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1.
J Biol Chem ; 294(16): 6506-6521, 2019 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-30808709

RESUMEN

Ether-a-go-go family (EAG) channels play a major role in many physiological processes in humans, including cardiac repolarization and cell proliferation. Cryo-EM structures of two of them, KV10.1 and human ether-a-go-go-related gene (hERG or KV11.1), have revealed an original nondomain-swapped structure, suggesting that the mechanism of voltage-dependent gating of these two channels is quite different from the classical mechanical-lever model. Molecular aspects of hERG voltage-gating have been extensively studied, indicating that the S4-S5 linker (S4-S5L) acts as a ligand binding to the S6 gate (S6 C-terminal part, S6T) and stabilizes it in a closed state. Moreover, the N-terminal extremity of the channel, called N-Cap, has been suggested to interact with S4-S5L to modulate channel voltage-dependent gating, as N-Cap deletion drastically accelerates hERG channel deactivation. In this study, using COS-7 cells, site-directed mutagenesis, electrophysiological measurements, and immunofluorescence confocal microscopy, we addressed whether these two major mechanisms of voltage-dependent gating are conserved in KV10.2 channels. Using cysteine bridges and S4-S5L-mimicking peptides, we show that the ligand/receptor model is conserved in KV10.2, suggesting that this model is a hallmark of EAG channels. Truncation of the N-Cap domain, Per-Arnt-Sim (PAS) domain, or both in KV10.2 abolished the current and altered channel trafficking to the membrane, unlike for the hERG channel in which N-Cap and PAS domain truncations mainly affected channel deactivation. Our results suggest that EAG channels function via a conserved ligand/receptor model of voltage gating, but that the N-Cap and PAS domains have different roles in these channels.


Asunto(s)
Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Activación del Canal Iónico , Modelos Moleculares , Animales , Células COS , Chlorocebus aethiops , Canal de Potasio ERG1/química , Canal de Potasio ERG1/genética , Canal de Potasio ERG1/metabolismo , Canales de Potasio Éter-A-Go-Go/química , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Humanos , Péptidos/química , Dominios Proteicos
2.
J Mol Cell Cardiol ; 99: 1-13, 2016 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-27590098

RESUMEN

Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K+ channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecular mechanism for QT prolongation in HIV-infected patients.


Asunto(s)
Potenciales de Acción , Fosfatidilinositol 4,5-Difosfato/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Animales , Células COS , Diferenciación Celular , Línea Celular , Canal de Potasio ERG1/metabolismo , Fenómenos Electrofisiológicos , Expresión Génica , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Canal de Potasio KCNQ1/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transfección , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética
3.
Sci Adv ; 8(8): eabk0052, 2022 Feb 25.
Artículo en Inglés | MEDLINE | ID: mdl-35196083

RESUMEN

FLNC truncating mutations (FLNCtv) are prevalent causes of inherited dilated cardiomyopathy (DCM), with a high risk of developing arrhythmogenic cardiomyopathy. We investigated the molecular mechanisms of mutant FLNC in the pathogenesis of arrhythmogenic DCM (a-DCM) using patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). We demonstrated that iPSC-CMs from two patients with different FLNCtv mutations displayed arrhythmias and impaired contraction. FLNC ablation induced a similar phenotype, suggesting that FLNCtv are loss-of-function mutations. Coimmunoprecipitation and proteomic analysis identified ß-catenin (CTNNB1) as a downstream target. FLNC deficiency induced nuclear translocation of CTNNB1 and subsequently activated the platelet-derived growth factor receptor alpha (PDGFRA) pathway, which were also observed in human hearts with a-DCM and FLNCtv. Treatment with the PDGFRA inhibitor, crenolanib, improved contractile function of patient iPSC-CMs. Collectively, our findings suggest that PDGFRA signaling is implicated in the pathogenesis, and inhibition of this pathway is a potential therapeutic strategy in FLNC-related cardiomyopathies.

4.
Sci Rep ; 10(1): 5852, 2020 04 03.
Artículo en Inglés | MEDLINE | ID: mdl-32246066

RESUMEN

Prokaryotic NaV channels are tetramers and eukaryotic NaV channels consist of a single subunit containing four domains. Each monomer/domain contains six transmembrane segments (S1-S6), S1-S4 being the voltage-sensor domain and S5-S6 the pore domain. A crystal structure of NaVMs, a prokaryotic NaV channel, suggests that the S4-S5 linker (S4-S5L) interacts with the C-terminus of S6 (S6T) to stabilize the gate in the open state. However, in several voltage-gated potassium channels, using specific S4-S5L-mimicking peptides, we previously demonstrated that S4-S5L/S6T interaction stabilizes the gate in the closed state. Here, we used the same strategy on another prokaryotic NaV channel, NaVSp1, to test whether equivalent peptides stabilize the channel in the open or closed state. A NaVSp1-specific S4-S5L peptide, containing the residues supposed to interact with S6T according to the NaVMs structure, induced both an increase in NaVSp1 current density and a negative shift in the activation curve, consistent with S4-S5L stabilizing the open state. Using this approach on a human NaV channel, hNaV1.4, and testing 12 hNaV1.4 S4-S5L peptides, we identified four activating S4-S5L peptides. These results suggest that, in eukaryotic NaV channels, the S4-S5L of DI, DII and DIII domains allosterically modulate the activation gate and stabilize its open state.


Asunto(s)
Péptidos/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Animales , Sitios de Unión , Células COS , Chlorocebus aethiops , Fenómenos Electrofisiológicos , Canales de Potasio con Entrada de Voltaje/metabolismo , Alineación de Secuencia , Relación Estructura-Actividad , Regulación hacia Arriba , Canales de Sodio Activados por Voltaje/genética
5.
Methods Mol Biol ; 1684: 305-319, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29058201

RESUMEN

Voltage-gated potassium channels play pivotal roles in excitable and non-excitable cells. For many decades, structural properties and molecular mechanisms of these channels were inferred from functional observations. At the turn of the twenty-first century, structural biology revealed major aspects in the structural basis of ion channel organization, permeation, and gating. Among the available tools, homology modeling associated with low resolution microscopy helps in delineating the different structural elements of voltage-gated channels. Here, we describe in detail the methodology of homology modeling, using the 3D structure of the Kv2.1ΔCTA ion channel as a reference.


Asunto(s)
Biología Computacional/métodos , Canales de Potasio Shab/química , Microscopía Electrónica , Modelos Moleculares , Conformación Proteica , Homología Estructural de Proteína
6.
Sci Rep ; 7(1): 113, 2017 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-28273916

RESUMEN

Delayed-rectifier potassium channels (hERG and KCNQ1) play a major role in cardiac repolarization. These channels are formed by a tetrameric pore (S5-S6) surrounded by four voltage sensor domains (S1-S4). Coupling between voltage sensor domains and the pore activation gate is critical for channel voltage-dependence. However, molecular mechanisms remain elusive. Herein, we demonstrate that covalently binding, through a disulfide bridge, a peptide mimicking the S4-S5 linker (S4-S5L) to the channel S6 C-terminus (S6T) completely inhibits hERG. This shows that channel S4-S5L is sufficient to stabilize the pore activation gate in its closed state. Conversely, covalently binding a peptide mimicking S6T to the channel S4-S5L prevents its inhibiting effect and renders the channel almost completely voltage-independent. This shows that the channel S4-S5L is necessary to stabilize the activation gate in its closed state. Altogether, our results provide chemical evidence that S4-S5L acts as a voltage-controlled ligand that binds S6T to lock the channel in a closed state, elucidating the coupling between voltage sensors and the gate in delayed rectifier potassium channels and potentially other voltage-gated channels.

7.
Biochimie ; 141: 80-85, 2017 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-28549840

RESUMEN

Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator (Tat) viral protein that is required for efficient HIV gene transcription. Extracellular Tat is able to enter uninfected cells. We recently reported that internalized Tat escapes endosomes to reach the cytosol and is then recruited to the plasma membrane by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). As a consequence, Tat strongly impairs different critical cellular functions in several cell types. Here we will review recent evidences showing that Tat, by affecting the interaction of key cellular effectors with PtdIns(4,5)P2, blocks exocytosis from neuroendocrine cells, perturbs the synaptic vesicle exo-endocytosis cycle, prevents efficient phagocytosis by macrophages, and alters potassium channel activity in cardiac cells. Potential mechanistic aspects of Tat effects on these cellular processes will be discussed.


Asunto(s)
Endosomas/metabolismo , VIH-1/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Endosomas/virología , Exocitosis , Humanos
8.
Front Pharmacol ; 6: 314, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26834636

RESUMEN

Mutations in Nav1.4 and Nav1.5 α-subunits have been associated with muscular and cardiac channelopathies, respectively. Despite intense research on the structure and function of these channels, a lot of information is still missing to delineate the various physiological and pathophysiological processes underlying their activity at the molecular level. Nav1.4 and Nav1.5 sequences are similar, suggesting structural and functional homologies between the two orthologous channels. This also suggests that any characteristics described for one channel subunit may shed light on the properties of the counterpart channel subunit. In this review article, after a brief clinical description of the muscular and cardiac channelopathies related to Nav1.4 and Nav1.5 mutations, respectively, we compare the knowledge accumulated in different aspects of the expression and function of Nav1.4 and Nav1.5 α-subunits: the regulation of the two encoding genes (SCN4A and SCN5A), the associated/regulatory proteins and at last, the functional effect of the same missense mutations detected in Nav1.4 and Nav1.5. First, it appears that more is known on Nav1.5 expression and accessory proteins. Because of the high homologies of Nav1.5 binding sites and equivalent Nav1.4 sites, Nav1.5-related results may guide future investigations on Nav1.4. Second, the analysis of the same missense mutations in Nav1.4 and Nav1.5 revealed intriguing similarities regarding their effects on membrane excitability and alteration in channel biophysics. We believe that such comparison may bring new cues to the physiopathology of cardiac and muscular diseases.

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