcohol by increasing their nicotine preference.

G protein-gated inwardly-rectifying potassium (GIRK) channels are potassium-selective ion channels. As their name suggests, GIRK channels are effectors of G(i/o) G protein-couple receptors whereby activation of these GPCRs leads to increased GIRK channel activity resulting in decreased cellular excitability. In this way, GIRK channels play diverse roles in physiology as effectors of G(i/o)-coupled GPCRs: peacemaking in the heart rate, modulation of hormone secretion in endocrine tissues, as well as numerous CNS functions including learning, memory and addiction/reward. Notably, GIRK channels are widely expressed along the spinothalamic tract and are positioned to play roles in both ascending and descending pain pathways. More notably, GIRK channel knockout and knock-down studies have found that GIRK channels play a major role in the action of opioid analgesics which act predominantly through G(i/o)-coupled, opioid-activated GPCRs (e.g. μ-opioid receptors). Recent advances in GIRK channel pharmacology have led to the development of small molecules that directly and selectively activate GIRK channels. Based on research implicating the involvement of GIRK channels in pain pathways and as effectors of opioid analgesics, we conducted a study to determine whether direct pharmacological activation of GIRK channels could produce analgesic efficacy and/or augment the analgesic efficacy morphine, an opioid receptor agonist capable of activating μ-opioid receptors as well as other opioid receptor subtypes. In the present study, we demonstrate that the small-molecule GIRK activator, VU0466551, has analgesic effects when dosed alone or in combination with sub-maximally effective doses of morphine.

Introduction and Hypothesis: Parasympathetic slowing of heart rate is largely mediated by muscarinic receptor (M2R) activation of the G protein gated inwardly-rectifying potassium (GIRK) channel on sinoatrial-nodal (SAN) cells and atrial myocytes. The strength of M2R-GIRK signaling is negatively regulated by regulator of G protein signaling 6, RGS6. Genetic ablation of RGS6 in mice gives rise to enhanced M2R-GIRK signaling in SAN cells, resulting in both exaggerated M2R-induced bradycardia and increased susceptibility to pacing-induced atrial fibrillation. Adenosine receptor (A1R) activation, which can provoke atrial arrhythmias in human patients, can also activate GIRK channels, however, the details of A1R-GIRK signaling are poorly understood. Here, we investigated A1R-GIRK signaling in mouse SAN cells and tested the hypothesis that RGS6 negatively regulates A1R-GIRK signaling. Methods and Results: Using whole-cell patch-clamp electrophysiology, we measured the efficacy and potency of M2R- and A1R- GIRK signaling in adult SAN cells from wildtype and Rgs6 -/- mice. We found SAN cells from Rgs6 -/- mice displayed both prolonged channel deactivation kinetics and increased channel sensitivity to CCh-induced currents as compared to wild-type SAN cells. Surprisingly, we found no difference in kinetics or channel sensitivity of A1R-GIRK responses in Rgs6 -/- SAN cells. We did observe, however, a striking, significant increase in the amplitude of the A1R-GIRK response in Rgs6 -/- SAN cells compared to wild-type controls. Furthermore, occlusion studies in wild-type SAN cells suggest that M2R activates nearly all of the GIRK channels present in SAN cells, while A1R activation results in only partial GIRK channel activation. Intriguingly, RGS6 ablation seems to allow a larger proportion of GIRK channels to be activated by A1R. Recordings from mice lacking cardiac GIRK channels confirm that M2R- and A1R- induced currents are GIRK-dependent. Conclusions: Our results suggest that M2R-GIRK and A1R-GIRK are coupled differently within mouse sino-atrial nodal cells, resulting in differential regulation by RGS6.

Drugs of abuse engage overlapping but distinct molecular and cellular mechanisms to enhance dopamine (DA) signaling in the mesocorticolimbic circuitry. DA neurons of the ventral tegmental area (VTA) are key substrates of drugs of abuse and have been implicated in addiction-related behaviors. Enhanced VTA DA neurotransmission evoked by drugs of abuse can engage inhibitory G-protein-dependent feedback pathways, mediated by GABABreceptors (GABABRs) and D2DA receptors (D2Rs). Chemogenetic inhibition of VTA DA neurons potently suppressed baseline motor activity, as well as the motor-stimulatory effect of cocaine and morphine, confirming the critical influence of VTA DA neurons and inhibitory G-protein signaling in these neurons on this addiction-related behavior. To resolve the relative influence of GABABR-dependent and D2R-dependent signaling pathways in VTA DA neurons on behavioral sensitivity to drugs of abuse, we developed a neuron-specific viral CRISPR/Cas9 approach to ablate D2R and GABABR in VTA DA neurons. Ablation of GABABR or D2R did not impact baseline physiological properties or excitability of VTA DA neurons, but it did preclude the direct somatodendritic inhibitory influence of GABABR or D2R activation. D2R ablation potentiated the motor-stimulatory effect of cocaine in male and female mice, whereas GABABR ablation selectively potentiated cocaine-induced activity in male subjects only. Neither D2R nor GABABR ablation impacted morphine-induced motor activity. Collectively, our data show that cocaine and morphine differ in the extent to which they engage inhibitory G-protein-dependent feedback pathways in VTA DA neurons and highlight key sex differences that may impact susceptibility to various facets of addiction.

Repeated exposure to psychostimulants induces locomotor sensitization and leads to persistent changes in the circuitry of the mesocorticolimbic dopamine (DA) system. G-protein-gated inwardly rectifying potassium (GIRK; also known as Kir3) channels mediate a slow IPSC and control the excitability of DA neurons. Repeated 5 d exposure to psychostimulants decreases the size of the GABABreceptor (GABABR)-activated GIRK currents (IBaclofen) in ventral tegmental area (VTA) DA neurons of mice, but the mechanism underlying this plasticity is poorly understood. Here, we show that methamphetamine-dependent attenuation of GABABR-GIRK currents in VTA DA neurons required activation of both D1R-like and D2R-like receptors. The methamphetamine-dependent decrease in GABABR-GIRK currents in VTA DA neurons did not depend on a mechanism of dephosphorylation of the GABABR2 subunit found previously for other neurons in the reward pathway. Rather, the presence of the GIRK3 subunit appeared critical for the methamphetamine-dependent decrease of GABABR-GIRK current in VTA DA neurons. Together, these results highlight different regulatory mechanisms in the learning-evoked changes that occur in the VTA with repeated exposure to psychostimulants.SIGNIFICANCE STATEMENTExposure to addictive drugs such as psychostimulants produces persistent adaptations in inhibitory circuits within the mesolimbic dopamine system, suggesting that addictive behaviors are encoded by changes in the reward neural circuitry. One form of neuroadaptation that occurs with repeated exposure to psychostimulants is a decrease in slow inhibition, mediated by a GABABreceptor and a potassium channel. Here, we examine the subcellular mechanism that links psychostimulant exposure with changes in slow inhibition and reveal that one type of potassium channel subunit is important for mediating the effect of repeated psychostimulant exposure. Dissecting out the components of drug-dependent plasticity and uncovering novel protein targets in the reward circuit may lead to the development of new therapeutics for treating addiction.

Background 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. Methods 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. Results 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-Ala2, N-MePhe4, 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%). Conclusions Overall, these results identify that GIRK channels contribute to respiratory inhibition by MOR, an essential step toward understanding respiratory depression by opioids.

BACKGROUND AND PURPOSE: G protein‐gated inwardly rectifying K(+) (K(ir)3) channels moderate the activity of excitable cells and have been implicated in neurological disorders and cardiac arrhythmias. Most neuronal K(ir)3 channels consist of K(ir)3.1 and K(ir)3.2 subtypes, while cardiac K(ir)3 channels consist of K(ir)3.1 and K(ir)3.4 subtypes. Previously, we identified a family of urea‐containing K(ir)3 channel activators, but these molecules exhibit suboptimal pharmacokinetic properties and modest selectivity for K(ir)3.1/3.2 relative to K(ir)3.1/3.4 channels. Here, we characterize a non‐urea activator, VU0810464, which displays nanomolar potency as a K(ir)3.1/3.2 activator, improved selectivity for neuronal K(ir)3 channels, and improved brain penetration. EXPERIMENTAL APPROACH: We used whole‐cell electrophysiology to measure the efficacy and potency of VU0810464 in neurons and the selectivity of VU0810464 for neuronal and cardiac K(ir)3 channel subtypes. We tested VU0810464 in vivo in stress‐induced hyperthermia and elevated plus maze paradigms. Parallel studies with ML297, the prototypical activator of K(ir)3.1‐containing K(ir)3 channels, were performed to permit direct comparisons. KEY RESULTS: VU0810464 and ML297 exhibited comparable efficacy and potency as neuronal K(ir)3 channel activators, but VU0810464 was more selective for neuronal K(ir)3 channels. VU0810464, like ML297, reduced stress‐induced hyperthermia in a K(ir)3‐dependent manner in mice. ML297, but not VU0810464, decreased anxiety‐related behaviour as assessed with the elevated plus maze test. CONCLUSION AND IMPLICATIONS: VU0810464 represents a new class of K(ir)3 channel activator with enhanced selectivity for K(ir)3.1/3.2 channels. VU0810464 may be useful for examining K(ir)3.1/3.2 channel contributions to complex behaviours and for probing the potential of K(ir)3 channel‐dependent manipulations to treat neurological disorders.