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1.
Nat Commun ; 15(1): 3849, 2024 May 08.
Artículo en Inglés | MEDLINE | ID: mdl-38719838

RESUMEN

Highly selective for K+ at neutral pH, the TWIK1 channel becomes permeable to Na+ upon acidification. Using molecular dynamics simulations, we identify a network of residues involved in this unique property. Between the open and closed states previously observed by electron microscopy, molecular dynamics simulations show that the channel undergoes conformational changes between pH 7.5-6 involving residues His122, Glu235, Lys246 and Phe109. A complex network of interactions surrounding the selectivity filter at high pH transforms into a simple set of stronger interactions at low pH. In particular, His122 protonated by acidification moves away from Lys246 and engages in a salt bridge with Glu235. In addition, stacking interactions between Phe109 and His122, which stabilize the selectivity filter in its K+-selective state at high pH, disappear upon acidification. This leads to dissociation of the Phe109 aromatic side chain from this network, resulting in the Na+-permeable conformation of the channel.

2.
J Biol Chem ; 298(10): 102447, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-36063992

RESUMEN

Two-pore domain K+ channels (K2P channels), active as dimers, produce inhibitory currents regulated by a variety of stimuli. Among them, TWIK1-related alkalinization-activated K+ channel 1 (TALK1), TWIK1-related alkalinization-activated K+ channel 2 (TALK2), and TWIK1-related acid-sensitive K+ channel 2 (TASK2) form a subfamily of structurally related K2P channels stimulated by extracellular alkalosis. The human genes encoding these proteins are clustered at chromosomal region 6p21 and coexpressed in multiple tissues, including the pancreas. The question whether these channels form functional heteromers remained open. By analyzing single-cell transcriptomic data, we show that these channels are coexpressed in insulin-secreting pancreatic ß-cells. Using in situ proximity ligation assay and electrophysiology, we show that they form functional heterodimers both upon heterologous expression and under native conditions in human pancreatic ß-cells. We demonstrate that heteromerization of TALK2 with TALK1 or with TASK2 endows TALK2 with sensitivity to extracellular alkalosis in the physiological range. We further show that the association of TASK2 with TALK1 and TALK2 increases their unitary conductance. These results provide a new example of heteromerization in the K2P channel family expanding the range of the potential physiological and pathophysiological roles of TALK1/TALK2/TASK2 channels, not only in insulin-secreting cells but also in the many other tissues in which they are coexpressed.


Asunto(s)
Alcalosis , Células Secretoras de Insulina , Canales de Potasio de Dominio Poro en Tándem , Humanos , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Células Secretoras de Insulina/metabolismo , Concentración de Iones de Hidrógeno , Insulina/metabolismo , Potasio/metabolismo
3.
Front Pharmacol ; 12: 795920, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34867429

RESUMEN

[This corrects the article DOI: 10.3389/fphar.2021.755826.].

4.
Cell Rep ; 37(9): 110070, 2021 11 30.
Artículo en Inglés | MEDLINE | ID: mdl-34852225

RESUMEN

Mechanoelectrical transduction is mediated by the opening of different types of force-sensitive ion channels, including Piezo1/2 and the TREK/TRAAK K2P channels. Piezo1 curves the membrane locally into an inverted dome that reversibly flattens in response to force application. Moreover, Piezo1 forms numerous preferential interactions with various membrane lipids, including cholesterol. Whether this structural architecture influences the functionality of neighboring membrane proteins is unknown. Here, we show that Piezo1/2 increase TREK/TRAAK current amplitude, slow down activation/deactivation, and remove inactivation upon mechanical stimulation. These findings are consistent with a mechanism whereby Piezo1/2 cause a local depletion of membrane cholesterol associated with a prestress of TREK/TRAAK channels. This regulation occurs in mouse fibroblasts between endogenous Piezo1 and TREK-1/2, both channel types acting in concert to delay wound healing. In conclusion, we demonstrate a community effect between different structural and functional classes of mechanosensitive ion channels.


Asunto(s)
Activación del Canal Iónico , Canales Iónicos/fisiología , Mecanotransducción Celular , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Colesterol/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Encía/citología , Encía/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canales de Potasio de Dominio Poro en Tándem/genética
5.
J Physiol ; 599(4): 1041-1055, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-33347640

RESUMEN

Potassium channels form the largest family of ion channels with more than 80 members involved in cell excitability and signalling. Most of them exist as homomeric channels, whereas specific conditions are required to obtain heteromeric channels. It is well established that heteromerization of voltage-gated and inward rectifier potassium channels affects their function, increasing the diversity of the native potassium currents. For potassium channels with two pore domains (K2P ), homomerization has long been considered the rule, their polymodal regulation by a wide diversity of physical and chemical stimuli being responsible for the adaptation of the leak potassium currents to cellular needs. This view has recently evolved with the accumulation of evidence of heteromerization between different K2P subunits. Several functional intragroup and intergroup heteromers have recently been identified, which contribute to the functional heterogeneity of this family. K2P heteromerization is involved in the modulation of channel expression and trafficking, promoting functional and signalling diversity. As illustrated in the Abstract Figure, heteromerization of TREK1 and TRAAK provides the cell with more possibilities of regulation. It is becoming increasingly evident that K2P heteromers contribute to important physiological functions including neuronal and cardiac excitability. Since heteromerization also affects the pharmacology of K2P channels, this understanding helps to establish K2P heteromers as new therapeutic targets for physiopathological conditions.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem , Neuronas/metabolismo , Potasio , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Transporte de Proteínas , Transducción de Señal
6.
Cell Rep Methods ; 1(8): None, 2021 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-34977850

RESUMEN

Ligand-gated ion channels (LGICs) are natural biosensors generating electrical signals in response to the binding of specific ligands. Creating de novo LGICs for biosensing applications is technically challenging. We have previously designed modified LGICs by linking G protein-coupled receptors (GPCRs) to the Kir6.2 channel. In this article, we extrapolate these design concepts to other channels with different structures and oligomeric states, namely a tetrameric viral Kcv channel and the dimeric mouse TREK-1 channel. After precise engineering of the linker regions, the two ion channels were successfully regulated by a GPCR fused to their N-terminal domain. Two-electrode voltage-clamp recordings showed that Kcv and mTREK-1 fusions were inhibited and activated by GPCR agonists, respectively, and antagonists abolished both effects. Thus, dissimilar ion channels can be allosterically regulated through their N-terminal domains, suggesting that this is a generalizable approach for ion channel engineering.


Asunto(s)
Técnicas Biosensibles , Canales Iónicos Activados por Ligandos , Animales , Ratones , Receptores Acoplados a Proteínas G/genética , Transducción de Señal , Canales Iónicos Activados por Ligandos/metabolismo , Ligandos
7.
J Biol Chem ; 295(2): 610-618, 2020 01 10.
Artículo en Inglés | MEDLINE | ID: mdl-31806709

RESUMEN

Two-pore domain K+ (K2P) channels have many important physiological functions. However, the functional properties of the TWIK-1 (K2P1.1/KCNK1) K2P channel remain poorly characterized because heterologous expression of this ion channel yields only very low levels of functional activity. Several underlying reasons have been proposed, including TWIK-1 retention in intracellular organelles, inhibition by posttranslational sumoylation, a hydrophobic barrier within the pore, and a low open probability of the selectivity filter (SF) gate. By evaluating these potential mechanisms, we found that the latter dominates the low intrinsic functional activity of TWIK-1. Investigating this further, we observed that the low activity of the SF gate appears to arise from the inefficiency of K+ in stabilizing an active (i.e. conductive) SF conformation. In contrast, other permeant ion species, such as Rb+, NH4+, and Cs+, strongly promoted a pH-dependent activated conformation. Furthermore, many K2P channels are activated by membrane depolarization via an SF-mediated gating mechanism, but we found here that only very strong nonphysiological depolarization produces voltage-dependent activation of heterologously expressed TWIK-1. Remarkably, we also observed that TWIK-1 Rb+ currents are potently inhibited by intracellular K+ (IC50 = 2.8 mm). We conclude that TWIK-1 displays unique SF gating properties among the family of K2P channels. In particular, the apparent instability of the conductive conformation of the TWIK-1 SF in the presence of K+ appears to dominate the low levels of intrinsic functional activity observed when the channel is expressed at the cell surface.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Cationes Monovalentes/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico , Simulación de Dinámica Molecular , Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/química , Conformación Proteica , Conformación Proteica en Hélice alfa , Rubidio/metabolismo , Xenopus
8.
Front Mol Neurosci ; 11: 301, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30233308

RESUMEN

TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions.

9.
J Med Chem ; 60(3): 1076-1088, 2017 02 09.
Artículo en Inglés | MEDLINE | ID: mdl-28051863

RESUMEN

The TWIK-related K+ channel, TREK-1, has recently emerged as an attractive therapeutic target for the development of a novel class of analgesic drugs, suggesting that activation of TREK-1 could result in pain inhibition. Here, we report the synthesis of a series of substituted acrylic acids (1-54) based on our previous work with caffeate esters. The analogues were evaluated for their ability to modulate TREK-1 channel by electrophysiology and for their in vivo antinociceptive activity (acetic acid-induced writhing and hot plate assays), leading to the identification of a series of novel molecules able to activate TREK-1 and displaying potent antinociceptive activity in vivo. Furyl analogue 36 is the most promising of the series.


Asunto(s)
Analgésicos/farmacología , Canales de Potasio de Dominio Poro en Tándem/agonistas , Animales
10.
Proc Natl Acad Sci U S A ; 113(17): E2460-8, 2016 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-27071086

RESUMEN

The venom peptide maurocalcin (MCa) is atypical among toxins because of its ability to rapidly translocate into cells and potently activate the intracellular calcium channel type 1 ryanodine receptor (RyR1). Therefore, MCa is potentially subjected to posttranslational modifications within recipient cells. Here, we report that MCa Thr(26) belongs to a consensus PKA phosphorylation site and can be phosphorylated by PKA both in vitro and after cell penetration in cellulo. Unexpectedly, phosphorylation converts MCa from positive to negative RyR1 allosteric modulator. Thr(26) phosphorylation leads to charge neutralization of Arg(24), a residue crucial for MCa agonist activity. The functional effect of Thr(26) phosphorylation is partially mimicked by aspartyl mutation. This represents the first case, to our knowledge, of both ex situ posttranslational modification and pharmacological reprogramming of a small natural cystine-rich peptide by target cells. So far, phosphorylated MCa is the first specific negative allosteric modulator of RyR1, to our knowledge, and represents a lead compound for further development of phosphatase-resistant analogs.


Asunto(s)
Venenos de Escorpión/metabolismo , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Homeostasis , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Venenos de Escorpión/farmacología
11.
J Mol Cell Cardiol ; 97: 24-35, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27103460

RESUMEN

The two-pore domain potassium (K(+)) channel TWIK-1 (or K2P1.1) contributes to background K(+) conductance in diverse cell types. TWIK-1, encoded by the KCNK1 gene, is present in the human heart with robust expression in the atria, however its physiological significance is unknown. To evaluate the cardiac effects of TWIK-1 deficiency, we studied zebrafish embryos after knockdown of the two KCNK1 orthologues, kcnk1a and kcnk1b. Knockdown of kcnk1a or kcnk1b individually caused bradycardia and atrial dilation (p<0.001 vs. controls), while ventricular stroke volume was preserved. Combined knockdown of both kcnk1a and kcnk1b resulted in a more severe phenotype, which was partially reversed by co-injection of wild-type human KCNK1 mRNA, but not by a dominant negative variant of human KCNK1 mRNA. To determine whether genetic variants in KCNK1 might cause atrial fibrillation (AF), we sequenced protein-coding regions in two independent cohorts of patients (373 subjects) and identified three non-synonymous variants, p.R171H, p.I198M and p.G236S, that were all located in highly conserved amino acid residues. In transfected mammalian cells, zebrafish and wild-type human TWIK-1 channels had a similar cellular distribution with predominant localization in the endosomal compartment. Two-electrode voltage-clamp experiments using Xenopus oocytes showed that both zebrafish and wild-type human TWIK-1 channels produced K(+) currents that are sensitive to external K(+) concentration as well as acidic pH. There were no effects of the three KCNK1 variants on cellular localization, current amplitude or reversal potential at pH7.4 or pH6. Our data indicate that TWIK-1 has a highly conserved role in cardiac function and is required for normal heart rate and atrial morphology. Despite the functional importance of TWIK-1 in the atrium, genetic variation in KCNK1 is not a common primary cause of human AF.


Asunto(s)
Remodelación Atrial/genética , Estudios de Asociación Genética , Atrios Cardíacos/metabolismo , Frecuencia Cardíaca/genética , Canales de Potasio de Dominio Poro en Tándem/genética , Adulto , Anciano , Animales , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Fibrilación Atrial/fisiopatología , Femenino , Expresión Génica , Técnicas de Inactivación de Genes , Variación Genética , Atrios Cardíacos/anatomía & histología , Atrios Cardíacos/patología , Humanos , Masculino , Persona de Mediana Edad , Mutación , Linaje , Canales de Potasio de Dominio Poro en Tándem/deficiencia , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Transporte de Proteínas , Factores de Riesgo , Pez Cebra
12.
Pflugers Arch ; 467(5): 1121-31, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25339226

RESUMEN

Among K2P channels, a few of them turned out to be difficult to express in heterologous systems and were coined "silent subunits". Recent studies have shed light on the mechanisms behind this apparent lack of channel activity at the plasma membrane. For TWIK1 and THIK2 channels, silence is related to a combination of intracellular retention and low intrinsic activity. TWIK1 is constitutively endocytosed from the plasma membrane before being transported to recycling endosomes, whereas THIK2 is restricted to endoplasmic reticulum. These intracellular localizations are related to trafficking signals located in the cytoplasmic parts of the channels. When these motifs are mutated or masked, channels are redistributed at the plasma membrane and produce measurable currents. However, these currents are of modest amplitude. This weak basal activity is due to a hydrophobic barrier in the deep pore that limits water and ions in the conduction pathway. Other silent channels KCNK7, TWIK2, and TASK5 are still under study. Expression and characterization of these K2P channels pave the way for a better understanding of the mechanisms controlling intracellular trafficking of membrane proteins, ion conduction, and channel gating.


Asunto(s)
Membrana Celular/metabolismo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Transporte de Proteínas/fisiología , Animales , Endocitosis/fisiología , Humanos
13.
J Biol Chem ; 289(41): 28202-12, 2014 Oct 10.
Artículo en Inglés | MEDLINE | ID: mdl-25148687

RESUMEN

Despite a high level of sequence homology, tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents, whereas THIK2 is silent. This lack of activity is due to a unique combination of intracellular retention and weak basal activity in the plasma membrane. Here, we designed THIK subunits containing dominant negative mutations (THIK1(DN) and THIK2(DN)). THIK2(DN) mutant inhibits THIK1 currents, whereas THIK1(DN) inhibits an activated form of THIK2 (THIK2-A155P-I158D). In situ proximity ligation assays and Förster/fluorescence resonance energy transfer (FRET) experiments support a physical association between THIK1 and THIK2. Next, we expressed covalent tandems of THIK proteins to obtain expression of pure heterodimers. Td-THIK1-THIK2 (where Td indicates tandem) produces K(+) currents of amplitude similar to Td-THIK1-THIK1 but with a noticeable difference in the current kinetics. Unlike Td-THIK2-THIK2 that is mainly detected in the endoplasmic reticulum, Td-THIK1-THIK2 distributes at the plasma membrane, indicating that THIK1 can mask the endoplasmic reticulum retention/retrieval motif of THIK2. Kinetics and unitary conductance of Td-THIK1-THIK2 are intermediate between THIK1 and THIK2. Altogether, these results show that THIK1 and THIK2 form active heteromeric channels, further expanding the known repertoire of K(+) channels.


Asunto(s)
Cloruros/química , Canales de Potasio de Dominio Poro en Tándem/química , Potasio/química , Secuencia de Aminoácidos , Animales , Cloruros/metabolismo , Perros , Regulación de la Expresión Génica , Células HEK293 , Humanos , Transporte Iónico , Cinética , Células de Riñón Canino Madin Darby , Potenciales de la Membrana , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oocitos/citología , Oocitos/fisiología , Técnicas de Placa-Clamp , Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
14.
Trends Pharmacol Sci ; 35(9): 461-9, 2014 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25023607

RESUMEN

K(+) channels play a key role in regulating cellular excitability. It was thought that the strong K(+) selectivity of these channels was static, only altered by mutations in their selectivity filter, which can cause severe genetic disorders. Recent studies demonstrate that selectivity of K(+) channels can also exhibit dynamic changes. Under acidic conditions or in low extracellular K(+) concentrations, the two-pore domain K(+) channel (K2P) TWIK1 becomes permeable to Na(+), shifting from an inhibitory role to an excitatory role. This phenomenon is responsible for the paradoxical depolarization of human cardiomyocytes in pathological hypokalemia, and therefore may contribute to cardiac arrhythmias. In other cell types, TWIK1 produces depolarizing leak currents under physiological conditions. Dynamic ion selectivity also occurs in other K2P channels. Here we review evidence that dynamic selectivity of K2P channels constitutes a new regulatory mechanism of cellular excitability, whose significance is only now becoming appreciated.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem/fisiología , Animales , Diferenciación Celular , Humanos , Concentración de Iones de Hidrógeno , Hipopotasemia/fisiopatología , Mutación , Miocitos Cardíacos/fisiología , Canales de Potasio de Dominio Poro en Tándem/genética
15.
ACS Chem Neurosci ; 5(9): 812-22, 2014 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-25028803

RESUMEN

Use of randomized peptide libraries to evolve molecules with new functions provides a means for developing novel regulators of protein activity. Despite the demonstrated power of such approaches for soluble targets, application of this strategy to membrane systems, such as ion channels, remains challenging. Here, we have combined libraries of a tethered protein scaffold with functional selection in yeast to develop a novel activator of the G-protein-coupled mammalian inwardly rectifying potassium channel Kir3.2 (GIRK2). We show that the novel regulator, denoted N5, increases Kir3.2 (GIRK2) basal activity by inhibiting clearance of the channel from the cellular surface rather than affecting the core biophysical properties of the channel. These studies establish the tethered protein display strategy as a means to create new channel modulators and highlight the power of approaches that couple randomized libraries with direct selections for functional effects. Our results further underscore the possibility for the development of modulators that influence channel function by altering cell surface expression densities rather than by direct action on channel biophysical parameters. The use of tethered library selection strategies coupled with functional selection bypasses the need for a purified target and is likely to be applicable to a range of membrane protein systems.


Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Animales , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Regulación de la Expresión Génica , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Ratones , Microinyecciones , Oocitos , Técnicas de Placa-Clamp , Biblioteca de Péptidos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Xenopus laevis
16.
J Biol Chem ; 288(49): 35081-92, 2013 Dec 06.
Artículo en Inglés | MEDLINE | ID: mdl-24163367

RESUMEN

The tandem pore domain halothane-inhibited K(+) channel 1 (THIK1) produces background K(+) currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K(+)-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Membrana Celular/metabolismo , Perros , Retículo Endoplásmico/metabolismo , Femenino , Silenciador del Gen , Humanos , Espacio Intracelular/metabolismo , Células de Riñón Canino Madin Darby , Potenciales de la Membrana , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos/metabolismo , Fosforilación , Canales de Potasio de Dominio Poro en Tándem/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Xenopus laevis
17.
Recent Pat CNS Drug Discov ; 8(3): 171-9, 2013 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-24050250

RESUMEN

Autoantibodies directed against ion channels and ionotropic receptors are associated with neuromuscular and neurological disorders. Their detection has proven to be useful for diagnosis, prognosis and treatment of these autoimmune syndromes. We have designed an ion channel chip for the systematic and rapid screening of antibodies directed against tens of different ion channels. The chip has been validated by confirming the presence of autoantibodies in patients with anti-NMDA receptor encephalitis. Such a chip will be useful for the diagnosis of already documented disorders, but also to identify new targets of autoimmunity and classification of the corresponding diseases. The article presents some promising patents on the Ion Channel Chip.


Asunto(s)
Autoanticuerpos , Enfermedades Autoinmunes/diagnóstico , Canales Iónicos , Enfermedades del Sistema Nervioso/diagnóstico , Patentes como Asunto , Enfermedades Autoinmunes/inmunología , Células HEK293 , Humanos , Enfermedades del Sistema Nervioso/inmunología , Pronóstico
18.
Proc Natl Acad Sci U S A ; 109(14): 5499-504, 2012 Apr 03.
Artículo en Inglés | MEDLINE | ID: mdl-22431633

RESUMEN

TWIK1 belongs to the family of background K(+) channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K(+) selective at neutral pH, it becomes permeable to Na(+) at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1(-/-) pancreatic ß cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.


Asunto(s)
Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Canales de Potasio/química , Homología de Secuencia de Aminoácido , Xenopus
19.
J Biol Chem ; 285(7): 4798-805, 2010 Feb 12.
Artículo en Inglés | MEDLINE | ID: mdl-19959478

RESUMEN

Tandem of P domains in a weak inwardly rectifying K(+) channel 1 (TWIK1) is a K(+) channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT(1)R or adrenoreceptor alpha2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G(i) protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.


Asunto(s)
Endocitosis/fisiología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Transporte de Proteínas/fisiología , Animales , Línea Celular , Membrana Celular/metabolismo , Perros , Electrofisiología , Endocitosis/genética , Humanos , Inmunohistoquímica , Microscopía Electrónica , Mutación , Fosforilación/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/genética , Transporte de Proteínas/genética , Receptores de Serotonina 5-HT1/metabolismo , Serotonina/farmacología
20.
Cell ; 139(3): 587-96, 2009 Oct 30.
Artículo en Inglés | MEDLINE | ID: mdl-19879844

RESUMEN

Autosomal-dominant polycystic kidney disease, the most frequent monogenic cause of kidney failure, is induced by mutations in the PKD1 or PKD2 genes, encoding polycystins TRPP1 and TRPP2, respectively. Polycystins are proposed to form a flow-sensitive ion channel complex in the primary cilium of both epithelial and endothelial cells. However, how polycystins contribute to cellular mechanosensitivity remains obscure. Here, we show that TRPP2 inhibits stretch-activated ion channels (SACs). This specific effect is reversed by coexpression with TRPP1, indicating that the TRPP1/TRPP2 ratio regulates pressure sensing. Moreover, deletion of TRPP1 in smooth muscle cells reduces SAC activity and the arterial myogenic tone. Inversely, depletion of TRPP2 in TRPP1-deficient arteries rescues both SAC opening and the myogenic response. Finally, we show that TRPP2 interacts with filamin A and demonstrate that this actin crosslinking protein is critical for SAC regulation. This work uncovers a role for polycystins in regulating pressure sensing.


Asunto(s)
Presión , Canales Catiónicos TRPP/metabolismo , Actinas/metabolismo , Animales , Proteínas Contráctiles/metabolismo , Filaminas , Mecanotransducción Celular , Ratones , Proteínas de Microfilamentos/metabolismo , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Presorreceptores/metabolismo
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