RESUMO
Ion channels play a pivotal role in regulating cellular excitability and signal transduction processes. Among the various ion channels, G-protein-coupled inwardly rectifying potassium (GIRK) channels serve as key mediators of neurotransmission and cellular responses to extracellular signals. GIRK channels are members of the larger family of inwardly-rectifying potassium (Kir) channels. Typically, GIRK channels are activated via the direct binding of G-protein ßγ subunits upon the activation of G-protein-coupled receptors (GPCRs). GIRK channel activation requires the presence of the lipid signaling molecule, phosphatidylinositol 4,5-bisphosphate (PIP2). GIRK channels are also modulated by endogenous proteins and other molecules, including RGS proteins, cholesterol, and SNX27 as well as exogenous compounds, such as alcohol. In the last decade or so, several groups have developed novel drugs and small molecules, such as ML297, GAT1508 and GiGA1, that activate GIRK channels in a G-protein independent manner. Here, we aim to provide a comprehensive overview focusing on the direct modulation of GIRK channels by G-proteins, PIP2, cholesterol, and novel modulatory compounds. These studies offer valuable insights into the underlying molecular mechanisms of channel function, and have potential implications for both basic research and therapeutic development.
RESUMO
G protein-gated inwardly rectifying potassium (GIRK) channels are essential regulators of cell excitability in the brain. While they are implicated in a variety of neurological diseases in both human and animal model studies, their therapeutic potential has been largely untapped. Here, we review recent advances in the development of small molecule compounds that specifically modulate GIRK channels and compare them with first-generation compounds that exhibit off-target activity. We describe the method of discovery of these small molecule modulators, their chemical features, and their effects in vivo. These studies provide a promising outlook on the future development of subunit-specific GIRK modulators to regulate neuronal excitability in a brain region-specific manner.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Neurônios , Animais , Encéfalo/metabolismo , Proteínas de Ligação ao GTP , Humanos , Neurônios/metabolismoRESUMO
G-protein-gated inwardly rectifying potassium (GIRK) channels are important for determining neuronal excitability. In addition to G proteins, GIRK channels are potentiated by membrane cholesterol, which is elevated in the brains of people with neurodegenerative diseases such as Alzheimer's dementia and Parkinson's disease. The structural mechanism of cholesterol modulation of GIRK channels is not well understood. In this study, we present cryo- electron microscopy (cryoEM) structures of GIRK2 in the presence and absence of the cholesterol analog cholesteryl hemisuccinate (CHS) and phosphatidylinositol 4,5-bisphosphate (PIP2). The structures reveal that CHS binds near PIP2 in lipid-facing hydrophobic pockets of the transmembrane domain. Our structural analysis suggests that CHS stabilizes PIP2 interaction with the channel and promotes engagement of the cytoplasmic domain onto the transmembrane region. Mutagenesis of one of the CHS binding pockets eliminates cholesterol-dependent potentiation of GIRK2. Elucidating the structural mechanisms underlying cholesterol modulation of GIRK2 channels could facilitate the development of therapeutics for treating neurological diseases. VIDEO ABSTRACT.
Assuntos
Colesterol/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Microscopia Crioeletrônica/métodos , Proteínas de Ligação ao GTP/metabolismo , Ativação do Canal Iônico/fisiologia , Camundongos , Ligação Proteica , SaccharomycetalesRESUMO
G-protein-gated inwardly rectifying K+ (GIRK) channels are essential effectors of inhibitory neurotransmission in the brain. GIRK channels have been implicated in diseases with abnormal neuronal excitability, including epilepsy and addiction. GIRK channels are tetramers composed of either the same subunit (e.g., homotetramers) or different subunits (e.g., heterotetramers). Compounds that specifically target subsets of GIRK channels in vivo are lacking. Previous studies have shown that alcohol directly activates GIRK channels through a hydrophobic pocket located in the cytoplasmic domain of the channel. Here, we report the identification and functional characterization of a GIRK1-selective activator, termed GiGA1, that targets the alcohol pocket. GiGA1 activates GIRK1/GIRK2 both in vitro and in vivo and, in turn, mitigates the effects of a convulsant in an acute epilepsy mouse model. These results shed light on the structure-based development of subunit-specific GIRK modulators that could provide potential treatments for brain disorders.
Assuntos
Encéfalo/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Animais , Epilepsia/metabolismo , Ativação do Canal Iônico/fisiologia , Camundongos KnockoutRESUMO
Mutations in the sodium channel genes SCN1A and SCN2A have been identified in monogenic childhood epilepsies, but SCN3A has not previously been investigated as a candidate gene for epilepsy. We screened a consecutive cohort of 18 children with cryptogenic partial epilepsy that was classified as pharmacoresistant because of nonresponse to carbamazepine or oxcarbazepine, antiepileptic drugs that bind sodium channels. The novel coding variant SCN3A-K354Q was identified in one patient and was not present in 295 neurological normal controls. Twelve novel SNPs were also detected. K354Q substitutes glutamine for an evolutionarily conserved lysine residue in the pore domain of SCN3A. Functional analysis of this mutation in the backbone of the closely related gene SCN5A demonstrated an increase in persistent current that is similar in magnitude to epileptogenic mutations of SCN1A and SCN2A. This observation of a potentially pathogenic mutation of SCN3A (Nav1.3) indicates that this gene should be further evaluated for its contribution to childhood epilepsy.
Assuntos
Química Encefálica/genética , Encéfalo/metabolismo , Epilepsia Parcial Complexa/genética , Epilepsia Parcial Complexa/metabolismo , Mutação/genética , Canais de Sódio/genética , Fatores Etários , Substituição de Aminoácidos/genética , Anticonvulsivantes/farmacologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiopatologia , Linhagem Celular , Pré-Escolar , Estudos de Coortes , Sequência Conservada/genética , Análise Mutacional de DNA , Resistência a Medicamentos/genética , Epilepsia Parcial Complexa/fisiopatologia , Frequência do Gene , Predisposição Genética para Doença/genética , Testes Genéticos , Humanos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.3 , Polimorfismo de Nucleotídeo Único/genética , Estrutura Terciária de Proteína/genética , Canais de Sódio/químicaRESUMO
Activation of G protein-gated inwardly rectifying potassium (GIRK) channels leads to a hyperpolarization of the neuron's membrane potential, providing an important component of inhibition in the brain. In addition to the canonical G protein-activation pathway, GIRK channels are activated by small molecules but less is known about the underlying gating mechanisms. One drawback to previous studies has been the inability to control intrinsic and extrinsic factors. Here we used a reconstitution strategy with highly purified mammalian GIRK2 channels incorporated into liposomes and demonstrate that cholesterol or intoxicating concentrations of ethanol, i.e., >20 mM, each activate GIRK2 channels directly, in the absence of G proteins. Notably, both activators require the membrane phospholipid PIP2 but appear to interact independently with different regions of the channel. Elucidating the mechanisms underlying G protein-independent pathways of activating GIRK channels provides a unique strategy for developing new types of neuronal excitability modulators.
Assuntos
Colesterol/farmacologia , Etanol/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Ativação Transcricional , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Proteínas de Ligação ao GTP/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lipossomos/metabolismo , Camundongos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Ligação ProteicaRESUMO
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 Gßγ subunits and ethanol.
RESUMO
Electrical activity in nerve, skeletal muscle, and heart requires finely tuned activity of voltage-gated Na+ channels that open and then enter a nonconducting inactivated state upon depolarization. Inactivation occurs when the gate, the cytoplasmic loop linking domains III and IV of the alpha subunit, occludes the open pore. Subtle destabilization of inactivation by mutation is causally associated with diverse human disease. Here we show for the first time that the inactivation gate is a molecular complex consisting of the III-IV loop and the COOH terminus (C-T), which is necessary to stabilize the closed gate and minimize channel reopening. When this interaction is disrupted by mutation, inactivation is destabilized allowing a small, but important, fraction of channels to reopen, conduct inward current, and delay cellular repolarization. Thus, our results demonstrate for the first time that physiologically crucial stabilization of inactivation of the Na+ channel requires complex interactions of intracellular structures and indicate a novel structural role of the C-T domain in this process.
Assuntos
Ativação do Canal Iônico/fisiologia , Fragmentos de Peptídeos/fisiologia , Canais de Sódio/fisiologia , Sequência de Aminoácidos , Linhagem Celular , Humanos , Ativação do Canal Iônico/genética , Substâncias Macromoleculares , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/fisiologia , Canais de Sódio/síntese química , Canais de Sódio/genéticaRESUMO
G protein-gated inwardly rectifying potassium (GIRK; Kir3) channels, which are members of the large family of inwardly rectifying potassium channels (Kir1-Kir7), regulate excitability in the heart and brain. GIRK channels are activated following stimulation of G protein-coupled receptors that couple to the G(i/o) (pertussis toxin-sensitive) G proteins. GIRK channels, like all other Kir channels, possess an extrinsic mechanism of inward rectification involving intracellular Mg(2+) and polyamines that occlude the conduction pathway at membrane potentials positive to E(K). In the past 17 years, more than 20 high-resolution atomic structures containing GIRK channel cytoplasmic domains and transmembrane domains have been solved. These structures have provided valuable insights into the structural determinants of many of the properties common to all inward rectifiers, such as permeation and rectification, as well as revealing the structural bases for GIRK channel gating. In this chapter, we describe advances in our understanding of GIRK channel function based on recent high-resolution atomic structures of inwardly rectifying K(+) channels discussed in the context of classical structure-function experiments.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potenciais da Membrana/fisiologia , Animais , Humanos , Potássio/metabolismoRESUMO
Local anesthetics (LAs) block Na(+) channels with a higher affinity for the fast or slow inactivated state of the channel. Their binding to the channel may stabilize fast inactivation or induce slow inactivation. We examined the role of the LA binding sites on domain IV, S6 (IVS6) of Na(+) channels in fast and slow inactivation by studying the gating properties of the mutants on IVS6 affecting LA binding. Mutation of the putative LA binding site, F1579C, inhibited fast and slow inactivation. Mutations of another putative LA binding site, Y1586C, and IVS6 residue involved in LA access and binding, I1575C, both enhanced fast and slow inactivation. None of the mutations affected channel activation. These results suggest that the LA binding site on IVS6 is involved in slow inactivation as well as fast inactivation, and these two gatings are coupled at the binding site.
Assuntos
Anestésicos Locais/metabolismo , Canais de Sódio/fisiologia , Animais , Sítios de Ligação , Linhagem Celular , Humanos , Ativação do Canal Iônico , Mutação , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Subunidades Proteicas , Ratos , Canais de Sódio/genética , Canais de Sódio/metabolismo , TransfecçãoRESUMO
The cardiac voltage-gated sodium channel (Na(V)1.5) underlies impulse conduction in the heart, and its depolarization-induced inactivation is essential in control of the duration of the QT interval of the electrocardiogram. Perturbation of Na(V)1.5 inactivation by drugs or inherited mutation can underlie and trigger cardiac arrhythmias. The carboxy terminus has an important role in channel inactivation, but complete structural information on its predicted structural domain is unknown. Here we measure interactions between the functionally critical distal carboxy terminus α-helix (H6) and the proximal structured EF-hand motif using transition-metal ion fluorescence resonance energy transfer. We measure distances at three loci along H6 relative to an intrinsic tryptophan, demonstrating the proximal-distal interaction in a contiguous carboxy terminus polypeptide. Using these data together with the existing Na(V)1.5 carboxy terminus nuclear magnetic resonance structure, we construct a model of the predicted structured region of the carboxy terminus. An arrhythmia-associated H6 mutant that impairs inactivation decreases fluorescence resonance energy transfer, indicating destabilization of the distal-proximal intramolecular interaction. These data provide a structural correlation to the pathological phenotype of the mutant channel.
Assuntos
Sistema de Condução Cardíaco/fisiologia , Síndrome do QT Longo/genética , Canais de Sódio/química , Canais de Sódio/genética , Motivos de Aminoácidos , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Coração/fisiologia , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação , Miocárdio/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5 , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Canais de Sódio/metabolismoRESUMO
Inherited mutations of SCN5A, the gene that encodes Na(V)1.5, the alpha subunit of the principle voltage-gated Na(+) channel in the heart, cause congenital Long QT Syndrome variant 3 (LQT-3) by perturbation of channel inactivation. LQT-3 mutations induce small, but aberrant, inward current that prolongs the ventricular action potential and subjects mutation carriers to arrhythmia risk dictated in part by the biophysical consequences of the mutations. Most previously investigated LQT-3 mutations are associated with increased arrhythmia risk during rest or sleep. Here we report a novel LQT-3 mutation discovered in a pediatric proband diagnosed with LQTS but who experienced cardiac events during periods of mild exercise as well as rest. The mutation, which changes a single amino acid (S1904L) in the Na(V)1.5 carboxy terminal domain, disrupts the channel inactivation gate complex and promotes late Na(+) channel currents, not by promoting a bursting mode of gating, but by increasing the propensity of the channel to reopen during prolonged depolarization. Incorporating a modified version of the Markov model of the Na(V)1.5 channel into a mathematical model of the human ventricular action potential predicts that the biophysical consequences of the S1904L mutation result in action potential prolongation that is seen for all heart rates but, in contrast to other previously-investigated LQT-3 mutant channels, is most pronounced at fast rates resulting in a drastic reduction in the cells ability to adapt APD to heart rate.
Assuntos
Arritmias Cardíacas/genética , Sistema de Condução Cardíaco/metabolismo , Ativação do Canal Iônico/genética , Síndrome do QT Longo/genética , Proteínas Musculares/genética , Mutação , Canais de Sódio/genética , Sódio/metabolismo , Potenciais de Ação , Substituição de Aminoácidos , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Criança , Eletrocardiografia , Predisposição Genética para Doença , Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca , Humanos , Cinética , Síndrome do QT Longo/complicações , Síndrome do QT Longo/metabolismo , Síndrome do QT Longo/fisiopatologia , Masculino , Cadeias de Markov , Modelos Cardiovasculares , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5 , Linhagem , Fenótipo , Conformação Proteica , Canais de Sódio/química , Canais de Sódio/metabolismoRESUMO
Perturbation of sodium channel inactivation, a finely tuned process that critically regulates the flow of sodium ions into excitable cells, is a common functional consequence of inherited mutations associated with epilepsy, skeletal muscle disease, autism, and cardiac arrhythmias. Understanding the structural basis of inactivation is key to understanding these disorders. Here we identify a novel role for a structural motif in the COOH terminus of the heart NaV1.5 sodium channel in determining channel inactivation. Structural modeling predicts an interhelical hydrophobic interface between paired EF hands in the proximal region of the NaV1.5 COOH terminus. The predicted interface is conserved among almost all EF hand-containing proteins and is the locus of a number of disease-associated mutations. Using the structural model as a guide, we provide biochemical and biophysical evidence that the structural integrity of this interface is necessary for proper Na+ channel inactivation gating. We thus demonstrate a novel role of the sodium channel COOH terminus structure in the control of channel inactivation and in pathologies caused by inherited mutations that disrupt it.
Assuntos
Arritmias Cardíacas/genética , Proteínas Musculares/química , Proteínas Musculares/fisiologia , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/fisiologia , Canais de Sódio/química , Canais de Sódio/fisiologia , Acrilamida/química , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Arritmias Cardíacas/congênito , Arritmias Cardíacas/metabolismo , Linhagem Celular , Biologia Computacional/métodos , Motivos EF Hand , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico/genética , Dados de Sequência Molecular , Proteínas Musculares/antagonistas & inibidores , Proteínas Musculares/genética , Canal de Sódio Disparado por Voltagem NAV1.5 , Fragmentos de Peptídeos/antagonistas & inibidores , Fragmentos de Peptídeos/genética , Estrutura Secundária de Proteína , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/fisiologia , Canais de Sódio/genética , Espectrometria de Fluorescência , Termodinâmica , Triptofano/químicaRESUMO
The role of the outermost three charged residues of Domain IV/S4 in controlling gating of Ca(v)3.2 was investigated using single substitutions of each arginine with glutamine, cysteine, histidine, and lysine in a Flp-In-293 cell line, in which expression levels could be compared. Channel density, based on gating charge measurements, was ~125,000 channels/cell (10 fC/pF), except for R2Q and R3C, which expressed at lower levels. Channels substituted at Arg-1715 (R1C, R1Q, R1H) demonstrated such modest changes that a role in voltage sensing could not be determined. Arg-1718 (R2) made a contribution to activation voltage sensing, and the channel was sensitive to the geometry of side-chain substitutions at this position. Arg-1721 (R3) substitutions produced complex kinetic changes that together suggested that geometry made a larger contribution than charge. Current decay at positive potentials (O-->I) exponentially approached a constant value for all mutants except R2K channels, which were biphasically dependent on potential. R2K channels also displayed slowed deactivation with reduced voltage dependence despite near control values for conductance. Voltage-dependent accessibility of R to C mutants, evaluated with intracellularly and extracellularly applied methanthiosulfonate (MTS) reagents, showed that both R2 and R3 were exposed only when cells were depolarized, although it was not necessary for channels to open. Together, the data indicate that Domain IV/S4 is an activation domain and is not involved in inactivation from the open state.
Assuntos
Arginina/fisiologia , Canais de Cálcio Tipo T/fisiologia , Ativação do Canal Iônico/fisiologia , Substituição de Aminoácidos/genética , Arginina/genética , Sítios de Ligação/genética , Canais de Cálcio Tipo T/genética , Linhagem Celular , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/farmacologia , Expressão Gênica/genética , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Mesilatos/farmacologia , Técnicas de Patch-Clamp , Reagentes de Sulfidrila/farmacologia , TransfecçãoRESUMO
A precise balance of ionic currents underlies normal cardiac excitation and relaxation. Disruption of this equilibrium by genetic defects, polymorphisms, therapeutic intervention, and structural abnormalities can cause arrhythmogenic phenotypes leading to syncope, seizures, and sudden cardiac death. Congenital defects result in an unpredictable expression of phenotypes with variable penetrance, even within single families. Additionally, phenotypically opposite and overlapping cardiac arrhythmogenic syndromes can even stem from the same mutation. Accordingly, the relationship between genetic mutations and clinical syndromes is becoming increasingly complex.
Assuntos
Arritmias Cardíacas/diagnóstico , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/genética , Humanos , Fenótipo , Polimorfismo Genético , Sódio/metabolismoRESUMO
The inner pore of the voltage-gated Na+ channel is predicted by the structure of bacterial potassium channels to be lined with the four S6 alpha-helical segments. Our previously published model of the closed pore based on the KcsA structure, and our new model of the open pore based on the MthK structure predict which residues in the mid-portion of S6 face the pore. We produced cysteine mutants of the mid-portion of domain IV-S6 (Ile-1575-Leu-1591) in NaV 1.4 and tested their accessibility to intracellularly and extracellularly placed positively charged methanethiosulfonate (MTS) reagents. We found that only two mutants, F1579C and V1583C, were accessible to both outside and inside 2-(aminoethyl)-methanethiosulfonate hydrobromide (MTSEA) Further study of those mutants showed that efficient closure of the fast inactivation gate prevented block by inside [2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET) at slow stimulation rates. When fast inactivation was inhibited by exposure to anthropleurin B (ApB), increasing channel open time, both mutants were blocked by inside MTSET at a rate that depended on the amount of time the channel was open. Consistent with the fast inactivation gate limiting access to the pore, in the absence of ApB, inside MTSET produced block when the cells were stimulated at 5 or 20 Hz. We therefore suggest that the middle of IV-S6 is an alpha-helix, and we propose a model of the open channel, based on MthK, in which Phe-1579 and Val-1583 face the pore.