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

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

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Human Adrenal Glomerulosa Cells Express K2P and GIRK Potassium Channels that are inhibited by AngII and ACTH

AJP Cell Physiology ◽

10.1152/ajpcell.00118.2021 ◽

2021 ◽

Author(s):

John J. Enyeart ◽

Judith A. Enyeart

Keyword(s):

G Protein ◽

Zona Glomerulosa ◽

Time Dependent ◽

Aldosterone Secretion ◽

Girk Channels ◽

Whole Cell ◽

Whole Cell Patch Clamp ◽

Girk Channel ◽

Inwardly Rectifying ◽

G Protein Coupled

In whole-cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two pore (K2P) leak-type and a G-protein-coupled, inwardly-rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly-rectifying current was activated by arachidonic acid and inhibited by angiotensin II (AngII), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by AngII, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by AngII or ACTH, and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by AngII and ACTH are likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.

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Abstract 17317: Differential Coupling and Regulation of GIRK-Dependent Muscarinic and Adenosine Receptor Signaling in Mouse Sino-Atrial Nodal Cells

Circulation ◽

10.1161/circ.138.suppl_1.17317 ◽

2018 ◽

Vol 138 (Suppl_1) ◽

Author(s):

Allison Anderson ◽

Kevin Wickman

Keyword(s):

G Protein ◽

Adenosine Receptor ◽

Protein Signaling ◽

Wild Type ◽

Girk Channels ◽

Genetic Ablation ◽

Whole Cell Patch Clamp ◽

Girk Channel ◽

Induced Currents ◽

Patch Clamp Electrophysiology

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

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Differential effects of inhibitory G protein isoforms on G protein-gated inwardly rectifying K+ currents in adult murine atria

AJP Cell Physiology ◽

10.1152/ajpcell.00271.2016 ◽

2018 ◽

Vol 314 (5) ◽

pp. C616-C626 ◽

Cited By ~ 4

Author(s):

Muriel Nobles ◽

David Montaigne ◽

Sonia Sebastian ◽

Lutz Birnbaumer ◽

Andrew Tinker

Keyword(s):

G Protein ◽

Knockout Mice ◽

Protein Isoforms ◽

Girk Channels ◽

Differential Effects ◽

Girk Channel ◽

Inhibitory G Protein ◽

Genetic Deletion ◽

The Right ◽

Inwardly Rectifying

G protein-gated inwardly rectifying K+ (GIRK) channels are the major inwardly rectifying K+ currents in cardiac atrial myocytes and an important determinant of atrial electrophysiology. Inhibitory G protein α-subunits can both mediate activation via acetylcholine but can also suppress basal currents in the absence of agonist. We studied this phenomenon using whole cell patch clamping in murine atria from mice with global genetic deletion of Gαi2, combined deletion of Gαi1/Gαi3, and littermate controls. We found that mice with deletion of Gαi2 had increased basal and agonist-activated currents, particularly in the right atria while in contrast those with Gαi1/Gαi3 deletion had reduced currents. Mice with global genetic deletion of Gαi2 had decreased action potential duration. Tissue preparations of the left atria studied with a multielectrode array from Gαi2 knockout mice showed a shorter effective refractory period, with no change in conduction velocity, than littermate controls. Transcriptional studies revealed increased expression of GIRK channel subunit genes in Gαi2 knockout mice. Thus different G protein isoforms have differential effects on GIRK channel behavior and paradoxically Gαi2 act to increase basal and agonist-activated GIRK currents. Deletion of Gαi2 is potentially proarrhythmic in the atria.

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Basal G-protein-gated inwardly rectifying potassium (Kir3/GirK) channels activity governs synaptic plasticity that supports dorsal hippocampus-dependent cognitive functions

10.1101/2020.12.11.421735 ◽

2020 ◽

Author(s):

Sara Temprano-Carazo ◽

Souhail Djebari ◽

Guillermo Iborra-Lazaro ◽

Irene Sanchez-Rodriguez ◽

Mauricio O. Nava-Mesa ◽

...

Keyword(s):

Synaptic Plasticity ◽

Learning And Memory ◽

G Protein ◽

Cognitive Functions ◽

Dorsal Hippocampus ◽

Hippocampal Slices ◽

Girk Channels ◽

Neural Excitability ◽

Girk Channel ◽

Inwardly Rectifying

G-protein-gated inwardly rectifying potassium (Kir3/GirK) channel is the effector of many G-protein-coupled receptors. Its dysfunction has been linked to the pathophysiology of Down syndrome, Alzheimer and Parkinson diseases, psychiatric disorders, epilepsy, drug addiction, or alcoholism. GirK channels are constitutively activated in the dorsal hippocampus contributing to resting membrane potential, and their synaptic activation compensates any excitation excess. Here, in order to elucidate the role of GirK channels activity in the maintenance of dorsal hippocampus-dependent cognitive functions, their involvement in controlling neuronal excitability at different levels of complexity was examined. For that purpose, basal GirK activity was pharmacologically modulated by two specific drugs: ML297, a GirK channel opener, and Tertiapin-Q, a GirK channel blocker. Ex vivo, using dorsal hippocampal slices, we studied pharmacological GirK modulation effect on synaptic plasticity processes induced in CA1 by Schaffer collateral stimulation. In vivo, we performed acute intracerebroventricular injections of both GirK modulators to study their contribution to CA3-CA1 synapse electrophysiological properties, synaptic plasticity, and learning and memory capabilities during hippocampal dependent tasks. We found that pharmacological disruption of basal GirK activity in dorsal hippocampus, causing either function gain or loss, induced learning and memory deficits by a mechanism involving neural excitability impairments and alterations in 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: The dorsal hippocampus mostly performs cognitive functions related to contextual/spatial associations. These functions rely on synaptic plasticity processes that are critically ruled by a finely tuned neural excitability. Being the downstream physiological effectors of a variety of G-coupled receptors, activation of G protein-gated inwardly rectifying K+ (GirK) channels induces neurons to hyperpolarize, contributing to neural excitability throughout the control of excitatory excess. Here, we demonstrate that modulation of basal GirK channels activity, causing either function gain or loss, transforms HFS-induced LTP into LTD, inducing deficits in dorsal hippocampus-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.

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Role of G protein–gated inwardly rectifying potassium channels in P2Y12 receptor–mediated platelet functional responses

Blood ◽

10.1182/blood-2004-01-0069 ◽

2004 ◽

Vol 104 (5) ◽

pp. 1335-1343 ◽

Cited By ~ 50

Author(s):

Haripriya Shankar ◽

Swaminathan Murugappan ◽

Soochong Kim ◽

Jianguo Jin ◽

Zhongren Ding ◽

...

Keyword(s):

Platelet Aggregation ◽

G Protein ◽

Shape Change ◽

Human Platelets ◽

P2y12 Receptor ◽

Functional Responses ◽

Girk Channels ◽

Girk Channel ◽

Inwardly Rectifying

Abstract The role of the Gi-coupled platelet P2Y12 receptor in platelet function has been well established. However, the functional effector or effectors contributing directly to αIIbβ3 activation in human platelets has not been delineated. As the P2Y12 receptor has been shown to activate G protein–gated, inwardly rectifying potassium (GIRK) channels, we investigated whether GIRK channels mediate any of the functional responses of the platelet P2Y12 receptor. Western blot analysis revealed that platelets express GIRK1, GIRK2, and GIRK4. In aspirin-treated and washed human platelets, 2 structurally distinct GIRK inhibitors, SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) and U50488H (trans-(±)-3,4-dichloro-N-methyl-N-[2-(pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate), inhibited adenosine diphosphate (ADP)–, 2-methylthioADP (2-MeSADP)–, U46619-, and low-dose thrombin–mediated platelet aggregation. However, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin, AYPGKF, or convulxin. Furthermore, the GIRK channel inhibitors reversed SFLLRN-induced platelet aggregation, inhibited the P2Y12-mediated potentiation of dense granule secretion and Akt phosphorylation, and did not affect the agonist-induced Gq-mediated platelet shape change and intracellular calcium mobilization. Unlike AR-C 69931MX, a P2Y12 receptor–selective antagonist, the GIRK channel blockers did not affect the ADP-induced adenlylyl cyclase inhibition, indicating that they do not directly antagonize the P2Y12 receptor. We conclude that GIRK channels are important functional effectors of the P2Y12 receptor in human platelets.

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De novo BK channel mutation causes epilepsy by regulating voltage-dependent, but not calcium-dependent, activation

10.1101/120543 ◽

2017 ◽

Author(s):

Xia Li ◽

Sibylle Poschmann ◽

Qiuyun Chen ◽

Nelly Jouayed Oundjian ◽

Francesca M. Snoeijen-Schouwenaars ◽

...

Keyword(s):

De Novo ◽

Neurological Diseases ◽

Calcium Sensitivity ◽

Bk Channels ◽

Bk Channel ◽

Common Disease ◽

Open Probability ◽

Calcium Dependent ◽

Functional Studies ◽

Variants Of Unknown Significance

AbstractEpilepsy is one of the most common neurological diseases. Here we report the first de novo mutation in the BK channel (p.N995S) that causes epilepsy in two independent families. The p.N995S mutant channel showed a markedly increased macroscopic potassium current mediated by increases in both channel open probability and channel open dwell time. Mutation p.N995S affects the voltage-activation pathway of BK channel, but does not affect the calcium sensitivity. Paxilline blocks potassium currents from both WT and mutant BK channels. We also identified two variants of unknown significance, p.E656A and p.N1159S in epilepsy patients. However, they do not affect BK channel functions, therefore, are unlikely to be a cause of disease. These results expand the BK channelopathy to a more common disease of epilepsy, suggest that the BK channel is a drug target for treatment of epilepsy, and highlight the importance of functional studies in the era of precision medicine.

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

Anesthesiology ◽

10.1097/aln.0000000000000984 ◽

2016 ◽

Vol 124 (3) ◽

pp. 641-650 ◽

Cited By ~ 49

Author(s):

Gaspard Montandon ◽

Jun Ren ◽

Nicole C. Victoria ◽

Hattie Liu ◽

Kevin Wickman ◽

...

Keyword(s):

Respiratory Rate ◽

G Protein ◽

Respiratory Depression ◽

Ventrolateral Medulla ◽

Wild Type ◽

Girk Channels ◽

Systemic Injection ◽

Baseline Rate ◽

Girk Channel ◽

Inwardly Rectifying

Abstract Background Drugs acting on μ-opioid receptors (MORs) are widely used as analgesics but present side effects including life-threatening respiratory depression. MORs are G-protein–coupled receptors inhibiting neuronal activity through calcium channels, adenylyl cyclase, and/or G-protein–gated inwardly rectifying potassium (GIRK) channels. The pathways underlying MOR-dependent inhibition of rhythmic breathing are unknown. Methods By using a combination of genetic, pharmacological, and physiological tools in rodents in vivo, the authors aimed to identify the role of GIRK channels in MOR-mediated inhibition of respiratory circuits. Results GIRK channels were expressed in the ventrolateral medulla, a neuronal population regulating rhythmic breathing, and GIRK channel activation with flupirtine reduced respiratory rate in rats (percentage of baseline rate in mean ± SD: 79.4 ± 7.4%, n = 7), wild-type mice (82.6 ± 3.8%, n = 3), but not in mice lacking the GIRK2 subunit, an integral subunit of neuronal GIRK channels (GIRK2−/−, 101.0 ± 1.9%, n = 3). Application of the MOR agonist [d-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO) to the ventrolateral medulla depressed respiratory rate, an effect partially reversed by the GIRK channel blocker Tertiapin-Q (baseline: 42.1 ± 7.4 breath/min, DAMGO: 26.1 ± 13.4 breath/min, Tertiapin-Q + DAMGO: 33.9 ± 9.8 breath/min, n = 4). Importantly, DAMGO applied to the ventrolateral medulla failed to reduce rhythmic breathing in GIRK2−/− mice (percentage of baseline rate: 103.2 ± 12.1%, n = 4), whereas it considerably reduced rate in wild-type mice (62.5 ± 17.7% of baseline, n = 4). Respiratory rate depression by systemic injection of the opioid analgesic fentanyl was markedly reduced in GIRK2−/− (percentage of baseline: 12.8 ± 15.8%, n = 5) compared with wild-type mice (72.9 ± 27.3%). Conclusions Overall, these results identify that GIRK channels contribute to respiratory inhibition by MOR, an essential step toward understanding respiratory depression by opioids.

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Molecular mechanisms of metabotropic GABAB receptor function

Science Advances ◽

10.1126/sciadv.abg3362 ◽

2021 ◽

Vol 7 (22) ◽

pp. eabg3362

Author(s):

Hamidreza Shaye ◽

Benjamin Stauch ◽

Cornelius Gati ◽

Vadim Cherezov

Keyword(s):

G Protein ◽

Molecular Mechanisms ◽

Gabab Receptor ◽

Neurological Diseases ◽

Activation Mechanism ◽

Allosteric Modulators ◽

Inhibitory Neurotransmitter ◽

Therapeutic Drugs ◽

G Protein Coupled ◽

The Brain

Metabotropic γ-aminobutyric acid G protein–coupled receptors (GABAB) represent one of the two main types of inhibitory neurotransmitter receptors in the brain. These receptors act both pre- and postsynaptically by modulating the transmission of neuronal signals and are involved in a range of neurological diseases, from alcohol addiction to epilepsy. A series of recent cryo-EM studies revealed critical details of the activation mechanism of GABAB. Structures are now available for the receptor bound to ligands with different modes of action, including antagonists, agonists, and positive allosteric modulators, and captured in different conformational states from the inactive apo to the fully active state bound to a G protein. These discoveries provide comprehensive insights into the activation of the GABAB receptor, which not only broaden our understanding of its structure, pharmacology, and physiological effects but also will ultimately facilitate the discovery of new therapeutic drugs and neuromodulators.

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Changes in Striatal Signaling Induce Remodeling of RGS Complexes Containing Gβ5 and R7BP Subunits

Molecular and Cellular Biology ◽

10.1128/mcb.01449-08 ◽

2009 ◽

Vol 29 (11) ◽

pp. 3033-3044 ◽

Cited By ~ 23

Author(s):

Garret R. Anderson ◽

Rafael Lujan ◽

Kirill A. Martemyanov

Keyword(s):

G Protein ◽

Cellular Response ◽

Neuronal Excitability ◽

Reward Processing ◽

Rgs Proteins ◽

Protein Signaling ◽

Striatal Neurons ◽

Selective Degradation ◽

Oxygenation Status ◽

Molecular Plasticity

ABSTRACT Neurotransmitter signaling via G protein coupled receptors is crucially controlled by regulators of G protein signaling (RGS) proteins that shape the duration and extent of the cellular response. In the striatum, members of the R7 family of RGS proteins modulate signaling via D2 dopamine and μ-opioid receptors controlling reward processing and locomotor coordination. Recent findings have established that R7 RGS proteins function as macromolecular complexes with two subunits: type 5 G protein β (Gβ5) and R7 binding protein (R7BP). In this study, we report that the subunit compositions of these complexes in striatum undergo remodeling upon changes in neuronal activity. We found that under normal conditions two equally abundant striatal R7 RGS proteins, RGS9-2 and RGS7, are unequally coupled to the R7BP subunit, which is present in complex predominantly with RGS9-2 rather than with RGS7. Changes in the neuronal excitability or oxygenation status resulting in extracellular calcium entry, uncouples RGS9-2 from R7BP, triggering its selective degradation. Concurrently, released R7BP binds to mainly intracellular RGS7 and recruits it to the plasma membrane and the postsynaptic density. These observations introduce activity-dependent remodeling of R7 RGS complexes as a new molecular plasticity mechanism in striatal neurons and suggest a general model for achieving rapid posttranslational subunit rearrangement in multisubunit complexes.

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Mal protein stabilizes luminal membrane PLC-β3 and negatively regulates ENaC in mouse cortical collecting duct cells

AJP Renal Physiology ◽

10.1152/ajprenal.00446.2018 ◽

2019 ◽

Vol 317 (4) ◽

pp. F986-F995

Author(s):

Kubra M. Tuna ◽

Bing-Chen Liu ◽

Qiang Yue ◽

Zinah M. Ghazi ◽

He-Ping Ma ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Expression ◽

Collecting Duct ◽

Renal Tubules ◽

Cortical Collecting Duct ◽

Open Probability ◽

Anionic Phospholipid ◽

Kinase Substrate ◽

Luminal Membrane ◽

Collecting Duct Cells

Abnormally high epithelial Na+ channel (ENaC) activity in the aldosterone-sensitive distal nephron and collecting duct leads to hypertension. Myelin and lymphocyte (Mal) is a lipid raft-associated protein that has been previously shown to regulate Na+-K-2Cl− cotransporter and aquaporin-2 in the kidney, but it is not known whether it regulates renal ENaC. ENaC activity is positively regulated by the anionic phospholipid phosphate phosphatidylinositol 4,5-bisphosphate (PIP2). Members of the myristoylated alanine-rich C-kinase substrate (MARCKS) family increase PIP2 concentrations at the plasma membrane, whereas hydrolysis of PIP2 by phospholipase C (PLC) reduces PIP2 abundance. Our hypothesis was that Mal protein negatively regulates renal ENaC activity by stabilizing PLC protein expression at the luminal plasma membrane. We investigated the association between Mal, MARCKS-like protein, and ENaC. We showed Mal colocalizes with PLC-β3 in lipid rafts and positively regulates its protein expression, thereby reducing PIP2 availability at the plasma membrane. Kidneys of 129Sv mice injected with MAL shRNA lentivirus resulted in increased ENaC open probability in split-open renal tubules. Overexpression of Mal protein in mouse cortical collecting duct (mpkCCD) cells resulted in an increase in PLC-β3 protein expression at the plasma membrane. siRNA-mediated knockdown of MAL in mpkCCD cells resulted in a decrease in PLC-β3 protein expression and an increase in PIP2 abundance. Moreover, kidneys from salt-loaded mice showed less Mal membrane protein expression compared with non-salt-loaded mice. Taken together, Mal protein may play an essential role in the negative feedback of ENaC gating in principal cells of the collecting duct.

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