GIRK Activation Research Articles

Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCβ. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCβ do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.

Increased GIRK channel activity prevents arrhythmia in mice with heart failure by enhancing ventricular repolarization

Ventricular arrhythmia causing sudden cardiac death is the leading mode of death in patients with heart failure. Yet, the mechanisms that prevent ventricular arrhythmias in heart failure are not well characterized. Using a mouse model of heart failure created by transverse aorta constriction, we show that GIRK channel, an important regulator of cardiac action potentials, is constitutively active in failing ventricles in contrast to normal cells. Evidence is presented indicating that the tonic activation of M2 muscarinic acetylcholine receptors by endogenously released acetylcholine contributes to the constitutive GIRK activity. This constitutive GIRK activity prevents the action potential prolongation in heart failure ventricles. Consistently, GIRK channel blockade with tertiapin-Q induces QT interval prolongation and increases the incidence of arrhythmia in heart failure, but not in control mice. These results suggest that constitutive GIRK channels comprise a key mechanism to protect against arrhythmia by providing repolarizing currents in heart failure ventricles.

Licorice metabolite 18β-glycyrrhetinic acid activates G protein-gated inwardly rectifying K+ channels.

Licorice (liquorice) is a common food additive and is used in Chinese medicine. Excess licorice intake can induce atrial fibrillation. Patients with atrial fibrillation possess constitutively activated G protein-gated inwardly rectifying K+ (GIRK) channels. Whether licorice affects GIRK channel activity is unknown. We aimed to clarify the effects of licorice ingredients on GIRK current and the mechanism of action. A major component of licorice, glycyrrhizic acid (GA), and its metabolite, 18β-glycyrrhetinic acid (18β-GA), were tested. We performed electrophysiological recordings in Xenopus oocytes to examine the effects of GA and 18β-GA on various GIRK subunits (Kir 3.1-Kir 3.4), mutagenesis analyses to identify the crucial residues for drug action and motion analysis in cultured rat atrial myocytes to clarify effects of 18β-GA on atrial functions. GA inhibited Kir 3.1-containing channels, while 18β-GA activated all Kir 3.x subunits. A pore helix residue Phe137 in Kir 3.1 was critical for GA-mediated inhibition, and the corresponding Ser148 in Kir 3.2 was critical for 18β-GA-mediated activation. 18β-GA activated GIRK channel in a Gβγ -independent manner, whereas phosphatidylinositol 4,5-bisphosphate (PIP2 ) was essential for activation. Glu236 located at the cytoplasmic pore of Kir 3.2 appeared to be important to interactions with 18β-GA. In rat atrial myocytes, 18β-GA suppressed spontaneous beating via activation of GIRK channels. GA acts as a novel GIRK inhibitor, and 18β-GA acts as a novel GIRK activator. 18β-GA alters atrial function via activation of GIRK channels. This study elucidates the pharmacological activity of licorice ingredients and provides information for drug design.

In Vitro Comparison of Ulotaront (SEP-363856) and Ralmitaront (RO6889450) - Two TAAR1 Agonist Candidate Antipsychotics.

Trace amine-associated receptor-1 (TAAR1) agonists have been proposed as potential antipsychotics, with ulotaront and ralmitaront having reached clinical trials. While ulotaront demonstrated efficacy in a recent Phase II trial, a corresponding study studies of ralmitaront failed to show efficacy as a monotherapy. In addition to TAAR1 agonism, ulotaront is a partial agonist at the serotonin 1A receptor (5-HT1AR). However, little is publicly known about ralmitaront. We compared ulotaront and ralmitaront at TAAR1, 5-HT1AR, and D2R using luciferase complementation-based G protein recruitment, cAMP accumulation, and GIRK channel activation assays. Ralmitaront showed lower efficacy at TAAR1 in G protein recruitment, cAMP accumulation, and GIRK activation assays. Moreover, ralmitaront lacked detectable activity at 5-HT1AR and D2R. Compared to ulotaront, ralmitaront shows lower efficacy and slower kinetics at TAAR1, and lacks efficacy at 5-HT1AR. These data may be relevant to understanding differences in clinical profiles of these two compounds.

Impairments in hippocampal oscillations accompany the loss of LTP induced by GIRK activity blockade

Learning and memory occurrence requires of hippocampal long-term synaptic plasticity and precise neural activity orchestrated by brain network oscillations, both processes reciprocally influencing each other. As G-protein-gated inwardly rectifying potassium (GIRK) channels rule synaptic plasticity that supports hippocampal-dependent memory, here we assessed their unknown role in hippocampal oscillatory activity in relation to synaptic plasticity induction.In alert male mice, pharmacological GIRK modulation did not alter neural oscillations before long-term potentiation (LTP) induction. However, after an LTP generating protocol, both gain- and loss-of basal GIRK activity transformed LTP into long-term depression, but only specific suppression of constitutive GIRK activity caused a disruption of network synchronization (δ, α, γ bands), even leading to long-lasting ripples and fast ripples pathological oscillations.Together, our data showed that constitutive GIRK activity plays a key role in the tuning mechanism of hippocampal oscillatory activity during long-term synaptic plasticity processes that underlies hippocampal-dependent cognitive functions.

Ionic signalling mechanisms involved in neurokinin-3 receptor-mediated augmentation of fear-potentiated startle response in the basolateral amygdala.

The tachykinin peptides include substance P (SP), neurokinin A and neurokinin B, which interact with three G-protein-coupled neurokinin receptors, NK1Rs, NK2Rs and NK3Rs, respectively. Whereas high densities of NK3Rs have been detected in the basolateral amygdala (BLA), the functions of NK3Rs in this brain region have not been determined. We found that activation of NK3Rs by application of the selective agonist, senktide, persistently excited BLA principal neurons. NK3R-elicited excitation of BLA neurons was mediated by activation of a non-selective cation channel and depression of the inwardly rectifying K+ (Kir) channels. With selective channel blockers and knockout mice, we further showed that NK3R activation excited BLA neurons by depressing the G protein-activated inwardly rectifying K+ (GIRK) channels and activating TRPC4 and TRPC5 channels. The effects of NK3Rs required the functions of phospholipase Cβ (PLCβ), but were independent of intracellular Ca2+ release and protein kinase C. PLCβ-mediated depletion of phosphatidylinositol 4,5-bisphosphate was involved in NK3R-induced excitation of BLA neurons. Microinjection of senktide into the BLA of rats augmented fear-potentiated startle (FPS) and this effect was blocked by prior injection of the selective NK3R antagonist SB218795, suggesting that activation of NK3Rs in the BLA increased FPS. We further showed that TRPC4/5 and GIRK channels were involved in NK3R-elicited facilitation of FPS. Our results provide a cellular and molecular mechanism whereby NK3R activation excites BLA neurons and enhances FPS. KEY POINTS: Activation of NK3 receptors (NK3Rs) facilitates the excitability of principal neurons in rat basolateral amygdala (BLA). NK3R-induced excitation is mediated by inhibition of GIRK channels and activation of TRPC4/5 channels. Phospholipase Cβ and depletion of phosphatidylinositol 4,5-bisphosphate are necessary for NK3R-mediated excitation of BLA principal neurons. Activation of NK3Rs in the BLA facilitates fear-potentiated startle response. GIRK channels and TRPC4/5 channels are involved in NK3R-mediated augmentation of fear-potentiated startle.

Exploring GIRK Functionality in Human and Mouse Peripheral Neurons

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

G-Protein-Gated Inwardly Rectifying Potassium (Kir3/GIRK) Channels Govern Synaptic Plasticity That Supports Hippocampal-Dependent Cognitive Functions in Male Mice.

The G-protein-gated inwardly rectifying potassium (Kir3/GIRK) channel is the effector of many G-protein-coupled receptors (GPCRs). Its dysfunction has been linked to the pathophysiology of Down syndrome, Alzheimer's and Parkinson's diseases, psychiatric disorders, epilepsy, drug addiction, or alcoholism. In the hippocampus, GIRK channels decrease excitability of the cells and contribute to resting membrane potential and inhibitory neurotransmission. Here, to elucidate the role of GIRK channels activity in the maintenance of hippocampal-dependent cognitive functions, their involvement in controlling neuronal excitability at different levels of complexity was examined in C57BL/6 male mice. For that purpose, GIRK activity in the dorsal hippocampus CA3-CA1 synapse was pharmacologically modulated by two drugs: ML297, a GIRK channel opener, and Tertiapin-Q (TQ), a GIRK channel blocker. Ex vivo, using dorsal hippocampal slices, we studied the effect of pharmacological GIRK modulation on synaptic plasticity processes induced in CA1 by Schaffer collateral stimulation. In vivo, we performed acute intracerebroventricular (i.c.v.) injections of the two GIRK modulators to study their contribution to electrophysiological properties and synaptic plasticity of dorsal hippocampal CA3-CA1 synapse, and to learning and memory capabilities during hippocampal-dependent tasks. We found that pharmacological disruption of GIRK channel activity by i.c.v. injections, causing either function gain or function loss, induced learning and memory deficits by a mechanism involving neural excitability impairments and alterations in the induction and maintenance of long-term synaptic plasticity processes. These results support the contention that an accurate control of GIRK activity must take place in the hippocampus to sustain cognitive functions.SIGNIFICANCE STATEMENT Cognitive processes of learning and memory that rely on hippocampal synaptic plasticity processes are critically ruled by a finely tuned neural excitability. G-protein-gated inwardly rectifying K+ (GIRK) channels play a key role in maintaining resting membrane potential, cell excitability and inhibitory neurotransmission. Here, we demonstrate that modulation of GIRK channels activity, causing either function gain or function loss, transforms high-frequency stimulation (HFS)-induced long-term potentiation (LTP) into long-term depression (LTD), inducing deficits in hippocampal-dependent learning and memory. Together, our data show a crucial GIRK-activity-mediated mechanism that governs synaptic plasticity direction and modulates subsequent hippocampal-dependent cognitive functions.

Long‐term synaptic depression and memory deficits are reversed by enhancement of GirK‐dependent signaling in a mouse model of early amyloidopathy

AbstractBackgroundHippocampal synaptic plasticity disruption by amyloid‐β (Aβ) peptides is 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.MethodHere, 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 Aβ 1‐42. In vitro electrophysiological recordings from slices showed that Aβ 1‐42 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.ResultRemarkably, selective pharmacological activation of GirK channels with ML297 rescued both HFS‐induced LTP and habituation memory from Aβ 1‐42 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 Aβ 1‐42. On the other hand, the molecular analysis of the recorded slices by western blot showed that expression of GIRK1/2 subunits, which form the prototypical GirK channel in the hippocampus, was not significantly regulated by Aβ 1‐42. However, immunohistochemical examination of our in vivo amyloidosis model showed Aβ 1‐42 to downregulate hippocampal GIRK1 subunit expression.ConclusionTogether, our results describe an Aβ‐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.

Presynaptic and Somatic Mechanisms of Opioid Depression in preBötC Inspiratory Circuits

Opioid‐induced respiratory depression (OIRD) is the primary cause of death from the overdose of opioid analgesics and drugs of abuse. We are interested in identifying molecular/cellular mechanisms underlying OIRD in central respiratory circuits, towards the aim of mitigating OIRD.Although multiple brainstem centers likely contribute to OIRD, we focused on the preBötzinger Complex (preBötC) since this locus is essential for generating rhythmic inspiratory drive. Using in vitro mouse brain slices containing the isolated preBötC, we confirmed that inspiratory burst frequency is depressed by bath‐applied DAMGO, a mu‐opioid receptor (MOR1) specific agonist. MOR1 is known to suppress excitability by signaling via the Gi/o pathway to suppress presynaptic calcium channels and activate GIRK K+ channels. The contribution of each effector pathway was tested with pharmacological and genetic tools. Unexpectedly, blockade of GIRK K+ channels (TertiapinQ) failed to rescue in vitro DAMGO‐depressed rhythms, and activation of GIRK channels (ML297) failed to mimic OIRD, suggesting a relatively modest role for MOR1‐mediated GIRK activation as a mechanism for OIRD. By contrast, slices derived from heterozygous CACNA1A (encoding P/Q‐type calcium channels) KO/+ animals were found to be 10‐fold sensitized to DAMGO depression, consistent with a major presynaptic component for OIRD. In addition, blockers of KCNQ K+ channels (XE991, Chromanol 293B) found in presynaptic terminals, rescued DAMGO‐depressed rhythms, and activators of KCNQ channels (ICA 69673, Retigabine) mimicked DAMGO‐mediated OIRD. Direct support for a presynaptic component of OIRD was provided by recording mEPSCs from genetically labelled Dbx1 neurons. Spontaneous excitatory mEPSCs frequency in TTX was suppressed by DAMGO (70% of cells) and restored by subsequent block of KCNQ channels (XE991) (~83% of cells), supporting the co‐localization of MOR1 and KCNQ K+ channels with P/Q‐type calcium channels to presynaptic terminals. In vivo plethysmography in unanesthetized animals combined with systemic injections with GIRK (ML297) and KCNQ (Retigabine) activators, along with morphine followed by KCNQ blocker (XE991), largely corroborated these in vitro findings. We hypothesize that preBötC OIRD is primarily due to MOR1‐mediated suppression of presynaptic calcium channels, modulated by co‐compartmentalized KCNQ K+ channels. By this model, decreased synaptic efficacy due to OIRD leads to collapse of the rhythmogenic capacity of the preBötC network. Although our results do not support a major role for GIRK K+ channels in OIRD, preliminary results suggest a secondary role for TWK‐type K+ leak channels in OIRD. Co‐expression of MOR1 and TREK2 in Xenopus oocytes produced currents that augmented ~2‐fold with DAMGO and blocked by fluoxetine. In vitro slice recordings revealed rescue of DAMGO‐mediated OIRD with fluoxetine and mimicking of OIRD with a TREK‐specific activator (BL 1249). Our results thus support a two‐tiered mechanism for OIRD: Primary suppression of network‐driven rhythmogenesis due to MOR1‐mediated reduction of excitatory synaptic transmission across the network, and a secondary role for intrinsic hyperpolarization driven by MOR1‐activated TREK K+ channels.Support or Funding InformationNIH R01HL126523, R01HL144801

Etrasimod, a Selective S1P1,4,5 Receptor Modulator, Exhibits Differential G Protein Signaling Compared to Other S1P Receptor Modulators

BackgroundEtrasimod (APD334) is an oral, selective sphingosine‐1‐phosphate (S1P) receptor modulator in development for atopic dermatitis and inflammatory bowel disease. In humans, S1P receptor modulators decrease heart rate at first‐dose due to initial on‐target S1P receptor 1 (S1P1) agonism in cardiac tissue. S1P1 agonism activates Gi and β‐arrestin signaling pathways: β‐arrestin activation leads to receptor internalization, the proposed mechanism by which S1P receptor modulators regulate lymphocyte trafficking; Gi‐coupled signaling activates G‐protein coupled inwardly rectifying potassium (GIRK) channels, that may contribute to the regulation of heart rate. Approved or investigational S1P receptor modulators have demonstrated different degrees of heart rate reduction and atrioventricular block in clinical studies. Such differences may be the result of differential signaling through the Gi coupled GIRK pathway.AimTo investigate S1P receptor expression in human sinoatrial (SA) node tissue, and the relative S1P1 mediated signaling events and GIRK channel activation of etrasimod and other S1P receptor modulators in the context of first dose heart rate effects.MethodsS1P receptor profiling was performed by qPCR in normal human primary SA node and right atrium samples. Potencies for multiple S1P receptor modulators to activate different S1P1 downstream signaling pathways (GTPγS, cAMP, β‐arrestin, S1P1 internalization) were measured in recombinant human‐S1P1 expressing cells. Gi mediated inhibition of cAMP was also measured in primary HUVEC. GIRK channel activation was measured in isolated adult human primary atrial myocytes.ResultsProfiling of S1P receptors in normal human atrial tissue and SA node demonstrated enriched expression of S1P1,2,3 with only minor expression of S1P4,5. Etrasimod was equipotent to other S1P receptor‐ modulators such as ozanimod, fingolimod, and siponimod in promoting β‐arrestin recruitment and S1P1 receptor internalization. Etrasimod was significantly less potent than ozanimod, fingolimod, and siponimod in assays measuring G‐protein activation (GTPγS binding and cAMP inhibition). In comparisons of GIRK activation, etrasimod was found to be a less potent activator of human atrial GIRK channels than ozanimod.ConclusionsWhen compared to other S1P1 modulators, the pharmacological profile of etrasimod at the S1P1 receptor suggests diminished capacity for signaling through Gi‐protein dependent pathways believed to be responsible for transient reductions in heart rate, while maintaining potent signaling in the β‐arrestin recruitment and receptor internalization related pathways underpinning the therapeutic efficacy of S1P1 modulators in immune‐mediated inflammatory disease settings.Support or Funding InformationThe study was sponsored by Arena Pharmaceuticals, Inc.

Inhibitory Mechanisms of G-protein-gated Inwardly Rectifying K+ Channel by Antihistamines

G-protein-gated inwardly rectifying K+ (GIRK) channels are important cellular modulators that regulate neuronal excitability, heart beat and islet hormone secretion. We previously found that some small-molecules modulate GIRK activity and one of them was a non-sedating antihistamine, terfenadine. This drug is known to induce arrhythmia by blocking human ether-a-go-go related gene channel. However, there is no information about the effects of terfenadine on GIRK channels. Here we performed electrophysiological experiments and molecular docking analyses to examine the drug effect on GIRK channel and revealed the structural determinant for the modulation. We found that GIRK1-containing channel is inhibited by terfenadine and some other non-sedating antihistamines. A Phe residue located in the center of the pore helix, position 137, in GIRK1 behind the selectivity filter is crucial for terfenadine-mediated inhibition. Mutations in the corresponding position 148 in GIRK2 alter the response to terfenadine and the side-chain volume is critical. This residue also contributes to the ion selectivity. Phosphoinositide-mediated channel activity is also inhibited by terfenadine. Our findings suggested that Phe137 in GIRK1 is responsible for the terfenadine-mediated inhibition. This study provides us with novel insights into the regulation of GIRK channel by the pore helix and information for drug design.

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‐β (Aβ) 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 Aβ1–42. In vitro electrophysiological recordings from slices showed that Aβ1–42 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 Aβ1–42 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 Aβ1–42. 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 Aβ1–42. However, immunohistochemical examination of our in vivo amyloidosis model showed Aβ1–42 to down‐regulate hippocampal GIRK1 subunit expression. Together, our results describe an Aβ‐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.

Presynaptic Mechanisms and KCNQ Potassium Channels Modulate Opioid Depression of Respiratory Drive.

Opioid-induced respiratory depression (OIRD) is the major cause of death associated with opioid analgesics and drugs of abuse, but the underlying cellular and molecular mechanisms remain poorly understood. We investigated opioid action in vivo in unanesthetized mice and in in vitro medullary slices containing the preBötzinger Complex (preBötC), a locus critical for breathing and inspiratory rhythm generation. Although hypothesized as a primary mechanism, we found that mu-opioid receptor (MOR1)-mediated GIRK activation contributed only modestly to OIRD. Instead, mEPSC recordings from genetically identified Dbx1-derived interneurons, essential for rhythmogenesis, revealed a prevalent presynaptic mode of action for OIRD. Consistent with MOR1-mediated suppression of presynaptic release as a major component of OIRD, Cacna1a KO slices lacking P/Q-type Ca2+ channels enhanced OIRD. Furthermore, OIRD was mimicked and reversed by KCNQ potassium channel activators and blockers, respectively. In vivo whole-body plethysmography combined with systemic delivery of GIRK- and KCNQ-specific potassium channel drugs largely recapitulated these in vitro results, and revealed state-dependent modulation of OIRD. We propose that respiratory failure from OIRD results from a general reduction of synaptic efficacy, leading to a state-dependent collapse of rhythmic network activity.

Receptor-specific regulation of atrial GIRK channel activity by different Ca2+-dependent PKC isoforms

G Protein-activated K+ channels (GIRK) channels are inhibited by depletion of PtdIns(4,5)P2(PIP2), and/or channel phosphorylation by proteinkinase C (PKC). By using FRET-based biosensors, expressed in HEK293 cells or in atrial myocytes, we quantified receptor-specific Gq-coupled receptor (GqPCR) signalling on the level of phospholipase C (PLC) activation by monitoring PIP2-depletion and diacylglycerol (DAG) formation. Simultaneous voltage-clamp experiments on GIRK channel activity were performed as a functional readout for Gq-coupled α1B- and ET-receptor-induced signalling. GqPCR-induced fast inhibition of GIRK channel activity is mediated by depletion of PIP2, whereas phosphorylation of GIRK channels results in delayed, but effective GIRK current inhibition.We demonstrate a receptor-induced inhibitory component on GIRK activity that is independent of PIP2-depletion, but attributed to the activation of Ca2+-dependent PKC isoforms. As a novel finding, we demonstrate receptor-dependent differences in GIRK inhibition according to receptor-specific activation of the Ca2+-dependent PKC isoforms PKCα and PKCβ. Pharmacological inhibition of PKCα, but not of PKCβ, abolishes GIRK inhibition induced by stimulation of α1B-receptors. In contrast, ET-R-induced reduction of GIRK activity is sensitive to pharmacological block of PKCβ, but not of PKCα. Coexpression of α1B-receptors (or ETB-R) and PKCα (or PKCβ) in HEK 293 cells increased homologous receptor desensitization as indicated by a rapid decline of the CKAR FRET signal monitoring receptor activity. These data suggest that receptor-species dependent differences in PKC isoform activation regulate both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism.

Structural mechanism underlying G protein family-specific regulation of G protein-gated inwardly rectifying potassium channel

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein βγ subunit (Gβγ), which is released from the heterotrimeric G protein (Gαβγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oβγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oβγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.

Peripheral Kappa Opioid Receptor (KOR)‐Mediated Antinociception Requires G Protein‐Gated Inward Rectifying Potassium (GIRK) Channels

Targeting peripherally expressed kappa opioid receptors (KOR) is a promising approach to treat inflammatory pain that would be devoid of CNS related adverse effects. KOR are known to couple to Gi‐protein‐mediated signaling pathways involved in the suppression of neuronal firing and nociception. In this study, we sought to determine the involvement of Gi protein coupled inward rectifying potassium (GIRK) channels in peripheral KOR‐mediated antinociceptive behavior in rats. Rats were injected intraplantarly (i.pl., 50ul) with PGE2 (0.3ug) with or without the KOR agonist, U50488 (0.1ug) and paw withdrawal latencies (PWL) to a thermal stimulus were determined over a 20 min period. Treatment with U50488 completely reduced the thermal allodynia produced by PGE2. Consistent with activation of Gi proteins by KOR, we found that the reduction of PGE2‐evoked thermal allodynia by U50488 was lost completely 24h after i.pl. injection of pertussis toxin. To determine if GIRK channels mediated the antinociceptive response to KOR, we measured the effect of U50488 on PGE2‐evoked thermal allodynia 15 minutes after i.pl. injection of either vehicle or tertiapin Q (TPNQ), an inhibitor that blocks channels containing GIRK1 and GIRK2 subunits. Administration of TPNQ had no effect on either baseline PWL or PGE2‐evoked thermal allodynia, however, U50488‐mediated reduction of PGE2‐evoked thermal allodynia was lost after TPNQ administration. We next tested if direct activation of GIRK channels by i.pl. injection of the GIRK activator, ML297, could produce antinociceptive effects. ML297 (33ug) produced a robust analgesic effect (+5 sec above baseline) compared to vehicle treated animals as well as prevented PGE2‐evoked thermal allodynia. To further corroborate the importance of GIRK channels in peripheral KOR mediated antinociception, we tested effects of siRNA‐mediated knockdown of GIRK2 channel subunits following local administration of GIRK2 Accell‐siRNA (Dharmacon) into the hindpaw. After two daily i.pl. injections of GIRK2 siRNA, neither baseline PWL nor PGE2‐evoked thermal allodynia were altered, however, U50488‐mediated reduction in PGE2‐evoked thermal allodynia was lost. We verified knockdown of the GIRK2 subunit in hindpaws by RT‐qPCR and immunohistochemistry. Compared to the contralateral (CONTRA) hindpaw, GIRK2 mRNA was decreased significantly in the ipsilateral (IPSI) hindpaw. Further, there was no difference between IPSI and CONTRA hindpaws in the immunofluorescence for PGP9.5, a marker used to identify neuronal processes, or DAPI, a cell nuclei marker. By contrast, GIRK2 immunofluorescence was significantly decreased in IPSI hindpaws compared to CONTRA hindpaws suggesting that the expression of GIRK2 was indeed reduced. Together these results suggest that peripherally mediated antinociception in response to activation of KOR is due, in large part, to activation of GIRK channels.Support or Funding InformationThis work is supported by NIH/NIDA RO1 DA 038645 and NIH/NIGMS RO1 GM 106035.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Novel sumanirole bivalent analogues as potent dopamine D2 receptor Go‐protein biased agonists

The dopamine D2 receptor (D2R) is a major target for therapeutic development of treatments for neuropsychiatric disorders. In particular, D2R agonists have been used for neurological disorders such as restless leg syndrome and Parkinson's Disease. However, current D2R agonist pharmacotherapies have drawbacks including the development of tolerance and other adverse side‐effects. To potentially improve D2R agonist‐based medications, we have designed bivalent ligands that have two pharmacophores tethered by a link. This strategy aims to improve selectivity, affinity, and cell signaling activation bias. Indeed, it has been demonstrated that the secondary pharmacophore plays a key role in inducing activation bias towards either G protein, or β‐arrestin pathways.Here we characterized a series of bivalent orthosteric‐arthosteric heteroaryl pharmacophores with assorted linkers and compared them with the potent orthosteric D2R‐selective agonist, sumanirole. Several bivalent analogues demonstrated modest increases in D2R affinity compared to sumanirole. D2R functional assays using bioluminescence resonance energy transfer (BRET) showed improved efficacy and potency for G protein coupling, but substantially diminished efficacy and potency for β‐arrestin recruitment. Analysis using an operational model of bias revealed significant G‐protein activation bias (>1000‐fold). Further, several compounds exhibited a G protein subtype (GoA over Gi1) activation bias. Finally, D2R‐mediated, G protein‐coupled inwardly‐rectifying potassium channel (GIRK) activity was measured in midbrain slices using electrophysiology. Recordings from D2R‐expressing dopaminergic cells in the ventral tegmental area confirmed prolonged GIRK activation by the biased bivalent compounds, compared to dopamine or sumanirole.Our novel sumanirole bivalent analogues demonstrate a proof of concept for G protein activation bias and thus may lead to D2R agonist therapeutics with reduced tolerance and improved efficacy. With these novel tools, the preferential targeting and activation of Go‐proteins, the most predominant Gi‐like protein family in the brain, are now conceivable in animal models. This advance may aid in treatments of basal ganglia disorders with reductions in untoward side‐effects in humans.Support or Funding InformationWork was supported by the Intramural funds of the National Institute on Drug AbuseThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Biased signaling of Ca2+-sensing receptors in cardiac myocytes regulates GIRK channel activity

Ca2+-sensing receptors (CaSRs) belong to the class C of G protein-coupled receptors and are activated by extracellular Ca2+. CaSRs display biased G protein signaling by coupling to different classes of heterotrimeric G proteins depending on agonist and cell type. In this study we used fluorescent biosensors to directly analyze G protein coupling to CaSRs and downstream signaling in living cells. In HEK 293 cells, CaSRs displayed biased signaling: elevation of extracellular Ca2+ or application of the alternative agonist spermine caused activation of Gi- and Gq-proteins. Adult cardiac myocytes express endogenous CaSRs, which have been implicated in regulating Ca2+ signaling and contractility. Biased signaling of CaSRs has not been investigated in these cells. To evaluate efficiencies of Gi- and Gq-signaling via CaSRs in rat atrial myocytes, we measured G protein-activated K+ (GIRK) channels. Activation of GIRK requires binding of Gβγ subunits released from Gi proteins, whereas Gq-signaling results in inhibition of GIRK channel activity. Stimulation of CaSRs by Ca2+ or spermine failed to directly activate Gi and GIRK channels. When GIRK channels were pre-activated via endogenous M2 receptors, stimulation of CaSRs caused pronounced inhibition of GIRK currents. This effect was specific to CaSR activation: GIRK current inhibition was sensitive to NPS-2143, a negative allosteric modulator of CaSRs, and abrogated by FR900359, a direct inhibitor of Gq. GIRK current inhibition was also sensitive to the PKC inhibitor chelerythrine, suggesting that following activation of CaSR and Gq, GIRK currents are modulated by PKC phosphorylation. We conclude from this data that cardiac CaSRs do not activate Gi and affect GIRK currents preferentially via the Gq/PKC pathway.

Correction to Analgesic Effects of the GIRK Activator, VU0466551, Alone and in Combination with Morphine in Acute and Persistent Pain Models.

Correction to Analgesic Effects of the GIRK Activator, VU0466551, Alone and in Combination with Morphine in Acute and Persistent Pain Models.