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1.
Sci Rep ; 6: 32444, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27580824

RESUMO

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90ß. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90ß inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90ß and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90ß play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.


Assuntos
Canais de Cloreto/genética , Proteínas de Choque Térmico HSP90/genética , Proteínas de Homeodomínio/genética , Chaperonas Moleculares/genética , Proteínas de Ligação a Tacrolimo/genética , Proteínas Supressoras de Tumor/genética , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/metabolismo , Proteínas Culina/genética , Proteínas Culina/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Regulação da Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Leupeptinas/farmacologia , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Miotonia Congênita/genética , Miotonia Congênita/metabolismo , Miotonia Congênita/patologia , Técnicas de Patch-Clamp , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Proteínas de Ligação a Tacrolimo/metabolismo , Transfecção , Proteínas Supressoras de Tumor/metabolismo , Ubiquitinação/efeitos dos fármacos
2.
J Gen Physiol ; 147(4): 291-308, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27022190

RESUMO

Electrical membrane properties of skeletal muscle fibers have been thoroughly studied over the last five to six decades. This has shown that muscle fibers from a wide range of species, including fish, amphibians, reptiles, birds, and mammals, are all characterized by high resting membrane permeability for Cl(-) ions. Thus, in resting human muscle, ClC-1 Cl(-) ion channels account for ∼80% of the membrane conductance, and because active Cl(-) transport is limited in muscle fibers, the equilibrium potential for Cl(-) lies close to the resting membrane potential. These conditions-high membrane conductance and passive distribution-enable ClC-1 to conduct membrane current that inhibits muscle excitability. This depressing effect of ClC-1 current on muscle excitability has mostly been associated with skeletal muscle hyperexcitability in myotonia congenita, which arises from loss-of-function mutations in the CLCN1 gene. However, given that ClC-1 must be drastically inhibited (∼80%) before myotonia develops, more recent studies have explored whether acute and more subtle ClC-1 regulation contributes to controlling the excitability of working muscle. Methods were developed to measure ClC-1 function with subsecond temporal resolution in action potential firing muscle fibers. These and other techniques have revealed that ClC-1 function is controlled by multiple cellular signals during muscle activity. Thus, onset of muscle activity triggers ClC-1 inhibition via protein kinase C, intracellular acidosis, and lactate ions. This inhibition is important for preserving excitability of working muscle in the face of activity-induced elevation of extracellular K(+) and accumulating inactivation of voltage-gated sodium channels. Furthermore, during prolonged activity, a marked ClC-1 activation can develop that compromises muscle excitability. Data from ClC-1 expression systems suggest that this ClC-1 activation may arise from loss of regulation by adenosine nucleotides and/or oxidation. The present review summarizes the current knowledge of the physiological factors that control ClC-1 function in active muscle.


Assuntos
Canais de Cloreto/metabolismo , Músculo Esquelético/metabolismo , Miotonia Congênita/metabolismo , Animais , Canais de Cloreto/genética , Humanos , Potenciais da Membrana , Músculo Esquelético/fisiologia , Músculo Esquelético/fisiopatologia , Miotonia Congênita/genética , Miotonia Congênita/fisiopatologia
3.
J Gen Physiol ; 146(6): 495-508, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26621774

RESUMO

Members of the CLC family of Cl(-) channels and transporters are homodimeric integral membrane proteins. Two gating mechanisms control the opening and closing of Cl(-) channels in this family: fast gating, which regulates opening and closing of the individual pores in each subunit, and slow (or common) gating, which simultaneously controls gating of both subunits. Here, we found that intracellularly applied Cd(2+) reduces the current of CLC-0 because of its inhibition on the slow gating. We identified CLC-0 residues C229 and H231, located at the intracellular end of the transmembrane domain near the dimer interface, as the Cd(2+)-coordinating residues. The inhibition of the current of CLC-0 by Cd(2+) was greatly enhanced by mutation of I225W and V490W at the dimer interface. Biochemical experiments revealed that formation of a disulfide bond within this Cd(2+)-binding site is also affected by mutation of I225W and V490W, indicating that these two mutations alter the structure of the Cd(2+)-binding site. Kinetic studies showed that Cd(2+) inhibition appears to be state dependent, suggesting that structural rearrangements may occur in the CLC dimer interface during Cd(2+) modulation. Mutations of I290 and I556 of CLC-1, which correspond to I225 and V490 of CLC-0, respectively, have been shown previously to cause malfunction of CLC-1 Cl(-) channel by altering the common gating. Our experimental results suggest that mutations of the corresponding residues in CLC-0 change the subunit interaction and alter the slow gating of CLC-0. The effect of these mutations on modulations of slow gating of CLC channels by intracellular Cd(2+) likely depends on their alteration of subunit interactions.


Assuntos
Cádmio/farmacologia , Canais de Cloreto/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Sequência de Aminoácidos , Sítios de Ligação , Canais de Cloreto/química , Canais de Cloreto/genética , Células HEK293 , Humanos , Dados de Sequência Molecular , Mutação , Ligação Proteica
4.
J Biol Chem ; 287(31): 25808-20, 2012 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-22689570

RESUMO

ClC-1 is the dominant sarcolemmal chloride channel and plays an important role in regulating membrane excitability that is underscored by ClC-1 mutations in congenital myotonia. Here we show that the coenzyme ß-nicotinamide adenine dinucleotide (NAD), an important metabolic regulator, robustly inhibits ClC-1 when included in the pipette solution in whole cell patch clamp experiments and when transiently applied to inside-out patches. The oxidized (NAD(+)) form of the coenzyme was more efficacious than the reduced (NADH) form, and inhibition by both was greatly enhanced by acidification. Molecular modeling, based on the structural coordinates of the homologous ClC-5 and CmClC proteins and in silico docking, suggest that NAD(+) binds with the adenine base deep in a cleft formed by ClC-1 intracellular cystathionine ß-synthase domains, and the nicotinamide base interacts with the membrane-embedded channel domain. Consistent with predictions from the models, mutation of residues in cystathionine ß-synthase and channel domains either attenuated (G200R, T636A, H847A) or abrogated (L848A) the effect of NAD(+). In addition, the myotonic mutations G200R and Y261C abolished potentiation of NAD(+) inhibition at low pH. Our results identify a new biological role for NAD and suggest that the main physiological relevance may be the exquisite sensitivity to intracellular pH that NAD(+) inhibition imparts to ClC-1 gating. These findings are consistent with the reduction of sarcolemmal chloride conductance that occurs upon acidification of skeletal muscle and suggest a previously unexplored mechanism in the pathophysiology of myotonia.


Assuntos
Canais de Cloreto/metabolismo , Músculo Esquelético/metabolismo , NAD/fisiologia , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Simulação por Computador , Humanos , Concentração de Íons de Hidrogênio , Líquido Intracelular , Cinética , Potenciais da Membrana , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/citologia , Mutagênese Sítio-Dirigida , NAD/farmacologia , Oócitos/metabolismo , Oócitos/fisiologia , Técnicas de Patch-Clamp , Ligação Proteica , Estrutura Terciária de Proteína , Xenopus
5.
Opt Lett ; 36(7): 1269-71, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21479054

RESUMO

Since its invention in 1930, Zernike phase contrast has been a pillar in optical microscopy and more recently in x-ray microscopy, in particular for low-absorption-contrast biological specimens. We experimentally demonstrate that hard-x-ray Zernike microscopy now reaches a lateral resolution below 30 nm while strongly enhancing the contrast, thus opening many new research opportunities in biomedicine and materials science.


Assuntos
Microscopia/métodos , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura , Ouro/química , Nanopartículas Metálicas , Camundongos , Poliestirenos/química , Raios X
6.
J Gen Physiol ; 137(4): 357-68, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21444658

RESUMO

The common gating of CLC-1 has been shown to be inhibited by intracellular adenosine triphosphate (ATP) in acidic pH conditions. Such modulation is thought to be mediated by direct binding of ATP to the cystathionine ß-synthase (CBS) domains at the C-terminal cytoplasmic region of CLC-1. Guided by the crystal structure of the C-terminal domain of CLC-5, we constructed a homology model of CLC-1's C terminus and mutated critical amino acid residues lining the potential ATP-binding site. The CLC-1 mutations V634A and E865A completely abolished the ATP inhibition of CLC-1, consistent with the loss of ATP binding seen with the corresponding mutations in CLC-5. Mutating two other residues, V613 and V860, also disrupted the ATP modulation of CLC-1. However, placing aromatic amino acids at position 634 increases the apparent ATP affinity. Mutant cycle analyses showed that the modulation effects of ATP and cytidine triphosphate on wild-type CLC-1 and the V634F mutant were nonadditive, suggesting that the side chain of amino acid at position 634 interacts with the base moiety of the nucleotide. The mutation effects of V634F and V613A on the ATP modulation were also nonadditive, which is consistent with the assertion suggested from the homology model that these two residues may both interact with the bound nucleotide. These results provide evidence for a direct ATP binding for modulating the function of CLC-1 and suggest an overall conserved architecture of the ATP-binding sites in CLC-1 and CLC-5. This study also demonstrates that CLC-1 is a convenient experimental model for studying the interaction of nucleotides/nucleosides with the CBS domain.


Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Canais de Cloreto/química , Cistationina beta-Sintase/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Humanos , Ativação do Canal Iônico , Cinética , Modelos Moleculares , Mutação , Xenopus/metabolismo
7.
Brain Res ; 1363: 159-69, 2010 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-20869350

RESUMO

Malignant glioma cells maintain an elevated intracellular pH (pH(i)) within hypoxic-ischemic tumor microenvironments through persistent activation of sodium-proton transport (McLean et al., 2000). Amiloride has been reported to selectively kill human malignant glioma cell lines but not primary astrocytes (Hegde et al., 2004). While amiloride reduces pH(i) of malignant gliomas by inhibiting isoform 1 of sodium-proton exchange (NHE1), direct acidification was shown to be cytostatic rather than cytotoxic. At cytotoxic concentrations, amiloride has multiple drug targets including inhibition of NHE1 and sodium-calcium exchange. Amiloride's glioma cytotoxicity can be explained, at least in part, by dual inhibition of NHE1 and of Na(+)-dependent calcium efflux by isoform 1.1 of the sodium-calcium exchanger (NCX1.1), which increases [Ca(2+)](i) and initiates glioma cell demise. As a result of persistent NHE1 activity, cytosolic free levels of sodium ([Na(+)](i)) in U87 and C6 glioma cells are elevated 3-fold, as compared with normal astrocytes. Basal cytosolic free calcium levels ([Ca(2+)](i)) also are increased 5-fold. 2', 4'-dichlorobenzamil (DCB) inhibits the sodium-dependent calcium transporter (NCX1.1) much more potently than NHE1. DCB was employed in a concentration-dependent fashion in glioma cells to selectively inhibit the forward mode of NCX1.1 at ≤1µM, while dually inhibiting both NHE1 and NCX1.1 at ≥20µM. DCB (1µM) was not cytotoxic to glioma cells, while DCB (20µM) further increased basal elevated levels of [Ca(2+)](i) in glioma cells that was followed by cell demise. Cariporide and SEA0400 are more selective inhibitors of NHE1 and NCX1.1 than amiloride or DCB, respectively. Individually, Cariporide and SEA0400 are not cytotoxic, but in combination induced glioma cell death. Like amiloride, the combination of Cariporide and SEA0400 produced glioma cell death in the absence of demonstrable caspase activation.


Assuntos
Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Cálcio/metabolismo , Morte Celular/fisiologia , Glioma/metabolismo , Sódio/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Compostos de Anilina/farmacologia , Animais , Astrócitos/citologia , Astrócitos/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Citosol/metabolismo , Glioma/patologia , Guanidinas/farmacologia , Humanos , Hipóxia-Isquemia Encefálica/metabolismo , Hipóxia-Isquemia Encefálica/patologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Espaço Pessoal , Éteres Fenílicos/farmacologia , Prótons , Ratos , Ratos Sprague-Dawley , Trocador de Sódio e Cálcio/antagonistas & inibidores , Trocador de Sódio e Cálcio/metabolismo , Trocador 1 de Sódio-Hidrogênio , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Sulfonas/farmacologia , Microambiente Tumoral/fisiologia
8.
Exp Eye Res ; 90(6): 771-9, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20346358

RESUMO

Transport of water and electrolytes is critical for corneal clarity. Recent studies indicate another important function of transport of ions and electrolytes - establishing wound electric fields that guide cell migration. We found chloride (Cl(-)) flux is a major component of the corneal wound electric current. In order to elucidate the mechanisms of Cl(-) transport, we studied Cl(-) channels and transporters in human corneal epithelial (HCE) cells. We tested a transformed human corneal epithelial cell line (tHCE), primary cultures of human corneal epithelial cells (pHCE), and human donor corneas. We first used RT-PCR to determine expression levels of mRNA of CLC (Cl(-) channels/transporters of CLC gene family) family members and CFTR (cystic fibrosis transmembrane conductance regulator) in HCE cells. We then confirmed protein expression and distribution of selected CLC family members and CFTR with Western blot and immunofluorescence confocal microscopy. Finally, Cl(-) currents were recorded with electrophysiological techniques. The mRNAs of CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, and CFTR were detected in the HCE cell line. CLC-1 and CLC-7 were not detectable. Western blot and immunostaining confirmed protein expression and distribution of CLC-2, CLC-3, CLC-4, CLC-6 and CFTR in human corneal epithelium. CLC-2 preferentially labeled the apical and basal layers, while CLC-3 and CLC-4 labeled only the superficial layer. CLC-6 and CFTR labeling showed a unique gradient with strong staining in apical layers which gradually decreased towards the basal layers. Corneal endothelium was positive for CLC-2, CLC-3, CLC-4, CLC-6 and possibly CFTR. Human corneal epithelial cells demonstrated voltage dependent Cl(-) currents. HCE cells express functional Cl(-) channels and transporters. CLC-2, CLC-3, CLC-4, CLC-6, and CFTR had distinct expression patterns in human corneal epithelium. Those molecules and their distribution may play important roles in maintaining resting Cl(-) fluxes and in regulating Cl(-) flux at corneal wounds, which may be a major contributor to wound electrical signaling.


Assuntos
Proteínas de Transporte de Ânions/genética , Canais de Cloreto/genética , Cloretos/metabolismo , Epitélio Corneano/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Transporte de Ânions/metabolismo , Western Blotting , Linhagem Celular Transformada , Canais de Cloreto/classificação , Canais de Cloreto/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Eletrofisiologia , Endotélio Corneano/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Transporte de Íons/fisiologia , Microscopia Confocal , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Ânion Dependentes de Voltagem
9.
Biophys J ; 98(3): 377-85, 2010 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-20141750

RESUMO

Using the substituted-cysteine-accessibility method, we previously showed that a cysteine residue introduced to the Y512 position of CLC-0 was more rapidly modified by a negatively charged methanethiosulfonate (MTS) reagent, 2-sulfonatoethyl MTS (MTSES), than by the positively charged 2-(trimethylammonium)ethyl MTS (MTSET). This result suggests that a positive intrinsic pore potential attracts the negatively charged MTS molecule. In this study, we further test this hypothesis of a positive pore potential in CLC-0 and find that the preference for the negatively charged MTS is diminished significantly in modifying the substituted cysteine at a deeper pore position, E166. To examine this conundrum, we study the rates of MTS inhibitions of the E166C current and those of the control mutant current from E166A. The results suggest that the inhibition of E166C by intracellularly applied MTS reagents is tainted by the modification of an endogenous cysteine, C229, located at the channel's dimer interface. After this endogenous cysteine is mutated, CLC-0 resumes its preference for selecting MTSES in modifying E166C, reconfirming the idea that the pore of CLC-0 is indeed built with a positive intrinsic potential. These experiments also reveal that MTS modification of C229 can inhibit the current of CLC-0 depending on the amino acid placed at position 166.


Assuntos
Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/química , Mesilatos/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Canais de Cloreto/genética , Cisteína/química , Cisteína/genética , Metanossulfonato de Etila/análogos & derivados , Metanossulfonato de Etila/química , Humanos , Indicadores e Reagentes/química , Cinética , Modelos Químicos , Técnicas de Patch-Clamp , Mutação Puntual , Estrutura Quaternária de Proteína/genética , Torpedo
10.
J Gen Physiol ; 132(4): 421-8, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18824589

RESUMO

The effect of intracellular adenosine triphosphate (ATP) on the "common gating" of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217-221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780-32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109-116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly--within 2-3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1.


Assuntos
Trifosfato de Adenosina/farmacologia , Canais de Cloreto/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Substâncias Redutoras/farmacologia , Animais , Linhagem Celular Transformada , Canais de Cloreto/química , Canais de Cloreto/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/fisiologia , Mercaptoetanol/farmacologia , Mesilatos/farmacologia , Oxirredução , Técnicas de Patch-Clamp , Fenantrolinas/farmacologia , Xenopus
11.
Physiol Rev ; 88(2): 351-87, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18391167

RESUMO

CLC-0 and cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channels play important roles in Cl(-) transport across cell membranes. These two proteins belong to, respectively, the CLC and ABC transport protein families whose members encompass both ion channels and transporters. Defective function of members in these two protein families causes various hereditary human diseases. Ion channels and transporters were traditionally viewed as distinct entities in membrane transport physiology, but recent discoveries have blurred the line between these two classes of membrane transport proteins. CLC-0 and CFTR can be considered operationally as ligand-gated channels, though binding of the activating ligands appears to be coupled to an irreversible gating cycle driven by an input of free energy. High-resolution crystallographic structures of bacterial CLC proteins and ABC transporters have led us to a better understanding of the gating properties for CLC and CFTR Cl(-) channels. Furthermore, the joined force between structural and functional studies of these two protein families has offered a unique opportunity to peek into the evolutionary link between ion channels and transporters. A promising byproduct of this exercise is a deeper mechanistic insight into how different transport proteins work at a fundamental level.


Assuntos
Canais de Cloreto/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Evolução Molecular , Família Multigênica , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Canais de Cloreto/química , Canais de Cloreto/genética , Regulador de Condutância Transmembrana em Fibrose Cística/química , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Especiação Genética , Humanos , Conformação Proteica
12.
J Gen Physiol ; 130(2): 217-21, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17664348

RESUMO

The CLC-1 Cl(-) channel is abundantly expressed on the plasma membrane of muscle cells, and the membrane potential of muscle cells is largely controlled by the activity of this Cl(-) channel. Previous studies showed that low intracellular pH increases the overall open probability of recombinant CLC-1 channels in various expression systems. Low intracellular pH, however, is known to inhibit the Cl(-) conductance on the native muscle membrane, contradicting the findings from the recombinant CLC-1 channels in expressed systems. Here we show that in the presence of physiological concentrations of ATP, reduction of the intracellular pH indeed inhibits the expressed CLC-1, mostly by decreasing the open probability of the common gate of the channel.


Assuntos
Trifosfato de Adenosina/fisiologia , Canais de Cloreto/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/efeitos dos fármacos , Citoplasma/fisiologia , Feminino , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Músculos/citologia , Músculos/fisiologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Xenopus laevis
13.
J Gen Physiol ; 127(4): 435-47, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16567465

RESUMO

The ClC family of Cl(-) channels and transporters comprises membrane proteins ubiquitously present in species ranging from prokaryotes to mammals. The recently solved structures of the bacterial ClC proteins have provided a good model to guide the functional experiments for the eukaryotic Cl(-) channels. Theoretical calculations based on the bacterial ClC structures have identified several residues critical for the Cl(-) binding energy in the Cl(-) transport pathway. It was speculated that the corresponding residues in eukaryotic Cl(-) channels might play similar roles for the channel functions. In this study, we made a series of mutations in three such residues in eukaryotic ClC Cl(-) channels (K149, G352, and H401 in ClC-0) and studied the functional consequences on the channel properties. A cysteine modification approach was also employed to evaluate the electrostatic effects of the charge placed at these three positions. The experimental results revealed that among the three residues tested, K149 plays the most important role in controlling both the gating and the permeation functions of ClC-0. On the other hand, mutations of H401 alter the channel conductance but not the gating properties, while mutations of G352 result in very little functional consequence. The mutation of K149 into a neutral residue leucine (K149L) shifts the activation curve and leads to flickery channel openings. The anion permeability ratios derived from bi-ionic experiments are also significantly altered in that the selectivity of Cl(-) over other anions is decreased. Furthermore, removing the positive charge at this position reduces and increases, respectively, the accessibility of the negatively and positively charged methane thiosulfonate reagents to the pore. The control of the accessibility to charged MTS reagents and the regulation of the anion permeation support the idea that K149 exerts an electrostatic effect on the channel function, confirming the prediction from computational studies.


Assuntos
Canais de Cloreto/fisiologia , Substituição de Aminoácidos , Animais , Permeabilidade da Membrana Celular , Canais de Cloreto/genética , Condutividade Elétrica , Humanos , Ativação do Canal Iônico , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Modelos Moleculares , Modelos Teóricos , Oócitos/fisiologia , Mutação Puntual , Conformação Proteica , Relação Estrutura-Atividade , Xenopus laevis
14.
Biophys J ; 88(6): 3936-45, 2005 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15778445

RESUMO

Oxidation and reduction (redox) are known to modulate the function of a variety of ion channels. Here, we report a redox regulation of the function of ClC-0, a chloride (Cl(-)) channel from the Torpedo electric organ. The study was motivated by the occasional observation of oocytes with hyperpolarization-activated Cl(-) current when these oocytes expressed ClC-0. We find that these atypical recording traces can be turned into typical ClC-0 current by incubating the oocyte in millimolar concentrations of reducing agents, suggesting that the channel function is regulated by oxidation and reduction. The redox control apparently results from an effect of oxidation on the slow (inactivation) gating: oxidation renders it more difficult for the channel to recover from the inactivated states. Introducing the point mutation C212S in ClC-0 suppresses the inactivation state, and this inactivation-suppressed mutant is no longer sensitive to the inhibition by oxidizing reagents. However, C212 is probably not the target for the redox reaction because the regulation of the inactivation gating by oxidation is still present in a pore mutant (K165C/K165 heterodimer) in which the C212S mutation is present. Taking advantage of the K165C/K165 heterodimer, we further explore the oxidation effect in ClC-0 by methane thiosulfonate (MTS) modifications. We found that trimethylethylammonium MTS modification of the introduced cysteine can induce current in the K165C/K165 heterodimer, an effect attributed to the recovery of the channel from the inactivation state. The current induction by MTS reagents is subjected to redox controls, and thus the extent of this current induction can serve as an indicator to report the oxidation state of the channel. These results together suggest that the inactivation gating of ClC-0 is affected by redox regulation. The finding also provides a convenient method to "cure" those atypical recording traces of ClC-0 expressed in Xenopus oocytes.


Assuntos
Canais de Cloreto/química , Canais de Cloreto/metabolismo , Torpedo/metabolismo , Animais , Fenômenos Biofísicos , Biofísica , Canais de Cloreto/antagonistas & inibidores , Canais de Cloreto/genética , Dimerização , Órgão Elétrico/metabolismo , Feminino , Técnicas In Vitro , Ativação do Canal Iônico , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Oxirredução , Mutação Puntual , Estrutura Quaternária de Proteína , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras , Torpedo/genética , Xenopus
15.
J Gen Physiol ; 122(2): 147-59, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12885876

RESUMO

ClC channels are a family of protein molecules containing two ion-permeation pores. Although these transmembrane proteins are important for a variety of physiological functions, their molecular operations are only superficially understood. High-resolution X-ray crystallography techniques have recently revealed the structures of two bacterial ClC channels, but whether vertebrate ClC channel pores are similar to those of bacterial homologues is not clear. To study the pore architecture of the Torpedo ClC-0 channel, we employed the substituted-cysteine-accessibility method (SCAM) and used charged methane thiosulfonate (MTS) compounds to modify the introduced cysteine. Several conclusions were derived from this approach. First, the MTS modification pattern from Y512C to E526C in ClC-0, which corresponds to residues forming helix R in bacterial ClC channels, is indeed consistent with the suggested helical structure. Second, the ClC-0 pore is more accessible to the negatively charged than to the positively charged MTS compound, a pore property that is regulated by the intrinsic electrostatic potential in the pore. Finally, attempts to modify the introduced cysteine at positions intracellular to the selectivity filter did not result in larger MTS modification rates for the open-state channel, suggesting that the fast gate of ClC-0 cannot be located at a position intracellular to the Cl- selectivity filter. Thus, the proposal that the glutamate side chain is the fast gate of the channel is applicable to ClC-0, revealing a structural and functional conservation of ClC channels between bacterial and vertebrate species.


Assuntos
Permeabilidade da Membrana Celular/fisiologia , Canais de Cloreto/química , Cisteína/química , Ativação do Canal Iônico/fisiologia , Substituição de Aminoácidos , Animais , Canais de Cloreto/fisiologia , Indicadores e Reagentes/química , Mesilatos/química , Mutação Puntual , Conformação Proteica , Relação Estrutura-Atividade , Ácidos Tiossulfônicos/química , Torpedo
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