Mechanism of PKCε regulation of cardiac GIRK channel gating.

G-protein-gated inwardly rectifying potassium (GIRK) channel activity is regulated by the membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PI 4,5P2). Constitutive activity of cardiac GIRK channels in atrial myocytes, that is implicated in atrial fibrillation (AF), is mediated via a protein kinase C-ε (PKCε)-dependent mechanism. The novel PKC isoform, PKCε, is reported to enhance the activity of cardiac GIRK channels. Here, we report that PKCε stimulation leads to activation of GIRK channels in mouse atria and in human stem cell-derived atrial cardiomyocytes (iPSCs). We identified residue GIRK4(S418) which when mutated to Ala abolished, or to Glu, mimicked the effects of PKCε on GIRK currents. PKCε strengthened the interactions of the cardiac GIRK isoforms, GIRK4 and GIRK1/4 with PIP2, an effect that was reversed in the GIRK4(S418A) mutant. This mechanistic insight into the PKCε-mediated increase in channel activity because of GIRK4(S418) phosphorylation, provides a precise druggable target to reverse AF-related pathologies due to GIRK overactivity.

Synaptotagmins 1 and 7 Play Complementary Roles in Somatodendritic Dopamine Release.

The molecular mechanisms underlying somatodendritic dopamine (DA) release remain unresolved, despite the passing of decades since its discovery. Our previous work showed robust release of somatodendritic DA in submillimolar extracellular Ca2+ concentration ([Ca2+]o). Here we tested the hypothesis that the high-affinity Ca2+ sensor synaptotagmin 7 (Syt7), is a key determinant of somatodendritic DA release and its Ca2+ dependence. Somatodendritic DA release from SNc DA neurons was assessed using whole-cell recording in midbrain slices from male and female mice to monitor evoked DA-dependent D2 receptor-mediated inhibitory currents (D2ICs). Single-cell application of an antibody to Syt7 (Syt7 Ab) decreased pulse train-evoked D2ICs, revealing a functional role for Syt7. The assessment of the Ca2+ dependence of pulse train-evoked D2ICs confirmed robust DA release in submillimolar [Ca2+]o in wild-type (WT) neurons, but loss of this sensitivity with intracellular Syt7 Ab or in Syt7 knock-out (KO) mice. In millimolar [Ca2+]o, pulse train-evoked D2ICs in Syt7 KOs showed a greater reduction in decreased [Ca2+]o than seen in WT mice; the effect on single pulse-evoked DA release, however, did not differ between genotypes. Single-cell application of a Syt1 Ab had no effect on train-evoked D2ICs in WT SNc DA neurons, but did cause a decrease in D2IC amplitude in Syt7 KOs, indicating a functional substitution of Syt1 for Syt7. In addition, Syt1 Ab decreased single pulse-evoked D2ICs in WT cells, indicating the involvement of Syt1 in tonic DA release. Thus, Syt7 and Syt1 play complementary roles in somatodendritic DA release from SNc DA neurons.SIGNIFICANCE STATEMENT The respective Ca2+ dependence of somatodendritic and axonal dopamine (DA) release differs, resulting in the persistence of somatodendritic DA release in submillimolar Ca2+ concentrations too low to support axonal release. We demonstrate that synaptotagmin7 (Syt7), a high-affinity Ca2+ sensor, underlies phasic somatodendritic DA release and its Ca2+ sensitivity in the substantia nigra pars compacta. In contrast, we found that synaptotagmin 1 (Syt1), the Ca2+ sensor underlying axonal DA release, plays a role in tonic, but not phasic, somatodendritic DA release in wild-type mice. However, Syt1 can facilitate phasic DA release after Syt7 deletion. Thus, we show that both Syt1 and Syt7 act as Ca2+ sensors subserving different aspects of somatodendritic DA release processes.

From Crosstalk to Synergism: The Combined Effect of Cholesterol and PI(4,5)P2 on Inwardly Rectifying Potassium Channels.

Inwardly rectifying potassium (Kir) channels are integral membrane proteins that control the flux of potassium ions across cell membranes and regulate membrane permeability. All eukaryotic Kir channels require the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) for activation. In recent years, it has become evident that the function of many members of this family of channels is also mediated by another essential lipid-cholesterol. Here, we focus on members of the Kir2 and Kir3 subfamilies and their modulation by these two key lipids. We discuss how PI(4,5)P2 and cholesterol bind to Kir2 and Kir3 channels and how they affect channel activity. We also discuss the accumulating evidence indicating that there is interplay between PI(4,5)P2 and cholesterol in the modulation of Kir2 and Kir3 channels. In particular, we review the crosstalk between PI(4,5)P2 and cholesterol in the modulation of the ubiquitously expressed Kir2.1 channel and the synergy between these two lipids in the modulation of the Kir3.4 channel, which is primarily expressed in the heart. Additionally, we demonstrate that there is also synergy in the modulation of Kir3.2 channels, which are expressed in the brain. These observations suggest that alterations in the relative levels PI(4,5)P2 and cholesterol may fine-tune Kir channel activity.

Selective Inhibition of Pulmonary Vein Excitability by Constitutively Active GIRK Channels Blockade in Rats.

Pulmonary veins (PV) are the main source of ectopy, triggering atrial fibrillation. This study investigated the roles of G protein-coupled inwardly rectifying potassium (GIRK) channels in the PV and the left atrium (LA) of the rat. Simultaneous intracellular microelectrode recording from the LA and the PV of the rat found that in the presence or absence of acetylcholine, the GIRK channel blocker tertiapin-Q induced AP duration elongation in the LA and the loss of over-shooting AP in the PV, suggesting the presence of constitutively active GIRK channels in these tissues. Patch-clamp recordings from isolated myocytes showed that tertiapin-Q inhibited a basal inwardly rectified background current in PV cells with little effect in LA cells. Experiments with ROMK1 and KCa1.1 channel blockers ruled out the possibility of an off-target effect. Western blot showed that GIRK4 subunit expression was greater in PV cardiomyocytes, which may explain the differences observed between PV and LA in response to tertiapin-Q. In conclusion, GIRK channels blockade abolishes AP only in the PV, providing a molecular target to induce electrical disconnection of the PV from the LA.

5HT1AR-FGFR1 Heteroreceptor Complexes Differently Modulate GIRK Currents in the Dorsal Hippocampus and the Dorsal Raphe Serotonin Nucleus of Control Rats and of a Genetic Rat Model of Depression.

The midbrain raphe serotonin (5HT) neurons provide the main ascending serotonergic projection to the forebrain, including hippocampus, which has a role in the pathophysiology of depressive disorder. Serotonin 5HT1A receptor (R) activation at the soma-dendritic level of serotonergic raphe neurons and glutamatergic hippocampal pyramidal neurons leads to a decrease in neuronal firing by activation of G protein-coupled inwardly-rectifying potassium (GIRK) channels. In this raphe-hippocampal serotonin neuron system, the existence of 5HT1AR-FGFR1 heteroreceptor complexes has been proven, but the functional receptor-receptor interactions in the heterocomplexes have only been investigated in CA1 pyramidal neurons of control Sprague Dawley (SD) rats. In the current study, considering the impact of the receptor interplay in developing new antidepressant drugs, the effects of 5HT1AR-FGFR1 complex activation were investigated in hippocampal pyramidal neurons and in midbrain dorsal raphe serotonergic neurons of SD rats and of a genetic rat model of depression (the Flinders Sensitive Line (FSL) rats of SD origin) using an electrophysiological approach. The results showed that in the raphe-hippocampal 5HT system of SD rats, 5HT1AR-FGFR1 heteroreceptor activation by specific agonists reduced the ability of the 5HT1AR protomer to open the GIRK channels through the allosteric inhibitory interplay produced by the activation of the FGFR1 protomer, leading to increased neuronal firing. On the contrary, in FSL rats, FGFR1 agonist-induced inhibitory allosteric action at the 5HT1AR protomer was not able to induce this effect on GIRK channels, except in CA2 neurons where we demonstrated that the functional receptor-receptor interaction is needed for producing the effect on GIRK. In keeping with this evidence, hippocampal plasticity, evaluated as long-term potentiation induction ability in the CA1 field, was impaired by 5HT1AR activation both in SD and in FSL rats, which did not develop after combined 5HT1AR-FGFR1 heterocomplex activation in SD rats. It is therefore proposed that in the genetic FSL model of depression, there is a significant reduction in the allosteric inhibition exerted by the FGFR1 protomer on the 5HT1A protomer-mediated opening of the GIRK channels in the 5HT1AR-FGFR1 heterocomplex located in the raphe-hippocampal serotonin system. This may result in an enhanced inhibition of the dorsal raphe 5HT nerve cell and glutamatergic hippocampal CA1 pyramidal nerve cell firing, which we propose may have a role in depression.

Protective effect of low-intensity treadmill exercise against acetylcholine-calcium chloride-induced atrial fibrillation in mice.

Atrial fibrillation (AF) is the most common supraventricular arrhythmia, and it corresponds highly with exercise intensity. Here, we induced AF in mice using acetylcholine (ACh)-CaCl2 for 7 days and aimed to determine the appropriate exercise intensity (no, low, moderate, high) to protect against AF by running the mice at different intensities for 4 weeks before the AF induction by ACh-CaCl2. We examined the AF-induced atrial remodeling using electrocardiogram, patch-clamp, and immunohistochemistry. After the AF induction, heart rate, % increase of heart rate, and heart weight/body weight ratio were significantly higher in all the four AF groups than in the normal control; highest in the high-ex AF and lowest in the low-ex (lower than the no-ex AF), which indicates that low-ex treated the AF. Consistent with these changes, G protein-gated inwardly rectifying K+ currents, which were induced by ACh, increased in an exercise intensity-dependent manner and were lower in the low-ex AF than the no-ex AF. The peak level of Ca2+ current (at 0 mV) increased also in an exercise intensity-dependent manner and the inactivation time constants were shorter in all AF groups except for the low-ex AF group, in which the time constant was similar to that of the control. Finally, action potential duration was shorter in all the four AF groups than in the normal control; shortest in the high-ex AF and longest in the low-ex AF. Taken together, we conclude that low-intensity exercise protects the heart from AF, whereas high-intensity exercise might exacerbate AF.

Reduced activity of GIRK1-containing heterotetramers is sufficient to affect neuronal functions, including synaptic plasticity and spatial learning and memory.

KEY POINTS: G-protein inwardly rectifying K+ (GIRK) channels consist of four homologous subunits (GIRK1-4) and are essential regulators of electrical excitability in the nervous system. GIRK2-null mice have been widely investigated for their distinct behaviour and altered depotentiation following long-term potentiation (LTP), whereas GIRK1 mice are less well characterized. Here we utilize a novel knockin mouse strain in which the GIRK1 subunit is fluorescently tagged with yellow fluorescent protein (YFP-GIRK1) and the GIRK1-null mouse line to investigate the role of GIRK1 in neuronal processes such as spatial learning and memory, locomotion and depotentiation following LTP. Neurons dissected from YFP-GIRK1 mice had significantly reduced potassium currents and this mouse line phenotypically resembled GIRK1-null mice, making it a 'functional knockdown' model of GIRK1-containing channels. YFP-GIRK1 and GIRK1-null mice had increased locomotion, reduced spatial learning and memory and blunted depotentiation following LTP. ABSTRACT: GIRK channels are essential for the slow inhibition of electrical activity in the nervous system and heart rate regulation via the parasympathetic system. The implications of individual GIRK isoforms in specific physiological activities are based primarily on studies conducted with GIRK-null mouse lines. Here we utilize a novel knockin mouse line in which YFP was fused in-frame to the N-terminus of GIRK1 (YFP-GIRK1) to correlate GIRK1 spatial distribution with physiological activities. These mice, however, displayed spontaneous seizure-like activity and thus were investigated for the origin of such activity. We show that GIRK tetramers containing YFP-GIRK1 are correctly assembled and trafficked to the plasma membrane, but are functionally impaired. A battery of behavioural assays conducted on YFP-GIRK1 and GIRK1-null (GIRK1-/- ) mice revealed similar phenotypes, including impaired nociception, reduced anxiety and hyperactivity in an unfamiliar environment. However, YFP-GIRK1 mice exhibited increased home-cage locomotion while GIRK1-/- mice did not. In addition, we show that the GIRK1 subunit is essential for intact spatial learning and memory and synaptic plasticity in hippocampal brain slices. This study expands our knowledge regarding the role of GIRK1 in neuronal processes and underlines the importance of GIRK1-containing heterotetramers.

The small molecule GAT1508 activates brain-specific GIRK1/2 channel heteromers and facilitates conditioned fear extinction in rodents.

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 nonfunctional 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 on 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 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. 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.

The Expression and Localisation of G-Protein-Coupled Inwardly Rectifying Potassium (GIRK) Channels Is Differentially Altered in the Hippocampus of Two Mouse Models of Alzheimer's Disease.

G protein-gated inwardly rectifying K+ (GIRK) channels are the main targets controlling excitability and synaptic plasticity on hippocampal neurons. Consequently, dysfunction of GIRK-mediated signalling has been implicated in the pathophysiology of Alzheimer´s disease (AD). Here, we provide a quantitative description on the expression and localisation patterns of GIRK2 in two transgenic mice models of AD (P301S and APP/PS1 mice), combining histoblots and immunoelectron microscopic approaches. The histoblot technique revealed differences in the expression of GIRK2 in the two transgenic mice models. The expression of GIRK2 was significantly reduced in the hippocampus of P301S mice in a laminar-specific manner at 10 months of age but was unaltered in APP/PS1 mice at 12 months compared to age-matched wild type mice. Ultrastructural approaches using the pre-embedding immunogold technique, demonstrated that the subcellular localisation of GIRK2 was significantly reduced along the neuronal surface of CA1 pyramidal cells, but increased in its frequency at cytoplasmic sites, in both P301S and APP/PS1 mice. We also found a decrease in plasma membrane GIRK2 channels in axon terminals contacting dendritic spines of CA1 pyramidal cells in P301S and APP/PS1 mice. These data demonstrate for the first time a redistribution of GIRK channels from the plasma membrane to intracellular sites in different compartments of CA1 pyramidal cells. Altogether, the pre- and post-synaptic reduction of GIRK2 channels suggest that GIRK-mediated alteration of the excitability in pyramidal cells could contribute to the cognitive dysfunctions as described in the two AD animal models.

Hippocampal long-term synaptic depression and memory deficits induced in early amyloidopathy are prevented by enhancing G-protein-gated inwardly rectifying potassium channel activity.

Hippocampal synaptic plasticity disruption by amyloid-ß (Aß) peptides + thought to be responsible for learning and memory impairments in Alzheimer's disease (AD) early stage. Failures in neuronal excitability maintenance seems to be an underlying mechanism. G-protein-gated inwardly rectifying potassium (GirK) channels control neural excitability by hyperpolarization in response to many G-protein-coupled receptors activation. Here, in early in vitro and in vivo amyloidosis mouse models, we study whether GirK channels take part of the hippocampal synaptic plasticity impairments generated by Aß1-42 . In vitro electrophysiological recordings from slices showed that Aß1-42 alters synaptic plasticity by switching high-frequency stimulation (HFS) induced long-term potentiation (LTP) to long-term depression (LTD), which led to in vivo hippocampal-dependent memory deficits. Remarkably, selective pharmacological activation of GirK channels with ML297 rescued both HFS-induced LTP and habituation memory from Aß1-42 action. Moreover, when GirK channels were specifically blocked by Tertiapin-Q, their activation with ML297 failed to rescue LTP from the HFS-dependent LTD induced by Aß1-42 . On the other hand, the molecular analysis of the recorded slices by western blot showed that the expression of GIRK1/2 subunits, which form the prototypical GirK channel in the hippocampus, was not significantly regulated by Aß1-42 . However, immunohistochemical examination of our in vivo amyloidosis model showed Aß1-42 to down-regulate hippocampal GIRK1 subunit expression. Together, our results describe an Aß-mediated deleterious synaptic mechanism that modifies the induction threshold for hippocampal LTP/LTD and underlies memory alterations observed in amyloidosis models. In this scenario, GirK activation assures memory formation by preventing the transformation of HFS-induced LTP into LTD.

Activating Transcription Factor 4 (ATF4) Regulates Neuronal Activity by Controlling GABABR Trafficking.

Activating Transcription Factor 4 (ATF4) has been postulated as a key regulator of learning and memory. We previously reported that specific hippocampal ATF4 downregulation causes deficits in synaptic plasticity and memory and reduction of glutamatergic functionality. Here we extend our studies to address ATF4's role in neuronal excitability. We find that long-term ATF4 knockdown in cultured rat hippocampal neurons significantly increases the frequency of spontaneous action potentials. This effect is associated with decreased functionality of metabotropic GABAB receptors (GABABRs). Knocking down ATF4 results in significant reduction of GABABR-induced GIRK currents and increased mIPSC frequency. Furthermore, reducing ATF4 significantly decreases expression of membrane-exposed, but not total, GABABR 1a and 1b subunits, indicating that ATF4 regulates GABABR trafficking. In contrast, ATF4 knockdown has no effect on surface expression of GABABR2s, several GABABR-coupled ion channels or ß2 and γ2 GABAARs. Pharmacologic manipulations confirmed the relationship between GABABR functionality and action potential frequency in our cultures. Specifically, the effects of ATF4 downregulation cited above are fully rescued by transcriptionally active, but not by transcriptionally inactive, shRNA-resistant, ATF4. We previously reported that ATF4 promotes stabilization of the actin-regulatory protein Cdc42 by a transcription-dependent mechanism. To test the hypothesis that this action underlies the mechanism by which ATF4 loss affects neuronal firing rates and GABABR trafficking, we downregulated Cdc42 and found that this phenocopies the effects of ATF4 knockdown on these properties. In conclusion, our data favor a model in which ATF4, by regulating Cdc42 expression, affects trafficking of GABABRs, which in turn modulates the excitability properties of neurons.SIGNIFICANCE STATEMENT GABAB receptors (GABABRs), the metabotropic receptors for the inhibitory neurotransmitter GABA, have crucial roles in controlling the firing rate of neurons. Deficits in trafficking/functionality of GABABRs have been linked to a variety of neurological and psychiatric conditions, including epilepsy, anxiety, depression, schizophrenia, addiction, and pain. Here we show that GABABRs trafficking is influenced by Activating Transcription Factor 4 (ATF4), a protein that has a pivotal role in hippocampal memory processes. We found that ATF4 downregulation in hippocampal neurons reduces membrane-bound GABABR levels and thereby increases intrinsic excitability. These effects are mediated by loss of the small GTPase Cdc42 following ATF4 downregulation. These findings reveal a critical role for ATF4 in regulating the modulation of neuronal excitability by GABABRs.

Hydrogen sulfide inhibits Kir2 and Kir3 channels by decreasing sensitivity to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2).

Inwardly rectifying potassium (Kir) channels establish and regulate the resting membrane potential of excitable cells in the heart, brain, and other peripheral tissues. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a key direct activator of ion channels, including Kir channels. The gasotransmitter carbon monoxide has been shown to regulate Kir channel activity by altering channel-PIP2 interactions. Here, we tested in two cellular models the effects and mechanism of action of another gasotransmitter, hydrogen sulfide (H2S), thought to play a key role in cellular responses under ischemic conditions. Direct administration of sodium hydrogen sulfide as an exogenous H2S source and expression of cystathionine γ-lyase, a key enzyme that produces endogenous H2S in specific brain tissues, resulted in comparable current inhibition of several Kir2 and Kir3 channels. This effect resulted from changes in channel-gating kinetics rather than in conductance or cell-surface localization. The extent of H2S regulation depended on the strength of the channel-PIP2 interactions. H2S regulation was attenuated when channel-PIP2 interactions were strengthened and was increased when channel-PIP2 interactions were weakened by depleting PIP2 levels. These H2S effects required specific cytoplasmic cysteine residues in Kir3.2 channels. Mutation of these residues abolished H2S inhibition, and reintroduction of specific cysteine residues back into the background of the cytoplasmic cysteine-lacking mutant rescued H2S inhibition. Molecular dynamics simulation experiments provided mechanistic insights into how potential sulfhydration of specific cysteine residues could lead to changes in channel-PIP2 interactions and channel gating.

Cholesterol Binding Sites in Inwardly Rectifying Potassium Channels.

Inwardly rectifying potassium (Kir) channels play a variety of critical cellular roles including modulating membrane excitability in neurons, cardiomyocytes and muscle cells, and setting the resting membrane potential, heart rate, vascular tone, insulin release, and salt flow across epithelia. These processes are regulated by a variegated list of modulators. In particular, in recent years, cholesterol has been shown to modulate a growing number of Kir channels. Subsequent to the discovery that members of the Kir2 subfamily were down-regulated by cholesterol, we have shown that members of several other Kir subfamilies were also modulated by cholesterol. However, not all cholesterol sensitive Kir channels were down-regulated by cholesterol. Our recent studies focused on three Kir channels: Kir2.1 (IRK1), Kir3.2^ (GIRK2^) and Kir3.4* (GIRK4*). Among these, Kir2.1 was down-regulated by cholesterol whereas Kir3.2^ and Kir3.4* were both up-regulated by cholesterol. Despite the opposite impact of cholesterol on these Kir3 channels compared to Kir2.1, putative cholesterol binding sites in all three channels were identified in equivalent transmembrane domains. Interestingly, however, there are intriguing differences in the specific residues that interact with the cholesterol molecule in these Kir channels. Here we compare and contrast the molecular characteristics of the putative cholesterol binding sites in the three channels, and discuss the potential implications of the differences for the impact of cholesterol on ion channels.

Role of GirK Channels in Long-Term Potentiation of Synaptic Inhibition in an In Vivo Mouse Model of Early Amyloid-ß Pathology.

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-ß (Aß) 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 Aß-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.

Sign Inversion in Photopharmacology: Incorporation of Cyclic Azobenzenes in Photoswitchable Potassium Channel Blockers and Openers.

Photopharmacology relies on ligands that change their pharmacodynamics upon photoisomerization. Many of these ligands are azobenzenes that are thermodynamically more stable in their elongated trans-configuration. Often, they are biologically active in this form and lose activity upon irradiation and photoisomerization to their cis-isomer. Recently, cyclic azobenzenes, so-called diazocines, have emerged, which are thermodynamically more stable in their bent cis-form. Incorporation of these switches into a variety of photopharmaceuticals could convert dark-active ligands into dark-inactive ligands, which is preferred in most biological applications. This "pharmacological sign-inversion" is demonstrated for a photochromic blocker of voltage-gated potassium channels, termed CAL, and a photochromic opener of G protein-coupled inwardly rectifying potassium (GIRK) channels, termed CLOGO.

µ-Opioid receptors inhibit the exercise pressor reflex by closing N-type calcium channels but not by opening GIRK channels in rats.

µ-Opioid G protein-coupled receptors (MOR) interact with ion channels to decrease neuronal excitability. In humans, intrathecal administration of the MOR agonist fentanyl inhibits the exercise pressor reflex, an effect that can be attributed to either the opening of inward rectifying potassium channels (GIRK) or the closing of N-type calcium channels. The purpose of this study was to determine if the highly selective MOR agonist [d-Ala2, N-MePhe4,Gly-ol]-enkephalin (DAMGO) attenuates the exercise pressor reflex and which of these two channels are responsible for this effect. In decerebrate rats, we determined the effect of intrathecal injection of either tertiapin-LQ, which blocks the GIRK channel or ω-conotoxin-GVIA, which blocks the N-type calcium channel on the exercise pressor reflex, which was evoked by contracting the triceps surae muscles. Initially, we established that intrathecal injection of DAMGO inhibited the exercise pressor reflex relative to no intrathecal injection or intrathecal saline injection ( P < 0.001, n = 5). We then found that intrathecal injection of two doses of tertiapin-LQ (1 and 10 µg) had no effect on the exercise pressor reflex ( n = 6 and n = 7, respectively; P > 0.05). Importantly, neither dose of tertiapin-LQ prevented the DAMGO-induced inhibition of the exercise pressor reflex. Last, we found that intrathecal injection of ω-conotoxin-GVIA markedly attenuated the exercise pressor reflex ( P < 0.001, n = 7). The cardioaccelerator response to contraction did not appear to be effected in any of the experiments. We conclude that N-type voltage-gated calcium channel inhibition appears to be the mechanism by which MOR activation inhibits the exercise pressor reflex in decerebrate rats.

Diverse actions of estradiol on anorexigenic and orexigenic hypothalamic arcuate neurons.

Contribution to Special Issue on Fast effects of steroids. There is now compelling evidence for membrane-associated estrogen receptors in hypothalamic neurons that are critical for the hypothalamic control of homeostatic functions. It has been known for some time that estradiol (E2) can rapidly alter hypothalamic neuronal activity within seconds, indicating that some cellular effects can occur via membrane initiated events. However, our understanding of how E2 signals via membrane-associated receptors and how these signals impact physiological functions is only just emerging. Thus, E2 can affect second messenger systems including calcium mobilization and a plethora of kinases to alter cell excitability and even gene transcription in hypothalamic neurons. One population of hypothalamic neurons, the anorexigenic proopiomelanocortin (POMC) neurons, has long been considered to be a target of E2's actions based on gene (Pomc) expression studies. However, we now know that E2 can rapidly alter POMC neuronal activity within seconds and activate several intracellular signaling cascades that ultimately affect gene expression, actions which are critical for maintaining sensitivity to insulin in metabolically stressed states. E2 also affects the orexigenic Neuropeptide Y/Agouti-related Peptide (NPY/AgRP) neurons in similarly rapid but antagonistic manner. Therefore, this review will summarize our current state of knowledge of how E2 signals via rapid membrane-initiated and intracellular signaling cascades in POMC and NPY/AgRP neurons to regulate energy homeostasis.

Lithium reduces the span of G protein-activated K+ (GIRK) channel inhibition in hippocampal neurons.

OBJECTIVES: Lithium (Li+ ) is one of the most widely used treatments for bipolar disorder (BD). However, the molecular and neuronal basis of BD, as well as the mechanisms of Li+ actions are poorly understood. Cellular and biochemical studies identified G proteins as being among the cellular targets for Li+ action, while genetic studies indicated an association with the KCNJ3 gene, which encodes the G protein-activated inwardly rectifying K+ (GIRK) channels. GIRK channels regulate neuronal excitability by mediating the inhibitory effects of multiple neurotransmitters and contribute to the resting potassium conductance. Here, we explored the effects of therapeutic dose of Li+ on neuronal excitability and the role of GIRK channels in Li+ actions. METHODS: Effects of Li+ on excitability were studied in hippocampal brain slices using whole-cell electrophysiological recordings. RESULTS: A therapeutic dose of Li+ (1 mM) dually regulated the function of GIRK channels in hippocampal slices. Li+ hyperpolarized the resting membrane potential of hippocampal CA1 pyramidal neurons and prolonged the latency to reach the action potential threshold and peak. These effects were abolished in the presence of tertiapin, a specific GIRK channel blocker, and at doses above the therapeutic window (2 mM). In contrast, Li+ reduced GIRK channel opening induced by GABAB receptor (GABAB R) activation, causing reduced hyperpolarization of the membrane potential, attenuated reduction of input resistance, and a smaller decrease of neuronal firing. CONCLUSIONS: A therapeutic dose of Li+ reduces the span of GIRK channel-mediated inhibition due to enhancement of basal GIRK currents and inhibition of GABAB R evoked responses, providing an important link between Li+ action, neuronal excitability, and cellular and genetic targets of BD.

G Protein-Gated Inwardly Rectifying Potassium Channel Subunit 3 Knock-Out Mice Show Enhanced Ethanol Reward.

BACKGROUND: G protein-gated inwardly rectifying potassium (GIRK) channels contribute to the effects of a number of drugs of abuse, including ethanol. However, the roles of individual subunits in the rewarding effects of ethanol are poorly understood. METHODS: We compare conditioned place preference (CPP) in GIRK3 subunit knock-out (GIRK3(-/-)), heterozygote (GIRK3(+/-)), and wild-type (WT) mice. In addition, the development of locomotor tolerance/sensitization and the effects of EtOH intoxication on associative learning (fear conditioning) are also assessed. RESULTS: Our data show significant EtOH CPP in GIRK3(-/-) and GIRK3(+/-) mice, but not in the WT littermates. In addition, we demonstrate that these effects are not due to differences in EtOH metabolism, the development of EtOH tolerance/sensitivity, or associative learning abilities. While there were no consistent genotype differences in the fear conditioning assay, our data do show a selective sensitization of the impairing effects of EtOH intoxication on contextual learning, but no effect on cued learning. CONCLUSIONS: These findings suggest that GIRK3 plays a role in EtOH reward. Furthermore, the selectivity of this effect suggests that GIRK channels could be an effective therapeutic target for the prevention and/or treatment of alcoholism.

Firing modes of dopamine neurons drive bidirectional GIRK channel plasticity.

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.