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The coupling of GABA(B) receptors to G-protein-gated inwardly rectifying potassium (GIRK) channels constitutes an important inhibitory pathway in the brain. Here, we examined the mechanism underlying desensitization of agonist-evoked currents carried by homomeric GIRK2 channels expressed in HEK-293T cells. The canonical GABA(B) receptor agonist bac...
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... Rgs4 protein is found to coimmunoprecipitate with GIRK1 and GIRK4 subunits (Adelfinger et al., 2014;Fujita, Inanobe, Chachin, Aizawa, & Kurachi, 2000). It decreases receptor sensitivity by stimulating GTP hydrolysis through interaction with G α subunit (Fowler, Aryal, Suen, & Slesinger, 2007;Mutneja, Berton, Suen, Luscher, & Slesinger, 2005;Ross & Wilkie, 2000). On the other hand, KCTD 8 , KCTD 12 and KCTD 16 proteins assembled into homo-and heterooligomers can interact with G protein associated to GABA B receptor, desensitizing differentially GABA B -GirK currents throughout the brain (Fritzius et al., 2017). ...
G protein-gated inwardly rectifying potassium channels (Kir3/GirK) are important for maintaining resting membrane potential, cell excitability and inhibitory neurotransmission. Coupled to numerous G protein-coupled receptors (GPCRs), they mediate the effects of many neurotransmitters, neuromodulators and hormones contributing to the general homeostasis and particular synaptic plasticity processes, learning, memory and pain signaling. A growing number of behavioral and genetic studies suggest a critical role for the appropriate functioning of the central nervous system, as well as their involvement in many neurologic and psychiatric conditions, such as neurodegenerative diseases, mood disorders, attention deficit hyperactivity disorder, schizophrenia, epilepsy, alcoholism and drug addiction. Hence, GirK channels emerge as a very promising tool to be targeted in the current scenario where these conditions already are or will become a global public health problem. This review examines recent findings on the physiology, function, dysfunction, and pharmacology of GirK channels in the central nervous system and highlights the relevance of GirK channels as a worthful potential target to improve therapies for related diseases.
... 107) RGS4 protein also induces a faster form of desensitization within a second of agonist application in vitro. 108) Transcription factors. ...
γ-aminobutyric acid type B (GABAB) receptors are broadly expressed in the nervous system and play an important role in neuronal excitability. GABAB receptors are G protein-coupled receptors that mediate slow and prolonged inhibitory action, via activation of Gαi/o-type proteins. GABAB receptors mediate their inhibitory action through activating inwardly rectifying K⁺ channels, inactivating voltage-gated Ca²⁺ channels, and inhibiting adenylate cyclase. Functional GABAB receptors are obligate heterodimers formed by the co-assembly of R1 and R2 subunits. It is well established that GABAB receptors interact not only with G proteins and effectors but also with various proteins. This review summarizes the structure, subunit isoforms, and function of GABAB receptors, and discusses the complexity of GABAB receptors, including how receptors are localized in specific subcellular compartments, the mechanism regulating cell surface expression and mobility of the receptors, and the diversity of receptor signaling through receptor crosstalk and interacting proteins.
... Murine and human GIRK2 are identical except for the first two amino acids, thus murine GIRK2(L173) is homologous to human GIRK2 (L171) (see Fig. 3A). We examined the effect of the GABA B receptor agonist, Baclofen, as well as activation with alcohol, e.g., propanol (PrOH), a G proteinindependent activator (Mutneja et al., 2005;Bodhinathan and Slesinger, 2013). In contrast to wildtype GIRK2, which showed large Baclofen-and PrOHinduced currents (Fig. 3B), we observed little change with Baclofen and a small inhibition with PrOH with the GIRK2 (L173R) mutant channel (Fig. 3C). ...
Here, we describe a fourth case of a human with a de novo KCNJ6 (GIRK2) mutation, who presented with clinical findings of severe hyperkinetic movement disorder and developmental delay, similar to Keppen-Lubinsky syndrome but without lipodystrophy. Whole exome sequencing of the patient's DNA revealed a heterozygous de novo variant in the KCNJ6 (c.512T>G, p.Leu171Arg). We conducted in vitro functional studies to determine if this Leu-to-Arg mutation alters the function of GIRK2 channels. Heterologous expression of the mutant GIRK2 channel alone produced an aberrant basal inward current that lacked G protein activation, lost K+ selectivity and gained Ca2+ permeability. Notably, the inward current was inhibited by the Na+ channel blocker QX-314, similar to the previously reported weaver mutation in murine GIRK2. Expression of a tandem dimer containing GIRK1 and GIRK2(p.Leu171Arg) did not lead to any currents, suggesting heterotetramers are not functional. In neurons expressing p.Leu171Arg GIRK2 channels, these changes in channel properties would be expected to generate a sustained depolarization, instead of the normal G protein-gated inhibitory response, which could be mitigated by expression of other GIRK subunits. The identification of the p.Leu171Arg GIRK2 mutation potentially expands the Keppen-Lubinsky syndrome phenotype to include severe dystonia and ballismus. Our study suggests screening for dominant KCNJ6 mutations in the evaluation of patients with severe movement disorders, which could provide evidence to support a causal role of KCNJ6 in neurological channelopathies.
... Fast activation and deactivation kinetics of GABA B R-activated K + currents in rat cerebellar granule cells suggested early on that native GABA B Rs are subject to modulation by RGS proteins (Doupnik et al., 2004). Likewise, experiments with the nonhydrolysable GTP analogue GTPγS or mutant Gα subunits that either sequester endogenous RGS proteins or render Gα insensitive to GAP activity suggested that endogenous RGS proteins regulate GABA B R responses in human embryonic kidney 293T cells (Mutneja, Berton, Suen, Luscher, & Slesinger, 2005). Additional experiments showed that RGS4 was responsible for enhancing the fast component of GABA B R-activated K + current desensitization in 293T cells. ...
GABAB receptors (GABABRs) regulate the excitability of most neurons in the central nervous system by modulating the activity of enzymes and ion channels. In the sustained presence of the neurotransmitter γ-aminobutyric acid, GABABRs exhibit a time-dependent decrease in the receptor response-a phenomenon referred to as homologous desensitization. Desensitization prevents excessive receptor influences on neuronal activity. Much work focused on the mechanisms of GABABR desensitization that operate at the receptor and control receptor expression at the plasma membrane. Over the past few years, it became apparent that GABABRs additionally evolved mechanisms for faster desensitization. These mechanisms operate at the G protein rather than at the receptor and inhibit G protein signaling within seconds of agonist exposure. The mechanisms for fast desensitization are ideally suited to regulate receptor-activated ion channel responses, which influence neuronal activity on a faster timescale than effector enzymes. Here, we provide an update on the mechanisms for fast desensitization of GABABR responses and discuss physiological and pathophysiological implications.
© 2015 Elsevier Inc. All rights reserved.
... Second, the kinetics for receptor agonist-mediated GIRK channel activation and deactivation are dramatically accelerated with moderate changes in steady-state peak current amplitude (Doupnik et al., 1997;Saitoh et al., 1997). And lastly, the time course for GIRK channel "rapid" desensitization during continuous receptor agonist activation is also significantly accelerated (Chuang et al., 1998;Doupnik et al., 1997;Mutneja, Berton, Suen, Luscher, & Slesinger, 2005;Saitoh et al., 1997). A summary list of reconstituted RGS protein-GIRK channel interactions is provided in Table 1. ...
... Heterologous expression of an engineered RGS-resistant Gαo subunit (G184S) in rat sympathetic neurons yield GIRK deactivation times markedly slower (>20 s) and provide an estimate of total endogenous RGS influence on GIRK gating kinetics ( Jeong & Ikeda, 2001). In other heterologous expression systems where "background" endogenous RGS protein levels are low, the time constant for GIRK deactivation is considerably larger (i.e., slower time course) ($10 s in CHO-K1 cells; >10 s in Xenopus oocytes) (Doupnik et al., 2004;Jaen & Doupnik, 2005Mutneja et al., 2005;Zhang et al., 2002). These quantitative considerations are likely to reflect the presence of additional RGS proteins in hippocampal and cerebellar neurons that contribute toward the accelerated RGS-dependent GIRK channel gating kinetics in a redundant manner (see Fig. 1). ...
... Evidence to date indicate robust functional redundancy in RGS protein regulation of neuronal GIRK channels (see Tables 1 and 2). The total influence of endogenous RGS proteins on GIRK channel gating, as revealed ( Jeong & Ikeda, 2001;Mutneja et al., 2005), indicate single RGS knockouts (i.e., RGS2, RGS4, RGS6, and RGS7) affect only a small subset of the total endogenous RGS influence on GIRK channel gating (Cifelli et al., 2008;Ostrovskaya et al., 2014; ...
Regulators of G protein signaling (RGS proteins) are key components of GPCR complexes, interacting directly with G protein α-subunits to enhance their intrinsic GTPase activity. The functional consequence is an accelerated termination of G protein effectors including certain ion channels. RGS proteins have a profound impact on the membrane-delimited gating behavior of G-protein-activated inwardly rectifying K+ (GIRK) channels as demonstrated in reconstitution assays and recent RGS knockout mice studies. Akin to GPCRs and G protein αβγ subunits, multiple RGS isoforms are expressed within single GIRK-expressing neurons, suggesting functional redundancy and/or specificity in GPCR-GIRK channel signaling. The extent and impact of RGS redundancy in neuronal GPCR–GIRK channel signaling is currently not fully appreciated; however, recent studies from RGS knockout mice are providing important new clues on the impact of individual endogenous RGS proteins and the extent of RGS functional redundancy. Incorporating “tools” such as engineered RGS-resistant Gαi/o subunits provide an important assessment method for determining the impact of all endogenous RGS proteins on a given GPCR response and an accounting benchmark to assess the impact of individual RGS knockouts on overall RGS redundancy within a given neuron. Elucidating the degree of regulation attributable to specific RGS proteins in GIRK channel function will aid in the assessment of individual RGS proteins as viable therapeutic targets in epilepsy, ataxia’s, memory disorders, and a growing list of neurological disorders.
... GABA-a receptors are the molecular base for inhibitory postsynaptic current. The GABA-b receptor is a metabotrophic receptor that enhances G protein-coupled inwardly-rectifying potassium channel (GIRK) channels, which provide a tonic hyperpolarizing current (Mutneja et al., 2005;Hearing et al., 2013). ...
Calcium is essential for both neurotransmitter release and muscle contraction. Given these important physiological processes, it seems reasonable to assume that hypocalcemia may lead to reduced neuromuscular excitability. Counterintuitively, however, clinical observation has frequently documented hypocalcemia's role in induction of seizures and general excitability processes such as tetany, Chvostek's sign, and bronchospasm. The mechanism of this calcium paradox remains elusive, and very few pathophysiological studies have addressed this conundrum. Nevertheless, several studies primarily addressing other biophysical issues have provided some clues. In this review, we analyze the data of these studies and propose an integrative model to explain this hypocalcemic paradox.
© The Author(s) 2015.
... These mechanisms operate at the G-protein rather than at the receptor. The ''Regulator of G-protein Signaling'' (RGS) protein 4 induces desensitization by accelerating the rate of GTP hydrolysis at the Ga subunit of the activated Gprotein [15][16][17]. Fast desensitization is additionally induced by the KCTD proteins 12 and 12b [18,19]. The KCTDs are auxiliary receptor subunits that constitutively associate with the C-terminal domain of GB2, in which mutation of Y902 to alanine (Y902A) completely abolishes association [18,20,21]. ...
GABAB receptors assemble from GABAB1 and GABAB2 subunits. GABAB2 additionally associates with auxiliary KCTD subunits (named after their K(+) channel tetramerization-domain). GABAB receptors couple to heterotrimeric G-proteins and activate inwardly-rectifying K(+) channels through the βγ subunits released from the G-protein. Receptor-activated K(+) currents desensitize in the sustained presence of agonist to avoid excessive effects on neuronal activity. Desensitization of K(+) currents integrates distinct mechanistic underpinnings. GABAB receptor activity reduces protein kinase-A activity, which reduces phosphorylation of serine-892 in GABAB2 and promotes receptor degradation. This form of desensitization operates on the time scale of several minutes to hours. A faster form of desensitization is induced by the auxiliary subunit KCTD12, which interferes with channel activation by binding to the G-protein βγ subunits. Here we show that the two mechanisms of desensitization influence each other. Serine-892 phosphorylation in heterologous cells rearranges KCTD12 at the receptor and slows KCTD12-induced desensitization. Likewise, protein kinase-A activation in hippocampal neurons slows fast desensitization of GABAB receptor-activated K(+) currents while protein kinase-A inhibition accelerates fast desensitization. Protein kinase-A fails to regulate fast desensitization in KCTD12 knock-out mice or knock-in mice with a serine-892 to alanine mutation, thus demonstrating that serine-892 phosphorylation regulates KCTD12-induced desensitization in vivo. Fast current desensitization is accelerated in hippocampal neurons carrying the serine-892 to alanine mutation, showing that tonic serine-892 phosphorylation normally limits KCTD12-induced desensitization. Tonic serine-892 phosphorylation is in turn promoted by assembly of receptors with KCTD12. This cross-regulation of serine-892 phosphorylation and KCTD12 activity sharpens the response during repeated receptor activation.
... However, it has not been specifically addressed whether this fast desensitization is due to the presence of KCTD12 in the receptor. In addition, desensitization of GABA B responses was shown to be regulated by GRK4 (10), RGS proteins (11)(12)(13)(14) or phosphorylation of the GABA B2 subunit (15,16). ...
GABAB receptors assemble from principle and auxiliary subunits. The principle subunits GABAB1 and GABAB2 form functional heteromeric GABAB(1,2) receptors that associate with homotetramers of auxiliary KCTD8, 12, 12b or 16 (named after their K+ channel tetramerization domain) subunits. These auxiliary subunits constitute receptor subtypes with distinct functional properties. KCTD12 and 12b generate desensitizing receptor responses while KCTD8 and 16 generate largely non-desensitizing receptor responses. The structural elements of the KCTDs underlying these differences in desensitization are unknown. KCTDs are modular proteins comprising a T1 tetramerization domain, which binds to GABAB2, and a H1 homology domain. KCTD8 and 16 contain an additional C-terminal H2 homology domain that is not sequence-related to the H1 domains. No functions are known for the H1 and H2 domains. Here we addressed which domains and sequence motifs in KCTD proteins regulate desensitization of the receptor response. We found that the H1 domains in KCTD12 and 12b mediate desensitization through a particular sequence motif, T/NFLEQ, which is not present in the H1 domains of KCTD8 and 16. In addition, the H2 domains in KCTD8 and 16 inhibit desensitization when expressed C-terminal to the H1 domains but not when expressed as a separate protein in trans. Intriguingly, the inhibitory effect of the H2 domain is sequence-independent, suggesting that the H2 domain sterically hinders desensitization by the H1 domain. Evolutionary analysis supports that KCTD12 and 12b evolved desensitizing properties by liberating their H1 domains from antagonistic H2 domains and acquisition of the T/NFLEQ motif.
... Therefore, it is possible that presynaptic GABA B receptors are less sensitive to prolonged ligand treatment. Other research has demonstrated that GABA B receptors do not internalize in response to agonist treatment (Fairfax et al., 2004;Mutneja et al., 2005;Perroy et al., 2003), but that agonist treatment does produce a decrease in cell surface-expressed receptors (Fairfax et al., 2004). Further, it would be interesting to determine if the total protein level differences observed translate to functional or even membrane-expressed differences in GABA B receptors. ...
The investigation of GABAergic systems in learning and extinction has principally focused on ionotropic GABA(A) receptors. Less well characterized is the metabotropic GABA(B) receptor, which when activated, induces a more sustained inhibitory effect and has been implicated in regulating oscillatory activity. Few studies have been carried out utilizing GABA(B) ligands in learning, and investigations of GABA(B) in extinction have primarily focused on interactions with drugs of abuse. The current study examined changes in GABA(B) receptor function using the GABA(B) agonist baclofen (2mg/mL) or the GABA(B) antagonist phaclofen (0.3mg/mL) on trace cued and contextual fear conditioning and extinction. The compounds were either administered during training and throughout extinction in Experiment 1, or starting 24h after training and throughout extinction in Experiment 2. All drugs were administered 1mL/kg via intraperitoneal injection. These studies demonstrated that the administration of baclofen during training and extinction trials impaired animals' ability to extinguish the fear association to the CS, whereas the animals that were administered baclofen starting 24h after training (Experiment 2) did display some extinction. Further, contextual fear extinction was impaired by baclofen in both experiments. Tissue analyses suggest the cued fear extinction deficit may be related to changes in the GABA(B2) receptor subunit in the amygdala. The data in the present investigation demonstrate that GABA(B) receptors play an important role in trace cued and contextual fear extinction, and may function differently than GABA(A) receptors in learning, memory, and extinction.
... To explain these discrepancies, it was proposed that receptor phosphorylation 23 , regulator of G protein signalling proteins (RGS proteins) 24,25 or distinct subunit combinations of downstream effector channels 13 modify receptor responses. These factors influenced GABA B R responses in various experimental systems, but it remained unclear whether they were responsible for modifying native GABA B R responses. ...
... A recent report showed that RGS7-Gβ5 complexes bind to GIRKs and accelerate GABA B R-mediated K + current kinetics 47 . Likewise, RGS4 protein expressed in HEK-293T cells desensitizes the K + current response 25 . RGS proteins promote entry into the G protein cycle and augment the GTPase activity of Gα. ...
... RGS proteins promote entry into the G protein cycle and augment the GTPase activity of Gα. This increase in GTPase activity not only accelerates activation kinetics but also produces fast K + current desensitization 25,48 . The effects of RGS proteins on K + current kinetics are thus similar to those that are observed with KCTD12 and KCTD12b. ...
GABA(B) receptors (GABA(B)Rs) are G protein-coupled receptors for GABA, the main inhibitory neurotransmitter in the CNS. In the past 5 years, notable advances have been made in our understanding of the molecular composition of these receptors. GABA(B)Rs are now known to comprise principal and auxiliary subunits that influence receptor properties in distinct ways. The principal subunits regulate the surface expression and the axonal versus dendritic distribution of these receptors, whereas the auxiliary subunits determine agonist potency and the kinetics of the receptor response. This Review summarizes current knowledge on how the subunit composition of GABA(B)Rs affects the distribution of these receptors, neuronal processes and higher brain functions.
