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GIRK Channels
Abstract
The ability of drug-associated cues to reinitiate drug craving and seeking, even after long periods of abstinence, has led to the hypothesis that addiction represents a form of pathological learning, in which drugs of abuse hijack normal learning and memory processes to support long-term addictive behaviors. In this chapter, we review evidence suggesting that G protein-gated inwardly rectifying potassium (GIRK/Kir3) channels are one mechanism through which numerous drugs of abuse can modulate learning and memory processes. We will examine the role of GIRK channels in two forms of experience-dependent long-term changes in neuronal function: homeostatic plasticity and synaptic plasticity. We will also discuss how drug-induced changes in GIRK-mediated signaling can lead to changes that support the development and maintenance of addiction.
... GIRK1, GIRK2, and GIRK3 subunits are distributed throughout the central nervous system, including in key regions that are involved in cognition, learning, memory, emotion, and motor function. [4][5][6][7] The GIRK2 subunit plays a role in regulating addictive substance-induced behaviors. GIRK2 knockout mice exhibit alterations of behavioral effects of alcohol, including an increase in alcohol consumption, a decrease in alcohol dependence, the stimulation of motor activity, and a decrease in the anxiolytic action of alcohol. ...
... The activity of GIRK channels is also partially involved in this process. 7 Based on the present results and previous findings, weaver mutant mice may exhibit impairments in reward-related learning and memory processes that are attributable to abnormal GIRK2 subunit function. Consequently, the absence of METH-induced CPP in weaver mutant mice might result from a failure to remember the drug-paired compartment. ...
Aims
G protein‐activated inwardly rectifying potassium (GIRK) channels are related to rewarding effects of addictive drugs. The GIRK2 subunit is thought to play key roles in the reward system. Weaver mutant mice exhibit abnormal GIRK2 function and different behaviors that are caused by several addictive substances compared with wild‐type mice. However, mechanisms of reward‐related alterations in weaver mutant mice remain unclear. The present study investigated changes in the rewarding effects of methamphetamine (METH) in weaver mutant mice.
Methods
The rewarding effects of METH (4.0 mg/kg) were investigated using the conditioned place preference (CPP) paradigm. Extracellular dopamine level in the nucleus accumbens (NAc) was measured by in vivo microdialysis. To identify brain regions that were associated with these changes in rewarding effects, METH‐induced alterations of Fos expression were investigated by immunohistochemical analysis.
Results
Weaver mutant mice exhibited a significant decrease in METH‐induced CPP and dopamine release in the NAc. Methamphetamine significantly increased Fos expression in the posterior NAc (pNAc) shell in wild‐type but not in weaver mutant mice.
Conclusions
Methamphetamine did not induce rewarding effects in weaver mutant mice. The pNAc shell exhibited a significant difference in neuronal activity between wild‐type and weaver mutant mice. The present results suggest that the absence of METH‐induced CPP in weaver mutant mice is probably related to an innate reduction of dopamine and decreased neural activity in the pNAc shell that is partially attributable to the change of GIRK channel function. GIRK channels, especially those containing the GIRK2 subunit, appear to be involved in METH dependence.
... In any case, Clarke et al. indeed found that NOR memory assessment induced an early depotentiation that could very likely be due to the natural GirK-mediated inhibitory LTP-like activity induced by NOR retrieval that we have found here for the first time. In fact, the presence of an inhibitory GirK-dependent LTP with a late appearance 48 h after an artificial HFS protocol has been previously demonstrated [21], suggesting that the plasticity of GirK channel signaling might be involved in the extinction of the fEPSP potentiation to basal amplitude levels [26,75]. There are factors that decrease the probability of reconsolidation taking place (age, sleep, memory strength, weak reactivation sessions, and predictable reactivated stimulus), as well as factors that promote reconsolidation (epigenetic priming, new information during reactivation, increased intensity of reactivation session, and plasticity enhancer strategies) [76][77][78]. ...
Synaptic plasticity is a cellular process involved in learning and memory by which specific patterns of neural activity adapt the synaptic strength and efficacy of the synaptic transmission. Its induction is governed by fine tuning between excitatory/inhibitory synaptic transmission. In experimental conditions, synaptic plasticity can be artificially evoked at hippocampal CA1 pyramidal neurons by repeated stimulation of Schaffer collaterals. However, long-lasting synaptic modifications studies during memory formation in physiological conditions in freely moving animals are very scarce. Here, to study synaptic plasticity phenomena during recognition memory in the dorsal hippocampus, field postsynaptic potentials (fPSPs) evoked at the CA3-CA1 synapse were recorded in freely moving mice during object-recognition task performance. Paired pulse stimuli were applied to Schaffer collaterals at the moment that the animal explored a new or a familiar object along different phases of the test. Stimulation evoked a complex synaptic response composed of an ionotropic excitatory glutamatergic fEPSP, followed by two inhibitory responses, an ionotropic, GABAA-mediated fIPSP and a metabotropic, G-protein-gated inwardly rectifying potassium (GirK) channel-mediated fIPSP. Our data showed the induction of LTP-like enhancements for both the glutamatergic and GirK-dependent components of the dorsal hippocampal CA3-CA1 synapse during the exploration of novel but not familiar objects. These results support the contention that synaptic plasticity processes that underlie hippocampal-dependent memory are sustained by fine tuning mechanisms that control excitatory and inhibitory neurotransmission balance.
... The study by Herman et al. (2015) exhibits the role of K IR 3.3/Kcnj9 as a critical gatekeeper of ethanol incentive salience and projects as a potential target for the treatment of excessive ethanol consumption [81]. Similar to Herman et al. (2015), knock-out (KO) of K IR 3.3/KCNJ9/protein enhanced ethanol conditioned place preference [94,95]. ...
Substance use disorders (SUDs) are ubiquitous throughout the world. However, much remains to be done to develop pharmacotherapies that are very efficacious because the focus has been mostly on using dopaminergic agents or opioid agonists. Herein we discuss the potential of using potassium channel activators in SUD treatment because evidence has accumulated to support a role of these channels in the effects of rewarding drugs. Potassium channels regulate neuronal action potential via effects on threshold, burst firing, and firing frequency. They are located in brain regions identified as important for the behavioral responses to rewarding drugs. In addition, their expression profiles are influenced by administration of rewarding substances. Genetic studies have also implicated variants in genes that encode potassium channels. Importantly, administration of potassium agonists have been shown to reduce alcohol intake and to augment the behavioral effects of opioid drugs. Potassium channel expression is also increased in animals with reduced intake of methamphetamine. Together, these results support the idea of further investing in studies that focus on elucidating the role of potassium channels as targets for therapeutic interventions against SUDs.
... Los receptores metabotrópicos muscarínicos acoplados a proteína G i están involucrados en la activación de canales rectificadores de entrada de potasio (GIRKs, por sus siglas en inglés) a través del dímero formado por las subunidades βγ de la proteína G [46]. Este dímero favorece la apertura del canal GIRK y el eflujo de cationes potasio (K + ), resultando en un proceso de hiperpolarización de la membrana celular [47]. Esta propiedad de los receptores muscarínicos del subtipo M 2 /M 4 ha sido emulada en células de riñón de embrión humano (HEK-239) mediante la expresión del DREADD de diseño M4. ...
En los años 80, tomó fuerza la ciencia dirigida al estudio del genoma humano, cuyo objetivo era mejorar la salud humana a través de la identificación de genes. Este estudio permitió el surgimiento de ciencias, entre ellas la Quimiogenómica y la Quimiogenética. La Quimiogenómica es un área interdisciplinaria que busca la sistematización del genoma al identificar, analizar, expandir y predecir las interacciones de los ligandos con las proteínas por medio de métodos in vitro e in silico. Por otro lado, la Quimiogenética se refiere al estudio de las características genéticas de un paciente para optimizar la Farmacoterapia y predecir la eficacia, los efectos secundarios y la dosificación de fármacos selectos. El conocimiento de las interacciones entre moléculas y proteínas específicas ayudaron al desarrollo de nuevas herramientas biotecnológicas y a la identificación de novedosas dianas farmacológicas que pudieran conducir a conocimientos más específicos. Algunas aplicaciones en estudio en el ámbito farmacéutico son los receptores diseñados exclusivamente activados por fármacos diseñados (DREADDs, por sus siglas en inglés). Con ellos se pretende que no se activen ante ligandos endógenos, sino en presencia de sintéticos. Actualmente existen muchas investigaciones en los cuales se estudian DREADDs para la identificación de neuronas y de vías de señalización involucradas en patologías como el miedo, la ansiedad, la depresión, la adicción y la obesidad.
... Their activation inhibits excitability, slowing the rate of pacemaker and atrial cell firing in the heart, inhibiting transmitter release by presynaptic neurons or opposing excitation of postsynaptic neurons. Polymorphisms and mutations in human GIRK channels have been linked to arrhythmias, hyperaldosteronism (and associated hypertension), sensitivity to analgesics, addiction, alcohol dependence, anxiety, and schizophrenia (7)(8)(9)(10). GIRK channels are activated by binding of the G protein βγ (Gβγ) subunits (1,(3)(4)(5)(6)(7)(11)(12)(13). ...
G protein–gated inwardly rectifying K ⁺ (GIRK) channels are targets of Gi/o protein signaling systems that inhibit cell excitability. GIRK channels exist as homotetramers (GIRK2 and GIRK4) or heterotetramers with non-functional homomeric subunits (GIRK1 and GIRK3). Although they have been implicated in multiple conditions, the lack of selective GIRK drugs that discriminate among the different GIRK channel subtypes has hampered investigations into their precise physiological relevance and therapeutic potential. Here, we report a highly specific, potent, and efficacious activator of brain GIRK1/2 channels. Using a chemical screen and electrophysiological assays, we found that this activator, the bromothiophene-substituted small molecule GAT1508, is specific for brain-expressed GIRK1/2 channels rather than for cardiac GIRK1/4 channels. Computational models predicted a GAT1508-binding site validated by experimental mutagenesis experiments, providing insights into how urea-based compounds engage distant GIRK1 residues required for channel activation. Furthermore, we provide computational and experimental evidence that GAT1508 is an allosteric modulator of channel–phosphatidylinositol 4,5-bisphosphate (PIP 2 ) interactions. Through brain slice electrophysiology, we show that subthreshold GAT1508 concentrations directly stimulate GIRK currents in the basolateral amygdala (BLA) and potentiate baclofen-induced currents. Of note, GAT1508 effectively extinguished conditioned fear in rodents and lacked cardiac and behavioral side effects, suggesting its potential for use in pharmacotherapy for post-traumatic stress disorder (PTSD). In summary, our findings indicate that the small molecule GAT1508 has high specificity for brain GIRK1/2 channel subunits, directly or allosterically activates GIRK1/2 channels in the BLA, and facilitates fear extinction in a rodent model.
For the past two decades several scholarly reviews have appeared on the inwardly rectifying potassium (Kir) channels. We would like to highlight two efforts in particular, which have provided comprehensive reviews of the literature up to 2010 (Hibino et al., Physiol Rev 90(1):291-366, 2010; Stanfield et al., Rev Physiol Biochem Pharmacol 145:47-179, 2002). In the past decade, great insights into the 3-D atomic resolution structures of Kir channels have begun to provide the molecular basis for their functional properties. More recently, computational studies are beginning to close the time domain gap between in silico dynamic and patch-clamp functional studies. The pharmacology of these channels has also been expanding and the dynamic structural studies provide hope that we are heading toward successful structure-based drug design for this family of K+ channels. In the present review we focus on placing the physiology and pharmacology of this K+ channel family in the context of atomic resolution structures and in providing a glimpse of the promising future of therapeutic opportunities.
Imbalances of excitatory/inhibitory synaptic transmission occur early in the pathogenesis of Alzheimer’s disease (AD), leading to hippocampal hyperexcitability and causing synaptic, network, and cognitive dysfunctions. G-protein-gated potassium (GirK) channels play a key role in the control of neuronal excitability, contributing to inhibitory signaling. Here, we evaluate the relationship between GirK channel activity and inhibitory hippocampal functionality in vivo. In a non-transgenic mouse model of AD, field postsynaptic potentials (fPSPs) from the CA3–CA1 synapse in the dorsal hippocampus were recorded in freely moving mice. Intracerebroventricular (ICV) injections of amyloid-β (Aβ) or GirK channel modulators impaired ionotropic (GABAA-mediated fPSPs) and metabotropic (GirK-mediated fPSPs) inhibitory signaling and disrupted the potentiation of synaptic inhibition. However, the activation of GirK channels prevented Aβ-induced changes in GABAA components. Our data shows, for the first time, the presence of long-term potentiation (LTP) for both the GABAA and GirK-mediated inhibitory postsynaptic responses in vivo. In addition, our results support the importance of an accurate level of GirK-dependent signaling for dorsal hippocampal performance in early amyloid pathology models by controlling the excess of excitation that disrupts synaptic plasticity processes.
Family, twin and adoption studies demonstrate clearly that alcohol dependence and alcohol use disorders are phenotypically complex and heritable. The heritability of alcohol use disorders is estimated at approximately 50–60% of the total phenotypic variability. Vulnerability to alcohol use disorders can be due to multiple genetic or environmental factors or their interaction which gives rise to extensive and daunting heterogeneity. This heterogeneity makes it a significant challenge in mapping and identifying the specific genes that influence alcohol use disorders. Genetic linkage and (candidate gene) association studies have been used now for decades to map and characterize genomic loci and genes that underlie the genetic vulnerability to alcohol use disorders. These approaches have been moderately successful in identifying several genes that contribute to the complexity of alcohol use disorders. Recently, genome-wide association studies have become one of the major tools for identifying genes for alcohol use disorders by examining correlations between millions of common single-nucleotide polymorphisms with diagnosis status. Genome-wide association studies are just beginning to uncover novel biology; however, the functional significance of results remains a matter of extensive debate and uncertainty. In this review, we present a select group of genome-wide association studies of alcohol dependence, as one example of a way to generate functional hypotheses, within the addiction cycle framework. This analysis may provide novel directions for validating the functional significance of alcohol dependence candidate genes.
Contextual stimuli associated with drug exposure can modulate various effects of drugs, but little is known about their role in relapse to drug seeking. Using a renewal procedure, the authors report that drug-associated contextual stimuli play a critical role in relapse to drug-seeking previously maintained by a heroin–cocaine mixture (speedball). Rats were trained to self-administer speedball, after which drug-reinforced behavior was extinguished over 20 days in the self-administration context or in a different context. On the test day, rats exposed to the drug-associated context, after extinction in a different context, reliably renewed drug seeking. The authors suggest that the renewal procedure can be used to study mechanisms underlying relapse to drug seeking elicited by drug-associated contextual stimuli.
Molecular cloning together with functional characterization has shown that the newly identified family of inwardly rectifying K ⁺ channels consists of several closely related members encoded by separate genes. In this report we demonstrate the differential mRNA expression and detailed cellular localization in the adult rat brain of seven members of the IRK and GIRK subfamilies. Using both radiolabeled cRNA riboprobes and specific oligonucleotide probes directed to nonconserved regions of both known and newly isolated rat brain cDNAs, in situ hybridization revealed wide distribution with partly overlapping expression of the mRNAs of IRK1–3 and GIRK1–4. Except for the low levels of GIRK4 transcripts observed, the overall distribution patterns of the other GIRK subunits were rather similar, with high levels of expression in the olfactory bulb, hippocampus, cortex, thalamus, and cerebellum. Marked differences in expression levels existed only in some thalamic, brainstem, and midbrain nuclei, e.g., the substantia nigra, superior colliculus, or inferior olive. In contrast, IRK subunits were expressed more differentially: all mRNAs were abundant in dentate gyrus, olfactory bulb, caudate putamen, and piriform cortex. IRK1 and IRK3 were restricted to these regions, but they were absent from most parts of the thalamus, cerebellum, and brainstem, where IRK2 was expressed predominantly. Because channel subunits may assemble as heteromultimers, additional functional characterization based on overlapping expression patterns may help to decipher the native K ⁺ channels in neurons and glial cells.
MEK (mitogen-activated protein kinase/extracellular signal-regulated kinasekinase) kinases (MEKKs) regulate c-Jun N-terminal kinase and extracellular response kinase pathways. The 14-3-3ζ and 14-3-3ε
isoforms were isolated in a two-hybrid screen for proteins interacting with the N-terminal regulatory domain of MEKK3. 14-3-3
proteins bound both the N-terminal regulatory and C-terminal kinase domains of MEKK3. The binding affinity of 14-3-3 for the
MEKK3 N terminus was 90 nm, demonstrating a high affinity interaction. 14-3-3 proteins also interacted with MEKK1 and MEKK2, but not MEKK4. Endogenous
14-3-3 protein and MEKK1 and MEKK2 were similarly distributed in the cell, consistent with their in vitrointeractions. MEKK1 and 14-3-3 proteins colocalized using two-color digital confocal immunofluorescence. Binding of 14-3-3
proteins mapped to the N-terminal 393 residues of 196-kDa MEKK1. Unlike MEKK2 and MEKK3, the C-terminal kinase domain of MEKK1
demonstrated little or no ability to interact with 14-3-3 proteins. MEKK1, but not MEKK2, -3 or -4, is a caspase-3 substrate
that when cleaved releases the kinase domain from the N-terminal regulatory domain. Functionally, caspase-3 cleavage of MEKK1
releases the kinase domain from the N-terminal 14-3-3-binding region, demonstrating that caspases can selectively alter protein
kinase interactions with regulatory proteins. With regard to MEKK1, -2 and -3, 14-3-3 proteins do not appear to directly influence
activity, but rather function as “scaffolds” for protein-protein interactions.
Significance
Almost 20% of women and 40% of men in the United States abuse alcohol or have experienced alcohol dependence in their lifetime. Though accidents, traffic fatalities, and violent crimes account for the majority of alcohol-involved mortalities, excessive, chronic drinking also causes often irreversible heart and liver damage. We identify regulator of G protein signaling 6 (RGS6) as a novel drug target with substantive potential clinical utility in the treatment of alcoholism and amelioration of the resultant hepatic and cardiac toxicity. Mice lacking RGS6 exhibit a reduction in voluntary alcohol consumption, conditioned reward and withdrawal. In addition, RGS6 −/− mice are largely protected from alcohol-induced cardiomyopathy, hepatic steatosis, gastrointestinal barrier dysfunction, and endotoxemia. Thus, targeting RGS6 could reduce alcohol cravings while simultaneously protecting the heart and liver from damage.
The dorsal and ventral hippocampi are functionally and anatomically distinct. Recently, we reported that dorsal CA1 neurons have a more hyperpolarized resting membrane potential (RMP), a lower input resistance (Rin), and fire fewer action potentials for a given current injection than ventral CA1 neurons. Differences in the hyperpolarizing HCN conductance between dorsal and ventral neurons have been reported, but these differences cannot fully account for the different resting properties of these neurons. Here we show that coupling of A1 adenosine receptors (A1ARs) to G-protein coupled inwardly rectifying potassium (GIRK) conductance contributes to the intrinsic membrane properties of dorsal CA1 neurons but not ventral CA1 neurons. The block of GIRKs with either barium or the more specific blocker tertiapin-Q revealed that there is more resting GIRK conductance in dorsal CA1 neurons compared to ventral CA1 neurons. We found that the higher resting GIRK conductance in dorsal CA1 neurons was mediated by tonic A1 adenosine receptor activation. These results demonstrate that the different resting membrane properties between dorsal and ventral CA1 neurons are due in part to higher adenosine A1 receptor-mediated GIRK activity in dorsal CA1 neurons.
Copyright © 2014, Journal of Neurophysiology.
A single injection of N-methyl-D-aspartate receptor (NMDAR) antagonists produces a rapid antidepressant response. Lasting changes in the synapse structure and composition underlie the effectiveness of these drugs. We recently discovered that rapid antidepressants cause a shift in the γ-aminobutyric acid receptor (GABA B R) signaling pathway, such that GABA B R activation shifts from opening inwardly rectifiying potassium channels (Kir/GIRK) to increasing resting dendritic calcium signal and mammalian Target of Rapamycin activity. However, little is known about the molecular and biochemical mechanisms that initiate this shift. Herein, we show that GABA B R signaling to Kir3 (GIRK) channels decreases with NMDAR blockade. Blocking NMDAR signaling stabilizes the adaptor protein 14-3-3η, which decouples GABA B R signaling from Kir3 and is required for the rapid antidepressant efficacy. Consistent with these results, we find that key proteins involved in GABA B R signaling bidirectionally change in a depression model and with rapid antidepressants. In socially defeated rodents, a model for depression, GABA B R and 14-3-3η levels decrease in the hippocampus. The NMDAR antagonists AP5 and Ro-25-6981, acting as rapid antidepressants, increase GABA B R and 14-3-3η expression and decrease Kir3.2. Taken together, these data suggest that the shift in GABA B R function requires a loss of GABA B R-Kir3 channel activity mediated by 14-3-3η. Our findings support a central role for 14-3-3η in the efficacy of rapid antidepressants and define a critical molecular mechanism for activity-dependent alterations in GABA B R signaling. Molecular Psychiatry advance online publication, 6 January 2015; doi:10.1038/mp.2014.165 INTRODUCTION
Background:
Methamphetamine is a psychomotor stimulant with abuse liability and a substrate for catecholamine uptake transporters. Acute methamphetamine elevates extracellular dopamine, which in the midbrain can activate D2 autoreceptors to increase a G-protein gated inwardly rectifying potassium (GIRK) conductance that inhibits dopamine neuron firing. These studies examined the neurophysiological consequences of methamphetamine self-administration on GIRK channel-mediated currents in dopaminergic neurons in the substantia nigra and ventral tegmental area.
Methods:
Male DBA/2J mice were trained to self-administer intravenous methamphetamine. A dose response was conducted as well as extinction and cue-induced reinstatement. In a second study, after at least 2 weeks of stable self-administration of methamphetamine, electrophysiological brain slice recordings were conducted on dopamine neurons from self-administering and control mice.
Results:
In the first experiment, ad libitum-fed, nonfood-trained mice exhibited a significant increase in intake and locomotion following self-administration as the concentration of methamphetamine per infusion was increased (0.0015–0.15mg/kg/infusion). Mice exhibited extinction in responding and cue-induced reinstatement. In the second experiment, dopamine cells in both the substantia nigra and ventral tegmental area from adult mice with a history of methamphetamine self-administration exhibited significantly smaller D2 and GABAB receptor-mediated currents compared with control mice, regardless of whether their daily self-administration sessions had been 1 or 4 hours. Interestingly, the effects of methamphetamine self-administration were not present when intracellular calcium was chelated by including BAPTA in the recording pipette.
Conclusions:
Our results suggest that methamphetamine self-administration decreases GIRK channel-mediated currents in dopaminergic neurons and that this effect may be calcium dependent.
Units of dendritic branches called dendritic spines represent more than simply decorative appendages of the neuron and actively participate in integrative functions of "spinous" nerve cells thereby contributing to the general phenomenon of synaptic plasticity. In animal models of drug addiction, spines are profoundly affected by treatments with drugs of abuse and represent important sub cellular markers which interfere deeply into the physiology of the neuron thereby providing an example of the burgeoning and rapidly increasing interest in "structural plasticity". Medium Spiny Neurons (MSNs) of the Nucleus Accumbens (Nacc) show a reduced number of dendritic spines and a decrease in TH-positive terminals upon withdrawal from opiates, cannabinoids and alcohol. The reduction is localized "strictly" to second order dendritic branches where dopamine (DA)-containing terminals, impinging upon spines, make synaptic contacts. In addition, long-thin spines seems preferentially affected raising the possibility that cellular learning of these neurons may be selectively hampered. These findings suggest that dendritic spines are affected by drugs widely abused by humans and provide yet another example of drug-induced aberrant neural plasticity with marked reflections on the physiology of synapses, system structural organization, and neuronal circuitry remodeling.
Significance
Many neurotransmitters dampen excitability in the heart and brain by activating G-protein–gated inwardly rectifying K ⁺ (GIRK) channels. The lack of selective pharmacological tools for GIRK channels has hindered investigations into their physiological and pathophysiological relevance. Here, we examined the mechanisms underlying the activation of GIRK channels by ML297, the prototypical member of a new family of small molecule GIRK channel modulators. ML297 activates GIRK channels via a unique mechanism that requires two amino acids specific to the GIRK1 subunit. In addition, ML297 reduces anxiety-related behavior in mice, in a GIRK1-dependent manner, without triggering sedation or addiction-related behavior. Thus, ML297 is a new tool for probing the therapeutic potential of GIRK channel modulation, which may benefit individuals with anxiety-related disorders.
Opioids are among the most effective drugs to treat severe pain. They produce their analgesic actions by specifically activating opioid receptors located along the pain perception pathway where they inhibit the flow of nociceptive information. This inhibition is partly accomplished by activation of hyperpolarizing G protein-coupled inwardly-rectifying potassium (GIRK or Kir3) channels. Kir3 channels control cellular excitability in the central nervous system and in the heart and, because of their ubiquitous distribution, they mediate the effects of a large range of hormones and neurotransmitters which, upon activation of corresponding G protein-coupled receptors (GPCRs) lead to channel opening. Here we analyze GPCR signaling via these effectors in reference to precoupling and collision models. Existing knowledge on signaling bias is discussed in relation to these models as a means of developing strategies to produce novel opioid analgesics with an improved side effects profile.
GABAB receptors assemble from GABAB1 and GABAB2 subunits. GABAB2 additionally associates with auxiliary KCTD subunits (named after their K(+) channel tetramerization-domain). GABAB receptors couple to heterotrimeric G-proteins and activate inwardly-rectifying K(+) channels through the βγ subunits released from the G-protein. Receptor-activated K(+) currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K(+) currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast desensitization of GABAB receptor-activated K(+) currents while protein kinase-A inhibition accelerates fast desensitization. Protein kinase-A fails to regulate fast desensitization in KCTD12 knock-out mice or knock-in mice with a serine-892 to alanine mutation, thus demonstrating that serine-892 phosphorylation regulates KCTD12-induced desensitization in vivo. Fast current desensitization is accelerated in hippocampal neurons carrying the serine-892 to alanine mutation, showing that tonic serine-892 phosphorylation normally limits KCTD12-induced desensitization. Tonic serine-892 phosphorylation is in turn promoted by assembly of receptors with KCTD12. This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation.
This review discusses the evidence for the hypothesis that the development of drug addiction can be understood in terms of interactions between Pavlovian and instrumental learning and memory mechanisms in the brain that underlie the seeking and taking of drugs. It is argued that these behaviours initially are goal-directed, but increasingly become elicited as stimulus-response habits by drug-associated conditioned stimuli that are established by Pavlovian conditioning. It is further argued that compulsive drug use emerges as the result of a loss of prefrontal cortical inhibitory control over drug seeking habits. Data are reviewed that indicate these transitions from use to abuse to addiction depend upon shifts from ventral to dorsal striatal control over behaviour, mediated in part by serial connectivity between the striatum and midbrain dopamine systems. Only some individuals lose control over their drug use, and the importance of behavioural impulsivity as a vulnerability trait predicting stimulant abuse and addiction in animals and humans, together with consideration of an emerging neuroendophenotype for addiction are discussed. Finally, the potential for developing treatments for addiction is considered in light of the neuropsychological advances that are reviewed, including the possibility of targeting drug memory reconsolidation and extinction to reduce Pavlovian influences on drug seeking as a means of promoting abstinence and preventing relapse.
G-protein-coupled inwardly rectifying potassium (GIRK) channels contribute to the resting membrane potential of many neurons, including dopamine (DA) neurons in the ventral tegmental area (VTA). VTA DA neurons are bistable, firing in two modes: one characterized by bursts of action potentials, the other by tonic firing at a lower frequency. Here we provide evidence that these firing modes drive bidirectional plasticity of GIRK channel-mediated currents. In acute midbrain slices of mice, we observed that in vitro burst activation of VTA DA neurons potentiated GIRK currents whereas tonic firing depressed these currents. This plasticity was not specific to the metabotropic receptor activating the GIRK channels, as direct activation of GIRK channels by nonhydrolyzable GTP also potentiated the currents. The plasticity of GIRK currents required NMDA receptor and CaMKII activation, and involved protein trafficking through specific PDZ domains of GIRK2c and GIRK3 subunit isoforms. Prolonged tonic firing may thus enhance the probability to switch into burst-firing mode, which then potentiates GIRK currents and favors the return to baseline. In conclusion, activity-dependent GIRK channel plasticity may represent a slow destabilization process favoring the switch between the two firing modes of VTA DA neurons.
Relapse is a widely recognized and difficult to treat feature of the addictions. Substantial evidence implicates cue-triggered activation of the mesolimbic dopamine system as an important contributing factor. Even drug cues presented outside of conscious awareness (i.e., subliminally) produce robust activation within this circuitry, indicating the sensitivity and vulnerability of the brain to potentially problematic reward signals. Because pharmacological agents that prevent these early cue-induced responses could play an important role in relapse prevention, we examined whether baclofen-a GABAB receptor agonist that reduces mesolimbic dopamine release and conditioned drug responses in laboratory animals-could inhibit mesolimbic activation elicited by subliminal cocaine cues in cocaine-dependent individuals. Twenty cocaine-dependent participants were randomized to receive baclofen (60 mg/d; 20 mg t.i.d.) or placebo. Event-related BOLD fMRI and a backward-masking paradigm were used to examine the effects of baclofen on subliminal cocaine (vs neutral) cues. Sexual and aversive cues were included to examine specificity. We observed that baclofen-treated participants displayed significantly less activation in response to subliminal cocaine (vs neutral) cues, but not sexual or aversive (vs neutral) cues, than placebo-treated participants in a large interconnected bilateral cluster spanning the ventral striatum, ventral pallidum, amygdala, midbrain, and orbitofrontal cortex (voxel threshold p < 0.005; cluster corrected at p < 0.05). These results suggest that baclofen may inhibit the earliest type of drug cue-induced motivational processing-that which occurs outside of awareness-before it evolves into a less manageable state.
Alcohol (ethanol)-induced behaviors may arise from direct interaction of alcohol with discrete protein cavities within brain proteins. Recent structural and biochemical studies have provided new insights into the mechanism of alcohol-dependent activation of G protein-gated inwardly rectifying potassium (GIRK) channels, which regulate neuronal responses in the brain reward circuit. GIRK channels contain an alcohol binding pocket formed at the interface of two adjacent channel subunits. Here, we discuss the physiochemical properties of the alcohol pocket and the roles of G protein βγ subunits and membrane phospholipid PIP2 in regulating the alcohol response of GIRK channels. Some of the features of alcohol modulation of GIRK channels may be common to other alcohol-sensitive brain proteins. We discuss the possibility of alcohol-selective therapeutics that block alcohol access to the pocket. Understanding alcohol recognition and modulation of brain proteins is essential for development of therapeutics for alcohol abuse and addiction.
DESPITE THE IMPORTANCE OF NUMEROUS PSYCHOSOCIAL FACTORS, AT ITS CORE, DRUG ADDICTION INVOLVES A BIOLOGICAL PROCESS: the ability of repeated exposure to a drug of abuse to induce changes in a vulnerable brain that drive the compulsive seeking and taking of drugs, and loss of control over drug use, that define a state of addiction. Here, we review the types of molecular and cellular adaptations that occur in specific brain regions to mediate addiction-associated behavioral abnormalities. These include alterations in gene expression achieved in part via epigenetic mechanisms, plasticity in the neurophysiological functioning of neurons and synapses, and associated plasticity in neuronal and synaptic morphology mediated in part by altered neurotrophic factor signaling. Each of these types of drug-induced modifications can be viewed as a form of "cellular or molecular memory." Moreover, it is striking that most addiction-related forms of plasticity are very similar to the types of plasticity that have been associated with more classic forms of "behavioral memory," perhaps reflecting the finite repertoire of adaptive mechanisms available to neurons when faced with environmental challenges. Finally, addiction-related molecular and cellular adaptations involve most of the same brain regions that mediate more classic forms of memory, consistent with the view that abnormal memories are important drivers of addiction syndromes. The goal of these studies which aim to explicate the molecular and cellular basis of drug addiction is to eventually develop biologically based diagnostic tests, as well as more effective treatments for addiction disorders.
G protein-dependent signaling pathways control the activity of excitable cells of the nervous system and heart, and are the targets of neurotransmitters, clinically relevant drugs, and drugs of abuse. G protein-gated inwardly rectifying potassium (K(+)) (Girk/Kir3) channels are a key effector in inhibitory signaling pathways. Girk-dependent signaling contributes to nociception and analgesia, reward-related behavior, mood, cognition, and heart-rate regulation, and has been linked to epilepsy, Down syndrome, addiction, and arrhythmias. We discuss recent advances in our understanding of Girk channel structure, organization in signaling complexes, and plasticity, as well as progress on the development of subunit-selective Girk modulators. These findings offer new hope for the selective manipulation of Girk channels to treat a variety of debilitating afflictions.
G protein-gated inward rectifier K+ (GIRK) channels mediate hyperpolarizing postsynaptic potentials in the nervous system and in the heart during activation of Galpha (i/o)-coupled receptors. In neurons and cardiac atrial cells the time course for receptor-mediated GIRK current deactivation is 20-40 times faster than that observed in heterologous systems expressing cloned receptors and GIRK channels, suggesting that an additional component(s) is required to confer the rapid kinetic properties of the native transduction pathway. We report here that heterologous expression of ``regulators of G protein signaling'' (RGS proteins), along with cloned G protein-coupled receptors and GIRK channels, reconstitutes the temporal properties of the native receptor --> GIRK signal transduction pathway. GIRK current waveforms evoked by agonist activation of muscarinic m2 receptors or serotonin 1A receptors were dramatically accelerated by coexpression of either RGS1, RGS3, or RGS4, but not RGS2. For the brain-expressed RGS4 isoform, neither the current amplitude nor the steady-state agonist dose-response relationship was significantly affected by RGS expression, although the agonist-independent ``basal'' GIRK current was suppressed by ≈ 40%. Because GIRK activation and deactivation kinetics are the limiting rates for the onset and termination of ``slow'' postsynaptic inhibitory currents in neurons and atrial cells, RGS proteins may play crucial roles in the timing of information transfer within the brain and to peripheral tissues.
Last evidences suggest that, in Alzheimer's disease (AD) early stage, Amyloid-β (Aβ) peptide induces an imbalance between excitatory and inhibitory neurotransmission systems resulting in the functional impairment of neural networks. Such alterations are particularly important in the septohippocampal system where learning and memory processes take place depending on accurate oscillatory activity tuned at fimbria-CA3 synapse. Here, the acute effects of Aβ on CA3 pyramidal neurons and their synaptic activation from septal part of the fimbria were studied in rats. A triphasic postsynaptic response defined by an excitatory potential (EPSP) followed by both early and late inhibitory potentials (IPSP) was evoked. The EPSP was glutamatergic acting on ionotropic receptors. The early IPSP was blocked by GABA A antagonists whereas the late IPSP was removed by GABA B antagonists. Aβ perfusion induced recorded cells to depolarize, increase their input resistance and decrease the late IPSP. Aβ action mechanism was localized at postsynaptic level and most likely linked to GABA B -related ion channels conductance decrease. In addition, it was found that the specific pharmacological modulation of the GABA B receptor effector, G-protein-coupled inward rectifier potassium (GirK) channels, mimicked all Aβ effects previously described. Thus, our findings suggest that Aβ altering GirK channels conductance in CA3 pyramidal neurons might have a key role in the septohippocampal activity dysfunction observed in AD.
GABAB receptors are the G-protein coupled receptors (GPCRs) for GABA, the main inhibitory neurotransmitter in the central nervous
system. Native GABAB receptors comprise principle and auxiliary subunits that regulate receptor properties in distinct ways. The principle subunits
GABAB1a, GABAB1b, and GABAB2 form fully functional heteromeric GABAB(1a,2) and GABAB(1b,2) receptors. Principal subunits regulate forward trafficking of the receptors from the endoplasmic reticulum to the plasma
membrane and control receptor distribution to axons and dendrites. The auxiliary subunits KCTD8, -12, -12b, and -16 are cytosolic
proteins that influence agonist potency and G-protein signaling of GABAB(1a,2) and GABAB(1b,2) receptors. Here, we used transfected cells to study assembly, surface trafficking, and internalization of GABAB receptors in the presence of the KCTD12 subunit. Using bimolecular fluorescence complementation and metabolic labeling, we
show that GABAB receptors associate with KCTD12 while they reside in the endoplasmic reticulum. Glycosylation experiments support that association
with KCTD12 does not influence maturation of the receptor complex. Immunoprecipitation and bioluminescence resonance energy
transfer experiments demonstrate that KCTD12 remains associated with the receptor during receptor activity and receptor internalization
from the cell surface. We further show that KCTD12 reduces constitutive receptor internalization and thereby increases the
magnitude of receptor signaling at the cell surface. Accordingly, knock-out or knockdown of KCTD12 in cultured hippocampal
neurons reduces the magnitude of the GABAB receptor-mediated K+ current response. In summary, our experiments support that the up-regulation of functional GABAB receptors at the neuronal plasma membrane is an additional physiological role of the auxiliary subunit KCTD12.
Using Down syndrome as a model for complex trait analysis, we sought to identify loci from chromosome 21q22.2 which, when present in an extra dose, contribute to learning abnormalities. We generated low-copy-number transgenic mice, containing four different yeast artificial chromosomes (YACs) that together cover approximately 2 megabases (Mb) of contiguous DNA from 21q22.2. We subjected independent lines derived from each of these YAC transgenes to a series of behavioural and learning assays. Two of the four YACs caused defects in learning and memory in the transgenic animals, while the other two YACs had no effect. The most severe defects were caused by a 570-kb YAC; the interval responsible for these defects was narrowed to a 180-kb critical region as a consequence of YAC fragmentation. This region contains the human homologue of a Drosophila gene, minibrain, and strongly implicates it in learning defects associated with Down syndrome.
Sorting nexin 27 (SNX27), a brain-enriched PDZ domain protein, regulates endocytic sorting and trafficking. Here we show that Snx27(-/-) mice have severe neuronal deficits in the hippocampus and cortex. Although Snx27(+/-) mice have grossly normal neuroanatomy, we found defects in synaptic function, learning and memory and a reduction in the amounts of ionotropic glutamate receptors (NMDA and AMPA receptors) in these mice. SNX27 interacts with these receptors through its PDZ domain, regulating their recycling to the plasma membrane. We demonstrate a concomitant reduced expression of SNX27 and CCAAT/enhancer binding protein β (C/EBPβ) in Down's syndrome brains and identify C/EBPβ as a transcription factor for SNX27. Down's syndrome causes overexpression of miR-155, a chromosome 21-encoded microRNA that negatively regulates C/EBPβ, thereby reducing SNX27 expression and resulting in synaptic dysfunction. Upregulating SNX27 in the hippocampus of Down's syndrome mice rescues synaptic and cognitive deficits. Our identification of the role of SNX27 in synaptic function establishes a new molecular mechanism of Down's syndrome pathogenesis.
In the hippocampus, the inhibitory neurotransmitter GABA shapes the activity of the output pyramidal neurons and plays important role in cognition. Most of its inhibitory effects are mediated by signaling from GABAB receptor to the G protein-gated Inwardly-rectifying K+ (GIRK) channels. Here, we show that RGS7, in cooperation with its binding partner R7BP, regulates GABABR-GIRK signaling in hippocampal pyramidal neurons. Deletion of RGS7 in mice dramatically sensitizes GIRK responses to GABAB receptor stimulation and markedly slows channel deactivation kinetics. Enhanced activity of this signaling pathway leads to decreased neuronal excitability and selective disruption of inhibitory forms of synaptic plasticity. As a result, mice lacking RGS7 exhibit deficits in learning and memory. We further report that RGS7 is selectively modulated by its membrane anchoring subunit R7BP, which sets the dynamic range of GIRK responses. Together, these results demonstrate a novel role of RGS7 in hippocampal synaptic plasticity and memory formation.
Alcohol exposure induces multiple neuroadaptive changes in the CNS that can have serious long-term consequences on CNS function including cognitive effects and attenuation of learning and memory. The cellular mechanisms underlying the CNS effects of alcohol have yet to be fully elucidated and are likely to depend on the pattern and dose of alcohol exposure. Using electrophysiological recordings from hippocampal slices obtained from control and chronic alcohol-treated rats, we have investigated the effects of a binge pattern of alcohol abuse on synaptic plasticity in the CNS. The alcohol-treated animals were exposed to ethanol vapor for 12–14 days using an intermittent exposure paradigm (14 h ethanol exposure/10 h ethanol withdrawal daily; blood alcohol levels ∼180 mg/dl), a paradigm that models human binge alcohol use. Induction of long-term potentiation (LTP) in the CA1 region of the hippocampus by tetanic stimulation of Schaffer collaterals was completely blocked in slices from the chronic alcohol-treated animals. LTP remained blocked 1 day after withdrawal of animals from alcohol, indicating that the neuroadaptive changes produced by alcohol were not readily reversible. Partial recovery was observed after withdrawal from alcohol for 5 days. Other measures of synaptic plasticity including posttetanic potentiation and paired-pulse facilitation were also altered by the intermittent alcohol treatment paradigm. The results suggest that alterations in synaptic plasticity induced by chronic intermittent ethanol consumption play an important role in the effects of binge alcohol use on learning and memory function.
Second order schedules of IV cocaine reinforcement in rats provide a reliable method for evaluating the effects of conditioned stimuli on cocaine-seeking behaviour, and for measuring the motivational aspects of cocaine reinforcement. In the procedure established here, each infusion of cocaine (0.25 mg/infusion) was initially made contingent on a lever press and was paired with a 20-s light conditioned stimulus (CS). When rats acquired stable rates of cocaine self-administration, the response requirement for cocaine was increased progressively to a second-order schedule of the type FI15 min(FR10:S), whereby the IV cocaine infusion was self-administered following the completion of the first FR10 responses (and CS presentation) after a 15-min fixed interval (FI) had elapsed. Evaluation of the animals' responding during the first, drug-free interval of each daily session provided a measure of cocaine-seeking behaviour, independent of other pharmacological effects of the self-administered drug. Thus, a dose-response study (dose range: 0.083, 0.25 and 0.50 mg/infusion) revealed that responding under this schedule during the initial, drug-free interval changed monotonically with dose, whereas an inverse relationship between cocaine dose and response level tended to appear during the rest of the session, after rats had self-administered the drug. Responding under this schedule was also shown to occur under the control of the CS, which had acquired conditioned reinforcing properties. Thus, a decrease in responding and an increase in the latency to initiate responding followed the omission of the CS for 3 consecutive days. In addition, extinction of cocaine-seeking behaviour was slower when contingent CS presentations occurred compared to extinction when the CS was not present. Furthermore, the reinstatement of responding for cocaine, which followed a brief period of non-contingent CS presentations, was retarded when this conditioned reinforcer had been extinguished together with cocaine. Finally, cocaine-seeking behaviour decreased markedly for the first 6 h that followed a 12-h period of continuous access to cocaine, when compared to responding 6 h after a 90-min session of limited access to the drug. Responding subsequently increased to baseline levels within 72 h. These results emphasise the utility of second-order schedules for studying drug-seeking behaviour and the importance of drug-associated cues in maintaining such responding for cocaine.
The pathological hallmark of Parkinson's disease (PD) is the degeneration of midbrain dopamine neurons. Cognitive dysfunction is a feature of PD patients even at the early stages of the disease. Electrophysiological studies on dopamine neurons in awake animals provide contradictory accounts of the role of dopamine. These studies have established that dopamine neurons convey a unique signal associated with rewards rather than cognitive functions. Emphasizing their role in reward processing leads to difficulty in developing hypothesis as to how cognitive impairments in PD are associated with the degeneration of dopamine circuitry. A hint to resolve this contradiction came from recent electrophysiological studies reporting that dopamine neurons transmit more diverse signals than previously thought. These studies suggest that dopamine neurons are divided into at least two functional subgroups, one signaling "motivational value" and the other signaling "salience." The former subgroup fits well with the conventional reward theory, whereas the latter subgroup has been shown to transmit signals related to salient but non-rewarding experiences such as aversive stimulations and cognitively demanding situations. This article reviews recent advances in understanding the non-reward functions of dopamine, and then discusses the possibility that cognitive dysfunction in PD is at least partially caused by the degeneration of the dopamine neuron subgroup signaling the salience of events in the environment. 2015 International Parkinson and Movement Disorder Society.
© 2015 International Parkinson and Movement Disorder Society.
Background
Alcohol affects many of the brain regions and neural processes that support learning and memory, and these effects are thought to underlie, at least in part, the development of addiction. Although much work has been done regarding the effects of alcohol intoxication on learning and memory, little is known about the effects of acute withdrawal from a single alcohol exposure.Methods
We assess the effects of acute ethanol withdrawal (6 hours postinjection with 4 g/kg ethanol) on 2 forms of fear conditioning (delay and trace fear conditioning) in C57BL/6J and DBA/2J mice. The influence of a number of experimental parameters (pre- and post training withdrawal exposure; foreground/background processing; training strength; and nonassociative effects) is also investigated.ResultsAcute ethanol withdrawal during training had a bidirectional effect on fear-conditioned responses, decreasing contextual responses and increasing cued responses. These effects were apparent for both trace and delay conditioning in DBA/2J mice and for trace conditioning in C57BL/6J mice; however, C57BL/6J mice were selectively resistant to the effects of acute withdrawal on delay cued responses.Conclusions
Our results show that acute withdrawal from a single, initial ethanol exposure is sufficient to alter long-term learning in mice. In addition, the differences between the strains and conditioning paradigms used suggest that specific learning processes can be differentially affected by acute withdrawal in a manner that is distinct from the reported effects of both alcohol intoxication and withdrawal following chronic alcohol exposure. Thus, our results suggest a unique effect of acute alcohol withdrawal on learning and memory processes.
Abnormal salience attribution is implicated in heroin addiction. Previously, combining functional magnetic resonance imaging (fMRI) and a drug cue-reactivity task, we demonstrated abnormal patterns of subjective response and brain reactivity in heroin-dependent individuals. However, whether the changes in cue-induced brain response were related to relapse was unknown. In a prospective study, we recruited 49 heroin-dependent patients under methadone maintenance treatment, a gold standard treatment (average daily dose 41.8 ± 16.0 mg), and 20 healthy subjects to perform the heroin cue-reactivity task during fMRI. The patients' subjective craving was evaluated. They participated in a follow-up assessment for 3 months, during which heroin use was assessed and relapse was confirmed by self-reported relapse or urine toxicology. Differences between relapsers and non-relapsers were analyzed with respect to the results from heroin-cue responses. Compared with healthy subjects, relapsers and non-relapsers commonly demonstrated significantly increased brain responses during the processing of heroin cues in the mesolimbic system, prefrontal regions and visuospatial-attention regions. However, compared with non-relapsers, relapsers demonstrated significantly greater cue-induced craving and the brain response mainly in the bilateral nucleus accumbens/subcallosal cortex and cerebellum. Although the cue-induced heroin craving was low in absolute measures, the change in craving positively correlated with the activation of the nucleus accumbens/subcallosal cortex among the patients. These findings suggest that in treatment-seeking heroin-dependent individuals, greater cue-induced craving and greater specific regional activations might be related to reward/craving and memory retrieval processes. These responses may predict relapse and represent important targets for the development of new treatment for heroin addiction.
Alcohol dependence is a complex condition with clear genetic factors. Some of the leading candidate genes code for subunits of the inhibitory GABAA and glycine receptors. These and related ion channels are also targets for the acute actions of alcohol, and there is considerable progress in understanding interactions of alcohol with these proteins at the molecular and even atomic levels. X-ray structures of open and closed states of ion channels combined with structural modeling and site-directed mutagenesis have elucidated direct actions of alcohol. Alcohol also alters channel function by translational and post-translational mechanisms, including phosphorylation and protein trafficking. Construction of mutant mice with either deletion of key proteins or introduction of alcohol-resistant channels has further linked specific proteins with discrete behavioral effects of alcohol. A combination of approaches, including genome wide association studies in humans, continues to advance the molecular basis of alcohol action on receptor structure and function.
Activation of K(+) channels by the G protein βγ subunits is an important signaling mechanism of G-protein-coupled receptors. Typically, receptor-activated K(+) currents desensitize in the sustained presence of agonists to avoid excessive effects on cellular activity. The auxiliary GABAB receptor subunit KCTD12 induces fast and pronounced desensitization of the K(+) current response. Using proteomic and electrophysiological approaches, we now show that KCTD12-induced desensitization results from a dual interaction with the G protein: constitutive binding stabilizes the heterotrimeric G protein at the receptor, whereas dynamic binding to the receptor-activated Gβγ subunits induces desensitization by uncoupling Gβγ from the effector K(+) channel. While receptor-free KCTD12 desensitizes K(+) currents activated by other GPCRs in vitro, native KCTD12 is exclusively associated with GABAB receptors. Accordingly, genetic ablation of KCTD12 specifically alters GABAB responses in the brain. Our results show that GABAB receptors are endowed with fast and reversible desensitization by harnessing KCTD12 that intercepts Gβγ signaling.
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.
Desensitization of μ-opioid receptors (MORs) develops over 5-15 min following application of some but not all opioid agonists and lasts for 10s of min following agonist removal. The decrease in function is receptor selective (homologous) and could result from (1) a reduction in receptor number or (2) a decrease in receptor coupling. The present investigation used photolysis of two caged opioid ligands in order to examine the kinetics of MOR-induced potassium conductance before and following MOR desensitization. Photolysis of a caged antagonist, caged-naloxone (CNV-NLX), blocked the current induced by a series of agonists and the time constant of decline was significantly decreased following desensitization. The increase in the rate of current decay was not observed following partial blockade of receptors with the irreversible antagonist, β-CNA. The time constant of current decay following desensitization was never more rapid than 1s, suggesting an increased agonist off rate rather than an increase in the rate of channel closure downstream of the receptor. The rate of GIRK current activation was examined using photolysis of a caged agonist, [Leu](5)enkephalin (CYLE). Following acute desensitization or partial irreversible block of MORs with β-CNA there was an increase in the time it took to reach a peak current. The decrease in the rate of agonist-induced GIRK conductance was receptor selective and dependent on receptor number. The results indicate that opioid receptor desensitization reduced the number of functional receptors and the remaining active receptors have a reduced agonist affinity.
Significance
G-protein–gated inward-rectifying K ⁺ (GIRK) channels control neuronal excitability in the brain’s reward circuit. Ethanol, a widely available drug of abuse, directly binds to a hydrophobic pocket in the GIRK channel and activates it. The molecular mechanism underlying ethanol activation of GIRK channel remained unknown. Here, we used specialized biophysical tools and discovered that ethanol associates directly with the GIRK channel, leading to enhanced interaction with a membrane phospholipid phosphatidylinositol 4,5-bisphosphate and activation of the channel. Most importantly, the alcohol-binding pocket in GIRK channels can be occupied by non-alcohol-like chemical groups that activate the channel. This study will enable the design of therapeutics to selectively block alcohol’s access to GIRK channel.
Repeated cocaine exposure triggers adaptations in layer 5/6 glutamatergic neurons in the medial prefrontal cortex (mPFC) that promote behavioral sensitization and drug-seeking behavior. While suppression of metabotropic inhibitory signaling has been implicated in these behaviors, underlying mechanisms are unknown. Here, we show that Girk/KIR3 channels mediate most of the GABAB receptor (GABABR)-dependent inhibition of layer 5/6 pyramidal neurons in the mPFC and that repeated cocaine suppresses this pathway. This adaptation was selective for GABABR-dependent Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex (PrLC) and involved a D1/5 dopamine receptor- and phosphorylation-dependent internalization of GABABR and Girk channels. Persistent suppression of Girk signaling in layer 5/6 of the dorsal mPFC enhanced cocaine-induced locomotor activity and occluded behavioral sensitization. Thus, the cocaine-induced suppression of GABABR-Girk signaling in layer 5/6 pyramidal neurons of the prelimbic cortex appears to represent an early adaptation critical for promoting addiction-related behavior.
Addiction is a chronic disorder marked by long-lasting maladaptive changes in behavior and in reward system function. However, the factors that contribute to the behavioral and biological changes that occur with addiction are complex and go beyond reward. Addiction involves changes in cognitive control and the development of disruptive drug-stimuli associations that can drive behavior. A reason for the strong influence drugs of abuse can exert on cognition may be the striking overlap between the neurobiological substrates of addiction and of learning and memory, especially areas involved in declarative memory. Declarative memories are critically involved in the formation of autobiographical memories, and the ability of drugs of abuse to alter these memories could be particularly detrimental. A key structure in this memory system is the hippocampus, which is critically involved in binding multimodal stimuli together to form complex long-term memories. While all drugs of abuse can alter hippocampal function, this review focuses on nicotine. Addiction to tobacco products is insidious, with the majority of smokers wanting to quit; yet the majority of those that attempt to quit fail. Nicotine addiction is associated with the presence of drug-context and drug-cue associations that trigger drug seeking behavior and altered cognition during periods of abstinence, which contributes to relapse. This suggests that understanding the effects of nicotine on learning and memory will advance understanding and potentially facilitate treating nicotine addiction. The following sections examine: 1) how the effects of nicotine on hippocampus-dependent learning change as nicotine administration transitions from acute to chronic and then to withdrawal from chronic treatment and the potential impact of these changes on addiction, 2) how nicotine usurps the cellular mechanisms of synaptic plasticity, 3) the physiological changes in the hippocampus that may contribute to nicotine withdrawal deficits in learning, and 4) the role of genetics and developmental stage (i.e., adolescence) in these effects.
This letter describes a multi-dimensional SAR campaign based on a potent, efficacious and selective GIRK1/2 activator (∼10-fold versus GIRK1/4 and inactive on nonGIRK 1-containing GIRKs, GIRK 2 or GIRK2/3). Further chemical optimization through an iterative parallel synthesis effort identified multiple 'molecular switches' that modulated the mode of pharmacology from activator to inhibitor, as well as engendering varying selectivity profiles for GIRK1/2 and GIRK1/4. Importantly, these compounds were all inactive on nonGIRK1 containing GIRK channels. However, SAR was challenging as subtle structural modifications had large effects on both mode of pharmacology and GIRK1/2 and GIRK1/4 channel selectivity.
Members of the Kir3.0 family of inwardly rectifying K ⁺ channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by G βγ subunits released in response to G i/o ‐coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the G q/11 ‐coupled M 1 and M 3 muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal‐type channels stably expressed in the human embryonic kidney cell line HEK293.
We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M 1 and M 3 receptors. This contrasts with the simple stimulation seen through activation of M 2 and M 4 receptors.
Channel stimulation via M 1 but not M 3 receptors was sensitive to pertussis toxin whereas channel inhibition through both M 1 and M 3 receptors was insensitive. In contrast over‐expression of the C‐terminus of phospholipase Cβ1 or a G q/11 ‐specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase.
The inhibitory effects of M 1 and M 3 receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4αphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M 3 receptor.
The receptor‐mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0‐669 n m ).
The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCδ and PKCε.
We also demonstrate that activation of such a pathway via both receptor‐mediated and receptor‐independent means profoundly attenuated subsequent channel stimulation by G i/o ‐coupled receptors.
Our data support a role for a Ca ²⁺ ‐independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M 1 and M 3 muscarinic receptor stimulation.
Drug addiction is a chronic brain disorder with the hallmark of a high rate of relapse to compulsive drug seeking and drug taking even after long-term abstinence. Addiction has been considered as an aberrant memory that has been termed "addiction memory." Drug-related memory plays a critical role in the maintenance of learned addictive behaviors and emergence of relapse. Disrupting these long-lasting memories by administering amnestic agents or other manipulations during specific phases of drug memory is a promising strategy for relapse prevention. Recent studies on the processes of drug addiction and relapse have demonstrated that the amygdala is involved in associative drug addiction learning processes. In this review, we focus on preclinical studies that used conditioned place preference and self-administration models to investigate the differential roles of the amygdala in each phase of drug-related memory, including acquisition, consolidation, retrieval, reconsolidation, and extinction. These studies indicate that the amygdala plays a critical role in both cue-associative learning and the expression of cue-induced relapse to drug-seeking behavior.
In the hippocampus, signalling through G protein-coupled receptors is modulated by Regulators of G protein Signalling (Rgs) proteins, which act to stimulate the rate of GTP hydrolysis, and consequently, G protein inactivation. The R7-Rgs subfamily selectively deactivates the Gi /o -class of Gα subunits that mediate the action of several GPCRs. Here, we used co-immunoprecipitation, electrophysiology and immunoelectron microscopy techniques to investigate the formation of macromolecular complexes and spatial relationship of Rgs7/Gβ5 complexes and its prototypical signalling partners, the GABAB receptor and Girk channel. Co-expression of recombinant GABAB receptors and Girk channels in combination with co-immunoprecipitation experiments established that the Rgs7/Gβ5 forms complexes with GABAB receptors or Girk channels. Using electrophysiological experiments, we found that GABAB -Girk current deactivation kinetics was markedly faster in cells co-expressing Rgs7/Gβ5. At the electron microscopic level, immunolabelling for Rgs7 and Gβ5 proteins was found primarily in the dendritic layers of the hippocampus and showed similar distribution patterns. Immunoreactivity was mostly localized along the extrasynaptic plasma membrane of dendritic shafts and spines of pyramidal cells and, to a lesser extent, to that of presynaptic terminals. Quantitative analysis of immunogold particles for Rgs7 and Gβ5 revealed an enrichment of the two proteins around excitatory synapses on dendritic spines, virtually identical to that of Girk2 and GABAB1 . These data support the existence of macromolecular complexes composed of GABAB receptor-G protein-Rgs7-Girk channels, in which Rgs7 and Gβ5 proteins may preferentially modulate GABAB receptor signalling through the deactivation of Girk channels on dendritic spines. In contrast, Rgs7 and Girk2 were associated but mainly segregated from GABAB1 in dendritic shafts, where Rgs7/Gβ5 signalling complexes might modulate Girk-dependent signalling via a different metabotropic receptor(s). © 2013 Wiley Periodicals, Inc.
The G-protein activated, inward-rectifying potassium (K+) channels, "GIRKs", are a family of ion channels (Kir3.1-Kir3.4) that has been the focus of intense research interest for nearly two decades. GIRKs are comprised of various homo and heterotetrameric combinations of four different subunits. These subunits are expressed in different combinations in a variety of regions throughout the central nervous system and in the periphery. The body of GIRK research implicates GIRK in processes as diverse as controlling heart rhythm, to effects on reward/addiction, to modulation of response to analgesics. Despite years of GIRK research, very few tools exist to selectively modulate GIRK channels' activity and until now no tools existed that potently and selectively activated GIRKs. Here we report the development and characterization of the first truly potent, effective, and selective GIRK activator, ML297 (VU0456810). We further demonstrate that ML297 is active in two in vivo models of epilepsy, a disease where up to 40% of patients remain with symptoms refractory to present treatments. The development of ML297 represents a truly significant advancement in our ability to selectively probe GIRK's role in physiology as well as providing the first tool for beginning to understand GIRK's potential as a target for a diversity of therapeutic indications.
Although stress has profound effects on motivated behavior, the underlying mechanisms responsible are incompletely understood.
In this study we elucidate a functional pathway in mouse brain that encodes the aversive effects of stress and mediates stress-induced
reinstatement of cocaine place preference (CPP). Activation of the dynorphin/kappa opioid receptor (KOR) system by either
repeated stress or agonist produces conditioned place aversion (CPA). Because KOR inhibition of dopamine release in the mesolimbic
pathway has been proposed to mediate the dysphoria underlying this response, we tested dopamine-deficient mice in this study
and found that KOR agonist in these mice still produced CPA. However, inactivation of serotonergic KORs by injection of the
KOR antagonist norBNI into the dorsal raphe nucleus (DRN), blocked aversive responses to the KOR agonist U50,488 and blocked
stress-induced reinstatement of CPP. KOR knockout (KO) mice did not develop CPA to U50,488; however, lentiviral re-expression
of KOR in the DRN of KOR KO mice restored place aversion. In contrast, lentiviral expression in DRN of a mutated form of KOR
that fails to activate p38 MAPK required for KOR-dependent aversion, did not restore place aversion. DRN serotonergic neurons
project broadly throughout the brain, but the inactivation of KOR in the nucleus accumbens (NAc) coupled with viral re-expression
in the DRN of KOR KO mice demonstrated that aversion was encoded by a DRN to NAc projection. These results suggest that the
adverse effects of stress may converge on the serotonergic system and offers an approach to controlling stress-induced dysphoria
and relapse.