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1.
Channels (Austin) ; 12(1): 262-275, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30001175

RESUMO

Degenerin/Epithelial Sodium Channels (DEG/ENaCs) are a large family of animal-specific non-voltage gated ion channels, with enriched expression in neuronal and epithelial tissues. While neuronal DEG/ENaCs were originally characterized as sensory receptor channels, recent studies indicate that several DEG/ENaC family members are also expressed throughout the central nervous system. Human genome-wide association studies have linked DEG/ENaC-coding genes with several neurologic and psychiatric disorders, including epilepsy and panic disorder. In addition, studies in rodent models further indicate that DEG/ENaC activity in the brain contributes to many behaviors, including those related to anxiety and long-term memory. Although the exact neurophysiological functions of DEG/ENaCs remain mostly unknown, several key studies now suggest that multiple family members might exert their neuronal function via the direct modulation of synaptic processes. Here, we review and discuss recent findings on the synaptic functions of DEG/ENaCs in both vertebrate and invertebrate species, and propose models for their possible roles in synaptic physiology.


Assuntos
Canais de Sódio Degenerina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Animais , Humanos , Transmissão Sináptica
2.
Biophys J ; 114(6): 1321-1335, 2018 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-29590590

RESUMO

The bile acid-sensitive ion channel is activated by amphiphilic substances such as bile acids or artificial detergents via membrane alterations; however, the mechanism of membrane sensitivity of the bile acid-sensitive ion channel is not known. It has also not been systematically investigated whether other members of the degenerin/epithelial Na+ channel (DEG/ENaC) gene family are affected by amphiphilic compounds. Here, we show that DEG/ENaCs ASIC1a, ASIC3, ENaC, and the purinergic receptor P2X2 are modulated by a large number of different, structurally unrelated amphiphilic substances, namely the detergents N-lauroylsarcosine, Triton X-100, and ß-octylglucoside; the fenamate flufenamic acid; the antipsychotic drug chlorpromazine; the natural phenol resveratrol; the chili pepper compound capsaicin; the loop diuretic furosemide; and the antiarrythmic agent verapamil. We determined the modification of membrane properties using large-angle x-ray diffraction experiments on model lipid bilayers, revealing that the amphiphilic compounds are positioned in a characteristic fashion either in the lipid tail group region or in the lipid head group region, demonstrating that they perturbed the membrane structure. Collectively, our results show that DEG/ENaCs and structurally related P2X receptors are modulated by diverse amphiphilic molecules. Furthermore, they suggest alterations of membrane properties by amphiphilic compounds as a mechanism contributing to modulation.


Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Canais de Sódio Degenerina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Animais , Ratos
3.
Pflugers Arch ; 470(7): 1087-1102, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29589117

RESUMO

The bile acid-sensitive ion channel (BASIC) is a member of the ENaC/degenerin family of ion channels. It is activated by bile acids and inhibited by extracellular Ca2+. The aim of this study was to explore the molecular mechanisms mediating these effects. The modulation of BASIC function by extracellular Ca2+ and tauro-deoxycholic acid (t-DCA) was studied in Xenopus laevis oocytes heterologously expressing human BASIC using the two-electrode voltage-clamp and outside-out patch-clamp techniques. Substitution of aspartate D444 to alanine or cysteine in the degenerin region of BASIC, a region known to be critically involved in channel gating, resulted in a substantial reduction of BASIC Ca2+ sensitivity. Moreover, mutating D444 or the neighboring alanine (A443) to cysteine significantly reduced the t-DCA-mediated BASIC stimulation. A combined molecular docking/simulation approach demonstrated that t-DCA may temporarily form hydrogen bonds with several amino acid residues including D444 in the outer vestibule of the BASIC pore or in the inter-subunit space. By these interactions, t-DCA may stabilize the open state of the channel. Indeed, single-channel recordings provided evidence that t-DCA activates BASIC by stabilizing the open state of the channel, whereas extracellular Ca2+ inhibits BASIC by stabilizing its closed state. In conclusion, our results highlight the potential role of the degenerin region as a critical regulatory site involved in the functional interaction of Ca2+ and t-DCA with BASIC.


Assuntos
Ácidos e Sais Biliares/metabolismo , Cálcio/metabolismo , Canais de Sódio Degenerina/metabolismo , Sequência de Aminoácidos , Animais , Bile/metabolismo , Humanos , Ativação do Canal Iônico/fisiologia , Simulação de Acoplamento Molecular/métodos , Oócitos/metabolismo , Xenopus laevis/metabolismo
4.
J Biol Chem ; 292(52): 21662-21675, 2017 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-29123030

RESUMO

The degenerin/epithelial sodium channel (DEG/ENaC) superfamily of ion channels contains subfamilies with diverse functions that are fundamental to many physiological and pathological processes, ranging from synaptic transmission to epileptogenesis. The absence in mammals of some DEG/ENaCs subfamily orthologues such as FMRFamide peptide-activated sodium channels (FaNaCs), which have been identified only in mollusks, indicates that the various subfamilies diverged early in evolution. We recently reported that the nonproton agonist 2-guanidine-4-methylquinazoline (GMQ) activates acid-sensing ion channels (ASICs), a DEG/ENaC subfamily mainly in mammals, in the absence of acidosis. Here, we show that GMQ also could directly activate the mollusk-specific FaNaCs. Differences in ion selectivity and unitary conductance and effects of substitutions at key residues revealed that GMQ and FMRFamide activate FaNaCs via distinct mechanisms. The presence of two activation mechanisms in the FaNaC subfamily diverging early in the evolution of DEG/ENaCs suggested that dual gating is an ancient feature in this superfamily. Notably, the GMQ-gating mode is still preserved in the mammalian ASIC subfamily, whereas FMRFamide-mediated channel gating was lost during evolution. This implied that GMQ activation may be essential for the functions of mammalian DEG/ENaCs. Our findings provide new insights into the evolution of DEG/ENaCs and may facilitate the discovery and characterization of their endogenous agonists.


Assuntos
Canais Epiteliais de Sódio/fisiologia , FMRFamida/metabolismo , FMRFamida/fisiologia , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Células CHO , Cricetulus , Cristalografia por Raios X/métodos , Canais de Sódio Degenerina/fisiologia , Guanidinas/farmacologia , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/fisiologia , Ligantes , Moluscos/metabolismo , Oócitos/fisiologia , Peptídeos/farmacologia , Quinazolinas/farmacologia , Xenopus laevis
5.
J Neurosci ; 37(12): 3171-3180, 2017 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-28213447

RESUMO

The protein family of degenerin/epithelial sodium channels (DEG/ENaCs) is composed of diverse animal-specific, non-voltage-gated ion channels that play important roles in regulating cationic gradients across epithelial barriers. Some family members are also enriched in neural tissues in both vertebrates and invertebrates. However, the specific neurophysiological functions of most DEG/ENaC-encoding genes remain poorly understood. The fruit fly Drosophila melanogaster is an excellent model for deciphering the functions of DEG/ENaC genes because its genome encodes an exceptionally large number of DEG/ENaC subunits termed pickpocket (ppk) 1-31 Here we demonstrate that ppk29 contributes specifically to the postsynaptic modulation of excitatory synaptic transmission at the larval neuromuscular junction. Electrophysiological data indicate that the function of ppk29 in muscle is necessary for normal postsynaptic responsivity to neurotransmitter release and for normal coordinated larval movement. The ppk29 mutation does not affect gross synaptic morphology and ultrastructure, which indicates that the observed phenotypes are likely due to defects in glutamate receptor function. Together, our data indicate that DEG/ENaC ion channels play a fundamental role in the postsynaptic regulation of excitatory neurotransmission.SIGNIFICANCE STATEMENT Members of the degenerin/epithelial sodium channel (DEG/ENaC) family are broadly expressed in epithelial and neuronal tissues. To date, the neurophysiological functions of most family members remain unknown. Here, by using the power of Drosophila genetics in combination with electrophysiological and behavioral approaches, we demonstrate that the DEG/ENaC-encoding gene pickpocket 29 contributes to baseline neurotransmission, possibly via the modulation of postsynaptic glutamate receptor functionality.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Ativação do Canal Iônico/fisiologia , Canais Iônicos/fisiologia , Junção Neuromuscular/fisiologia , Sódio/metabolismo , Animais , Células Cultivadas , Canais de Sódio Degenerina/fisiologia , Canais Epiteliais de Sódio/fisiologia , Transmissão Sináptica/fisiologia
6.
Pflugers Arch ; 467(1): 39-48, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25053538

RESUMO

Mechanosensory neurons, whose activity is controlled by mechanical force, underlie the senses of touch, hearing, and proprioception, yet despite their importance, the molecular basis of mechanotransduction is poorly understood. Genetic studies in Caenorhabditis elegans have provided a useful approach for identifying potential components of mechanotransduction complexes that might be conserved in more complex organisms. This review describes the mechanosensory systems of C. elegans, including the sensory neurons and circuitry involved in body touch, nose touch, and proprioception. In addition, the roles of genes encoding known and potential mechanosensory receptors, including members of the broadly conserved transient receptor potential (TRP) and degerin/epithelial Na(+) channel (DEG/ENaC) channel families, are discussed.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Mecanorreceptores/fisiologia , Mecanotransdução Celular/fisiologia , Propriocepção/fisiologia , Tato/fisiologia , Animais , Canais de Sódio Degenerina/metabolismo , Modelos Biológicos , Canais de Receptores Transientes de Potencial/metabolismo
7.
Curr Biol ; 24(24): 2920-5, 2014 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25454784

RESUMO

The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the degenerin/epithelial sodium channel (DEG/ENaC) family. PPK is specifically expressed in nociceptive, class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses. In this study, in a genome-wide genetic screen for other ion channel subunits required for mechanical nociception, we identify a gene that we name balboa (also known as CG8546, ppk26). Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK. Moreover, laser-capture isolation of RNA from larval neurons and microarray analyses reveal that balboa is also highly enriched in nociceptive neurons. The requirement for Balboa and PPK in mechanical nociception behaviors and their specific expression in larval nociceptors led us to hypothesize that these DEG/ENaC subunits form an ion channel complex in vivo. In nociceptive neurons, Balboa::GFP proteins distribute uniformly throughout dendrites but remarkably localize to discrete foci when ectopically expressed in other neuron subtypes (where PPK is not expressed). Indeed, ectopically coexpressing ppk transforms this punctate Balboa::GFP expression pattern to the uniform distribution observed in its native cell type. Furthermore, ppk-RNAi in class IV neurons alters the broad Balboa::GFP pattern to a punctate distribution. Interestingly, this interaction is mutually codependent as balboa-RNAi eliminates Venus::PPK from the sensory dendrites of nociceptors. Finally, using a GFP-reconstitution approach in transgenic larvae, we directly detect in vivo physical interactions among PPK and Balboa subunits. Combined, our results indicate a critical mechanical nociception function for heteromeric PPK and Balboa channels in vivo.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Canais Epiteliais de Sódio/genética , Nociceptividade , Canais de Sódio/genética , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/crescimento & desenvolvimento , Animais Geneticamente Modificados/fisiologia , Canais de Sódio Degenerina/genética , Canais de Sódio Degenerina/metabolismo , Dendritos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Canais Epiteliais de Sódio/metabolismo , Larva/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Análise de Sequência de Proteína , Canais de Sódio/metabolismo
8.
Cell Rep ; 9(4): 1446-58, 2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25456135

RESUMO

A major gap in our understanding of sensation is how a single sensory neuron can differentially respond to a multitude of different stimuli (polymodality), such as propio- or nocisensation. The prevailing hypothesis is that different stimuli are transduced through ion channels with diverse properties and subunit composition. In a screen for ion channel genes expressed in polymodal nociceptive neurons, we identified Ppk26, a member of the trimeric degenerin/epithelial sodium channel (DEG/ENaC) family, as being necessary for proper locomotion behavior in Drosophila larvae in a mutually dependent fashion with coexpressed Ppk1, another member of the same family. Mutants lacking Ppk1 and Ppk26 were defective in mechanical, but not thermal, nociception behavior. Mutants of Piezo, a channel involved in mechanical nociception in the same neurons, did not show a defect in locomotion, suggesting distinct molecular machinery for mediating locomotor feedback and mechanical nociception.


Assuntos
Comportamento Animal , Canais de Sódio Degenerina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Canais Epiteliais de Sódio/metabolismo , Locomoção , Canais de Sódio/metabolismo , Animais , Membrana Celular/metabolismo , Dendritos/metabolismo , Mutação/genética , Nociceptividade , Ligação Proteica , Subunidades Proteicas/metabolismo , Temperatura
9.
BMC Biol ; 12: 84, 2014 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-25312679

RESUMO

BACKGROUND: It is generally the case that fast transmission at neural synapses is mediated by small molecule neurotransmitters. The simple nervous system of the cnidarian Hydra, however, contains a large repertoire of neuropeptides and it has been suggested that neuropeptides are the principal transmitters of Hydra. An ion channel directly gated by Hydra-RFamide neuropeptides has indeed been identified in Hydra - the Hydra Na+ channel (HyNaC) 2/3/5, which is expressed at the oral side of the tentacle base. Hydra-RFamides are more widely expressed, however, being found in neurons of the head and peduncle region. Here, we explore whether further peptide-gated HyNaCs exist, where in the animal they are expressed, and whether they are all gated by Hydra-RFamides. RESULTS: We report molecular cloning of seven new HyNaC subunits - HyNaC6 to HyNaC12, all of which are members of the DEG/ENaC gene family. In Xenopus oocytes, these subunits assemble together with the four already known subunits into thirteen different ion channels that are directly gated by Hydra-RFamide neuropeptides with high affinity (up to 40 nM). In situ hybridization suggests that HyNaCs are expressed in epitheliomuscular cells at the oral and the aboral side of the tentacle base and at the peduncle. Moreover, diminazene, an inhibitor of HyNaCs, delayed tentacle movement in live Hydra. CONCLUSIONS: Our results show that Hydra has a large variety of peptide-gated ion channels that are activated by a restricted number of related neuropeptides. The existence and expression pattern of these channels, and behavioral effects induced by channel blockers, suggests that Hydra co-opted neuropeptides for fast neuromuscular transmission.


Assuntos
Canais de Sódio Degenerina/fisiologia , Células Epiteliais/metabolismo , Hydra/genética , Neuropeptídeos/fisiologia , Transmissão Sináptica , Sequência de Aminoácidos , Animais , Clonagem Molecular , Canais de Sódio Degenerina/genética , Hydra/fisiologia , Hibridização In Situ , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/fisiologia , Oócitos , Filogenia , Alinhamento de Sequência , Sinapses/genética , Sinapses/fisiologia , Xenopus
10.
Pflugers Arch ; 466(2): 253-63, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23842738

RESUMO

The human bile acid-sensitive ion channel (hBASIC) is a cation channel of the degenerin/epithelial Na(+) channel gene family that is expressed in the intestinal tract and can be activated by bile acids. Here, we show that in addition to its sensitivity for bile acids, hBASIC shares further key features with its rat ortholog: it is blocked by extracellular divalent cations, is inhibited by micromolar concentrations of the diarylamidine diminazene, and activated by millimolar concentrations of flufenamic acid. Furthermore, we demonstrate that two major bile acids present in human bile, chenodeoxycholic acid and deoxycholic acid, activate hBASIC in a synergistic manner. In addition, we determined the single-channel properties of hBASIC in outside-out patch clamp recordings, revealing a single-channel conductance of about 11 pS and a high Na(+) selectivity. Deoxycholic acid activates hBASIC in patch clamp recordings mainly by reducing the single-channel closed time. In summary, we provide a thorough functional characterization of hBASIC.


Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Ácidos e Sais Biliares/farmacologia , Canais de Sódio Degenerina/fisiologia , Canais Iônicos Sensíveis a Ácido/efeitos dos fármacos , Cátions Bivalentes/farmacologia , Canais de Sódio Degenerina/efeitos dos fármacos , Diminazena/farmacologia , Canais Epiteliais de Sódio/efeitos dos fármacos , Canais Epiteliais de Sódio/fisiologia , Ácido Flufenâmico/farmacologia , Humanos , Ativação do Canal Iônico/fisiologia , Técnicas de Patch-Clamp
11.
FASEB J ; 27(12): 5034-45, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24018065

RESUMO

Epithelial Na(+) channel (ENaC)/degenerin family members are involved in mechanosensation, blood pressure control, pain sensation, and the expression of fear. Several of these channel types display a form of desensitization that allows the channel to limit Na(+) influx during prolonged stimulation. We used site-directed mutagenesis and chemical modification, functional analysis, and molecular dynamics simulations to investigate the role of the lower palm domain of the acid-sensing ion channel 1, a member of the ENaC/degenerin family. The lower palm domains of this trimeric channel are arranged around a central vestibule, at ∼20 Šabove the plasma membrane and are covalently linked to the transmembrane channel parts. We show that the lower palm domains approach one another during desensitization. Residues in the palm co-determine the pH dependence of desensitization, its kinetics, and the stability of the desensitized state. Mutations of palm residues impair desensitization by preventing the closing movement of the palm. Overexpression of desensitization-impaired channel mutants in central neurons allowed--in contrast to overexpression of wild type--a sustained signaling response to rapid pH fluctuations. We identify and describe here the function of an important regulatory domain that most likely has a conserved role in ENaC/degenerin channels.


Assuntos
Canais de Sódio Degenerina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ativação do Canal Iônico , Sequência de Aminoácidos , Animais , Canais de Sódio Degenerina/química , Canais de Sódio Degenerina/genética , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Concentração de Íons de Hidrogênio , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Mutação Puntual , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Xenopus
12.
Curr Mol Pharmacol ; 6(1): 44-9, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23547934

RESUMO

The epithelial sodium channel/degenerin (ENaC/deg) family of ion channels is formed by a large number of genes with variable tissue expression patterns and physiological roles. ENaC is a non-voltage gated, constitutively active channel highly selective for sodium. ENaC is formed by three homologous subunits, α, ß and γ, and a fourth subunit (δ) has been found in human and monkeys that can substitute α to form functional channels. The best-characterized role of ENaC is to serve as a rate-limiting step in transepithelial sodium reabsorption in the distal part of the kidney tubule and other tight epithelia. However, ENaC subunits are also found in the peripheral and central nervous system, where their functional roles are only beginning to be understood. In this review, we mainly focus on the putative pathophysiological roles of ENaC channels in the central nervous system and their potential value as drug targets in neurodegenerative disorders and the central control of blood pressure.


Assuntos
Encéfalo/metabolismo , Canais Epiteliais de Sódio/metabolismo , Canais Iônicos Sensíveis a Ácido/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Canais de Sódio Degenerina/metabolismo , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Humanos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo
13.
G3 (Bethesda) ; 3(3): 441-50, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23449991

RESUMO

Degenerin/epithelial sodium channels (DEG/ENaC) represent a large family of animal-specific membrane proteins. Although the physiological functions of most family members are not known, some have been shown to act as nonvoltage gated, amiloride-sensitive sodium channels. The DEG/ENaC family is exceptionally large in genomes of Drosophila species relative to vertebrates and other insects. To elucidate the evolutionary history of the DEG/ENaC family in Drosophila, we took advantage of the genomic and genetic information available for 12 Drosophila species that represent all the major species groups in the Drosophila clade. We have identified 31 family members (termed pickpocket genes) in Drosophila melanogaster, which can be divided into six subfamilies, which are represented in all 12 species. Structure prediction analyses suggested that some subunits evolved unique structural features in the large extracellular domain, possibly supporting mechanosensory functions. This finding is further supported by experimental data that show that both ppk1 and ppk26 are expressed in multidendritic neurons, which can sense mechanical nociceptive stimuli in larvae. We also identified representative genes from five of the six DEG/ENaC subfamilies in a mosquito genome, suggesting that the core DEG/ENaC subfamilies were already present early in the dipteran radiation. Spatial and temporal analyses of expression patterns of the various pickpocket genes indicated that paralogous genes often show very different expression patterns, possibly indicating that gene duplication events have led to new physiological or cellular functions rather than redundancy. In summary, our analyses support a rapid early diversification of the DEG/ENaC family in Diptera followed by physiological and/or cellular specialization. Some members of the family may have diversified to support the physiological functions of a yet unknown class of ligands.


Assuntos
Canais de Sódio Degenerina/genética , Drosophila melanogaster/genética , Canais Epiteliais de Sódio/genética , Genoma de Inseto , Sequência de Aminoácidos , Animais , Culicidae/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Evolução Molecular , Duplicação Gênica , Perfilação da Expressão Gênica , Variação Genética , Larva/genética , Larva/fisiologia , Mecanotransdução Celular , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Família Multigênica , Neurônios/fisiologia , Filogenia , Canais de Sódio/genética , Canais de Sódio/metabolismo , Especificidade da Espécie , Sintenia
14.
J Biol Chem ; 288(13): 9418-27, 2013 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-23404498

RESUMO

The voltage-gated K(+) (Kv) channel blocker 4-aminopyridine (4-AP) is used to target symptoms of the neuroinflammatory disease multiple sclerosis (MS). By blocking Kv channels, 4-AP facilitates action potential conduction and neurotransmitter release in presynaptic neurons, lessening the effects of demyelination. Because they conduct inward Na(+) and Ca(2+) currents that contribute to axonal degeneration in response to inflammatory conditions, acid-sensing ion channels (ASICs) contribute to the pathology of MS. Consequently, ASICs are emerging as disease-modifying targets in MS. Surprisingly, as first demonstrated here, 4-AP inhibits neuronal degenerin/epithelial Na(+) (Deg/ENaC) channels, including ASIC and BLINaC. This effect is specific for 4-AP compared with its heterocyclic base, pyridine, and the related derivative, 4-methylpyridine; and akin to the actions of 4-AP on the structurally unrelated Kv channels, dose- and voltage-dependent. 4-AP has differential actions on distinct ASICs, strongly inhibiting ASIC1a channels expressed in central neurons but being without effect on ASIC3, which is enriched in peripheral sensory neurons. The voltage dependence of the 4-AP block and the single binding site for this inhibitor are consistent with 4-AP binding in the pore of Deg/ENaC channels as it does Kv channels, suggesting a similar mechanism of inhibition in these two classes of channels. These findings argue that effects on both Kv and Deg/ENaC channels should be considered when evaluating the actions of 4-AP. Importantly, the current results are consistent with 4-AP influencing the symptoms of MS as well as the course of the disease because of inhibitory actions on Kv and ASIC channels, respectively.


Assuntos
4-Aminopiridina/farmacologia , Canais de Sódio Degenerina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Esclerose Múltipla/metabolismo , Neurônios/metabolismo , Animais , Animais Recém-Nascidos , Células CHO , Cricetinae , Drosophila melanogaster , Hipocampo/metabolismo , Concentração Inibidora 50 , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/metabolismo , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Ratos , Ratos Sprague-Dawley
15.
J Neurosci ; 33(3): 936-49, 2013 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-23325233

RESUMO

Neuronal DEG/ENaC (degenerin and epithelial Na(+) channel) Na(+) channels have been implicated in touch sensation. For example, MEC-4 is expressed in touch neurons in Caenorhabditis elegans and mediates gentle-touch response. Similarly, homologous mammalian ASIC2 and ASIC3 are expressed in sensory neurons and produce touch phenotypes when knocked out in mice. Here, we show that novel DEG/ENaC subunits DELM-1 and DELM-2 (degenerin-like channel mechanosensory linked-1 and degenerin-like channel mechanosensory linked-2) are expressed in glia associated with touch neurons in C. elegans and that their knock-out causes defects in mechanosensory behaviors related to nose touch and foraging, which are mediated by OLQ and IL1 sensory neurons. Cell-specific rescue supports that DELM-1 and DELM-2 are required cell-autonomously in glia to orchestrate mechanosensory behaviors. Electron microscopy reveals that in delm-1 knock-outs, OLQ and IL1 sensory neurons and associated glia are structurally normal. Furthermore, we show that knock-out of DELM-1 and DELM-2 does not disrupt the expression or cellular localization of TRPA-1, a TRP channel needed in OLQ and IL1 neurons for touch behaviors. Rather, rescue of the delm-1 nose-touch-insensitive phenotype by expression of a K(+) channel in socket glia and of a cationic channel in OLQ neurons suggests that DELM channels set basal neuronal excitability. Together, our data show that DELM-1 and DELM-2 are expressed in glia associated with touch neurons where they are not needed for neuronal structural integrity or cellular distribution of neuronal sensory channels, but rather for their function.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Canais de Sódio Degenerina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Neuroglia/metabolismo , Percepção do Tato/fisiologia , Tato/fisiologia , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Canais de Sódio Degenerina/genética , Canais Epiteliais de Sódio/genética , Mecanotransdução Celular/fisiologia , Neurônios/metabolismo
16.
Br J Pharmacol ; 168(7): 1584-96, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22928819

RESUMO

BACKGROUND AND PURPOSE: Chondrocytes exist within cartilage and serve to maintain the extracellular matrix. It has been postulated that osteoarthritic (OA) chondrocytes lose the ability to regulate their volume, affecting extracellular matrix production. In previous studies, we identified expression of epithelial sodium channels (ENaC) in human chondrocytes, but their function remained unknown. Although ENaC typically has Na(+) transport roles, it is also involved in the cell volume regulation of rat hepatocytes. ENaC is a member of the degenerin (Deg) family, and ENaC/Deg-like channels have a low conductance and high sensitivity to benzamil. In this study, we investigated whether canine chondrocytes express functional ENaC/Deg-like ion channels and, if so, what their function may be. EXPERIMENTAL APPROACH: Canine chondrocytes were harvested from dogs killed for unassociated welfare reasons. We used immunohistochemistry and patch-clamp electrophysiology to investigate ENaC expression and video microscopy to analyse the effects of pharmacological inhibition of ENaC/Deg on cell volume regulation. KEY RESULTS: Immunofluorescence showed that canine chondrocytes expressed ENaC protein. Single-channel recordings demonstrated expression of a benzamil-sensitive Na(+) conductance (9 pS), and whole-cell experiments show this to be approximately 1.5 nS per cell with high selectivity for Na(+) . Benzamil hyperpolarized chondrocytes by approximately 8 mV with a pD2 8.4. Chondrocyte regulatory volume decrease (RVI) was inhibited by benzamil (pD2 7.5) but persisted when extracellular Na(+) ions were replaced by Li(+) . CONCLUSION AND IMPLICATIONS: Our data suggest that benzamil inhibits RVI by reducing the influx of Na(+) ions through ENaC/Deg-like ion channels and present ENaC/Deg as a possible target for pharmacological modulation of chondrocyte volume.


Assuntos
Amilorida/análogos & derivados , Condrócitos/efeitos dos fármacos , Canais de Sódio Degenerina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Amilorida/farmacologia , Animais , Tamanho Celular/efeitos dos fármacos , Condrócitos/citologia , Condrócitos/metabolismo , Cães , Imunofluorescência , Técnicas In Vitro , Técnicas de Patch-Clamp
17.
J Gen Physiol ; 140(4): 391-402, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23008433

RESUMO

Degenerin/epithelial Na(+) channels (DEG/ENaCs) are Na(+) channels that are blocked by the diuretic amiloride. In general, they are impermeable for Ca(2+) or have a very low permeability for Ca(2+). We describe here, however, that a DEG/ENaC from the cnidarian Hydra magnipapillata, the Hydra Na(+) channel (HyNaC), is highly permeable for Ca(2+) (P(Ca)/P(Na) = 3.8). HyNaC is directly gated by Hydra neuropeptides, and in Xenopus laevis oocytes expressing HyNaCs, RFamides elicit currents with biphasic kinetics, with a fast transient component and a slower sustained component. Although it was previously reported that the sustained component is unselective for monovalent cations, the selectivity of the transient component had remained unknown. Here, we show that the transient current component arises from secondary activation of the Ca(2+)-activated Cl(-) channel (CaCC) of Xenopus oocytes. Inhibiting the activation of the CaCC leads to a simple on-off response of peptide-activated currents with no apparent desensitization. In addition, we identify a conserved ring of negative charges at the outer entrance of the HyNaC pore that is crucial for the high Ca(2+) permeability, presumably by attracting divalent cations to the pore. At more positive membrane potentials, the binding of Ca(2+) to the ring of negative charges increasingly blocks HyNaC currents. Thus, HyNaC is the first member of the DEG/ENaC gene family with a high Ca(2+) permeability.


Assuntos
Cálcio/metabolismo , Canais de Sódio Degenerina/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ativação do Canal Iônico , Animais , Permeabilidade da Membrana Celular , Canais de Cloreto/antagonistas & inibidores , Cloretos/metabolismo , Canais de Sódio Degenerina/química , Canais Epiteliais de Sódio/química , Hydra , Potenciais da Membrana , Neuropeptídeos/metabolismo , Nitrobenzoatos/farmacologia , Estrutura Terciária de Proteína , Agonistas de Canais de Sódio/metabolismo , Eletricidade Estática , Xenopus
18.
Eur J Hum Genet ; 20(1): 84-90, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21811305

RESUMO

Panic disorder (PD) is a mental disorder with recurrent panic attacks that occur spontaneously and are not associated to any particular object or situation. There is no consensus on what causes PD. However, it is recognized that PD is influenced by environmental factors, as well as genetic factors. Despite a significant hereditary component, genetic studies have only been modestly successful in identifying genes of importance for the development of PD. In this study, we conducted a genome-wide scan using microsatellite markers and PD patients and control individuals from the isolated population of the Faroe Islands. Subsequently, we conducted a fine mapping, which revealed the amiloride-sensitive cation channel 1 (ACCN1) located on chromosome 17q11.2-q12 as a potential candidate gene for PD. The further analyses of the ACCN1 gene using single-nucleotide polymorphisms (SNPs) revealed significant association with PD in an extended Faroese case-control sample. However, analyses of a larger independent Danish case-control sample yielded no substantial significant association. This suggests that the possible risk alleles associated in the isolated population are not those involved in the development of PD in a larger outbred population.


Assuntos
Canais Epiteliais de Sódio/genética , Estudo de Associação Genômica Ampla , Proteínas do Tecido Nervoso/genética , Transtorno de Pânico/genética , Canais Iônicos Sensíveis a Ácido , Alelos , Estudos de Casos e Controles , Mapeamento Cromossômico , Cromossomos Humanos Par 17/genética , Canais de Sódio Degenerina , Dinamarca/epidemiologia , Dinamarca/etnologia , Grupos Étnicos/genética , Genótipo , Humanos , Repetições de Microssatélites , Transtorno de Pânico/diagnóstico , Transtorno de Pânico/epidemiologia , Polimorfismo de Nucleotídeo Único , Grupos Populacionais/genética
19.
Adv Genet ; 76: 1-26, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22099690

RESUMO

All animals use a sophisticated array of receptor proteins to sense their external and internal environments. Major advances have been made in recent years in understanding the molecular and genetic bases for sensory transduction in diverse modalities, indicating that both metabotropic and ionotropic pathways are important in sensory functions. Here, I review the historical background and recent advances in understanding the roles of a relatively newly discovered family of receptors, the degenerin/epithelial sodium channels (DEG/ENaC). These animal-specific cation channels show a remarkable sequence and functional diversity in different species and seem to exert their functions in diverse sensory modalities. Functions for DEG/ENaC channels have been implicated in mechanosensation as well as chemosensory transduction pathways. In spite of overall sequence diversity, all family members share a unique protein topology that includes just two transmembrane domains and an unusually large and highly structured extracellular domain, that seem to be essential for both their mechanical and chemical sensory functions. This review will discuss many of the recent discoveries and controversies associated with sensory function of DEG/ENaC channels in both vertebrate and invertebrate model systems, covering the role of family members in taste, mechanosensation, and pain.


Assuntos
Canais Epiteliais de Sódio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Sensação/fisiologia , Canais Iônicos Sensíveis a Ácido , Animais , Canais de Sódio Degenerina , Canais Epiteliais de Sódio/química , Canais Epiteliais de Sódio/genética , Humanos , Mecanotransdução Celular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Dor/metabolismo , Dor/fisiopatologia , Sistema Nervoso Periférico/metabolismo , Sistema Nervoso Periférico/fisiopatologia
20.
Pharmacogenomics ; 12(11): 1559-69, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21961650

RESUMO

AIMS: Bipolar disorder (BD) is a lifelong psychiatric illness characterized by manic and depressive episodes affecting 1-5% of the general population. Among mood-stabilizing treatments, lithium represents the mainstay in the therapeutic management of BD. However, besides the relatively high rate of excellent responders, a significant fraction of patients present patterns of partial or nonresponse to lithium. This variability might be influenced by genetic factors, even though findings have so far been inconclusive. Here, we present the results of an exploratory genome-wide scan followed by extended genotyping carried out on a sample of 204 Sardinian BD patients characterized for lithium response. MATERIALS & METHODS: Phenotypic assessment of lithium response was made using the retrospective criteria of long-term treatment response scale. Using Affymetrix(®) 6.0 SNP arrays, we genotyped a subsample of 52 BD patients evenly distributed at the extreme ends of the treatment response scale. The associated SNPs were then prioritized and selected for validation and extended genotyping in the whole sample of BD patients characterized for lithium response. Association was also tested using the scale for a quantitative trait analysis. RESULTS: Our findings showed that several SNPs were nominally associated (p ≤ 10(-5)) with lithium response in the subgroup of 52 BD subjects. Some association signals were then confirmed in the extended sample. The strongest association, also supported by the quantitative trait analysis, was shown for a SNP located in intron 1 of the ACCN1 gene, encoding for a cation channel with high affinity for sodium and permeable to lithium. CONCLUSION: Our results indicate that ACCN1 gene is a potential candidate for response to lithium treatment that would serve as a genetic marker of lithium efficacy for BD patients.


Assuntos
Antimaníacos/uso terapêutico , Biomarcadores Farmacológicos , Transtorno Bipolar/tratamento farmacológico , Canais Epiteliais de Sódio/genética , Compostos de Lítio/uso terapêutico , Proteínas do Tecido Nervoso/genética , Canais Iônicos Sensíveis a Ácido , Adulto , Canais de Sódio Degenerina , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Itália , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Característica Quantitativa Herdável
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