RESUMO
UNLABELLED: The alphaviral6kgene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the6kproteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel6kin-frame deletion mutant. Comprehensive microscopic analysis revealed that the6kproteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the6kproteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the6kdeletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequentin vivostudies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the6kproteins may contribute to alphaviral disease manifestations and suggest that manipulation of the6kgene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE: Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV6kdeletion mutant. The absence of the6kgene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.
Assuntos
Infecções por Alphavirus/virologia , Ross River virus/genética , Ross River virus/imunologia , Proteínas Estruturais Virais/genética , Infecções por Alphavirus/imunologia , Infecções por Alphavirus/fisiopatologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Vírus Chikungunya/imunologia , Chlorocebus aethiops , Cricetinae , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Mutação , Fases de Leitura , Ross River virus/patogenicidade , Deleção de Sequência , Células Vero , Carga Viral , Proteínas Estruturais Virais/análise , Vacinas Virais/administração & dosagem , Vacinas Virais/genética , Vacinas Virais/imunologia , Replicação ViralRESUMO
Native GABA(A) channels display a single-channel conductance ranging between approximately 10 and 90 pS. Diazepam increases the conductance of some of these native channels but never those of recombinant receptors, unless they are coexpressed with GABARAP. This trafficking protein clusters recombinant receptors in the membrane, suggesting that high-conductance channels arise from receptors that are at locally high concentrations. The amphipathic (MA) helix that is present in the large cytoplasmic loop of every subunit of all ligand-gated ion channels mediates protein-protein interactions. Here we report that when applied to inside-out patches, a peptide mimicking the MA helix of the gamma2 subunit (gamma(381-403)) of the GABA(A) receptor abrogates the potentiating effect of diazepam on both endogenous receptors and recombinant GABA(A) receptors coexpressed with GABARAP, by substantially reducing their conductance. The protein interaction disrupted by the peptide did not involve GABARAP, because a shorter peptide (gamma(386-403)) known to compete with the gamma2-GABARAP interaction did not affect the conductance of recombinant alphabetagamma receptors coexpressed with GABARAP. The requirement for receptor clustering and the fact that the gamma2 MA helix is able to self-associate support a mechanism whereby adjacent GABA(A) receptors interact via their gamma2-subunit MA helices, altering ion permeation through each channel. Alteration of ion-channel function arising from dynamic interactions between ion channels of the same family has not been reported previously and highlights a novel way in which inhibitory neurotransmission in the brain may be differentially modulated.
Assuntos
Receptores de GABA-A/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Encéfalo , Diazepam/farmacologia , Eletrofisiologia , Regulação da Expressão Gênica/fisiologia , Camundongos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Receptores de GABA-A/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Ligand-gated ion channels respond to specific neurotransmitters by transiently opening an integral membrane ion-selective pore, allowing ions to move down their electrochemical gradient. A distinguishing feature of all members of the ligand-gated ion channel superfamily is the presence of a 13-amino acid disulfide loop (Cys-loop) in the extracellular ligand-binding domain. Structural data derived from the acetylcholine receptor place this loop at the interface between the ligand-binding domain and the transmembrane pore-forming domain where it is ideally located to participate in coupling ligand binding to channel opening. We have introduced specific mutations into a conserved motif at the mid-point of the Cys-loop of the GABA A receptor subunits alpha1, beta2 and gamma2S where the sequence reads aromatic, proline, aliphatic (ArProAl motif). Receptors carrying a mutation in the Cys-loop of one of their subunits were expressed in L929 cells and responses to both GABA and drugs were assessed using the whole-cell patch clamp technique. Drug potentiation and direct activation were significantly enhanced by mutations in this Cys-loop but these effects were subunit-dependent. Currents in response to agonists were larger when mutations were carried in the alpha and beta subunits but not in the gamma subunit. In contrast, potentiation of current responses by diazepam, etomidate and pentobarbital were all enhanced when mutations were carried in the alpha and gamma subunits, but not the beta subunit. Since the disruption of interactions mediated through the ArProAl motif enhances the mutant receptor's response to both agonist and drugs we suggest that this motif in the Cys-loop of the wild-type receptor participates in interactions that create activation barriers to conformational changes during channel gating.
Assuntos
Cistina/química , Moduladores GABAérgicos/farmacologia , Receptores de GABA-A/química , Receptores de GABA-B/química , Ácido gama-Aminobutírico/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Cistina/genética , Diazepam/farmacologia , Sinergismo Farmacológico , Etomidato/farmacologia , Humanos , Ativação do Canal Iônico , Dados de Sequência Molecular , Técnicas de Patch-Clamp , Pentobarbital/farmacologia , Prolina/química , Prolina/genética , Conformação Proteica , Receptores de GABA-A/efeitos dos fármacos , Receptores de GABA-A/genética , Receptores de GABA-B/efeitos dos fármacos , Receptores de GABA-B/genéticaRESUMO
The effects of the neuroactive steroids alphaxalone and pregnanolone on single GABA(A) receptor channels were tested in cell-attached and inside-out patches from cultured newborn rat hippocampal neurons. The conductance of these single channels ranged between 10 and 80 pS when exposed to low (0.5-3 microM) GABA concentrations. These GABA concentrations activated low-conducting channels (<40 pS) in 78% of the patches, 22% of patches had channels with a maximum conductance above 40 pS. Alphaxalone at concentrations above 1 microM, and pregnanolone at concentrations above 0.1 microM, significantly increased the conductance of initially low-conducting single channels activated by GABA up to seven-fold and at all concentrations tested, both drugs increased open probability and mean open time and decreased closed probability and mean closed time of channels. Both steroids at higher concentrations could directly activate high conductance (>40 pS) chloride channels. Both the directly activated channels and those channels that had been previously affected by alphaxalone were modulated by diazepam, a benzodiazepine drug that is known to specifically modulate GABA(A) channels. The present study is the first one to show that neurosteroids can significantly increase single GABA(A) channel conductance, thus enlarging our current knowledge on the molecular mechanism of action of these compounds.
Assuntos
Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Pregnanodionas/farmacologia , Pregnanolona/farmacologia , Receptores de GABA-A/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Combinação de Medicamentos , Moduladores GABAérgicos , Hipocampo/metabolismo , Canais Iônicos/efeitos dos fármacos , Neurônios/metabolismo , RatosRESUMO
Members of the glutathione transferase (GST) structural family are novel regulators of cardiac ryanodine receptor (RyR) calcium channels. We present the first detailed report of the effect of endogenous muscle GST on skeletal and cardiac RyRs. An Mu class glutathione transferase is specifically expressed in human muscle. An hGSTM2-2-like protein was isolated from rabbit skeletal muscle and sheep heart, at concentrations of approximately 17-93 microM. When added to the cytoplasmic side of RyRs, hGSTM2-2 and GST isolated from skeletal or cardiac muscle, modified channel activity in an RyR isoform-specific manner. High activity skeletal RyR1 channels were inactivated at positive potentials or activated at negative potentials by hGSTM2-2 (8-30 microM). Inactivation became faster as the positive voltage was increased. Channels recovered from inactivation when the voltage was reversed, but recovery times were significantly slowed in the presence of hGSTM2-2 and muscle GSTs. Low activity RyR1 channels were activated at both potentials. In contrast, hGSTM2-2 and GSTs isolated from muscle (1-30 microM) in the cytoplasmic solution, caused a voltage-independent inhibition of cardiac RyR2 channels. The results suggest that the major GST isoform expressed in muscle regulates Ca2+ signalling in skeletal and cardiac muscle and conserves Ca2+ stores in the sarcoplasmic reticulum.
Assuntos
Glutationa Transferase/metabolismo , Músculo Esquelético/enzimologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Animais , Citosol/efeitos dos fármacos , Citosol/enzimologia , Glutationa Transferase/isolamento & purificação , Glutationa Transferase/farmacologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Bicamadas Lipídicas , Masculino , Músculo Esquelético/efeitos dos fármacos , Miocárdio/metabolismo , Isoformas de Proteínas/isolamento & purificação , Isoformas de Proteínas/metabolismo , Coelhos , Ovinos , Fatores de TempoRESUMO
The single channel properties of recombinant gamma-aminobutyric acid type A (GABA(A))alphabetagamma receptors co-expressed with the trafficking protein GABARAP were investigated using membrane patches in the outside-out patch clamp configuration from transiently transfected L929 cells. In control cells expressing alphabetagamma receptors alone, GABA activated single channels whose main conductance was 30 picosiemens (pS) with a subconductance state of 20 pS, and increasing the GABA concentration did not alter their conductance. In contrast, when GABA(A) receptors were co-expressed with GABARAP, the GABA-activated single channels displayed multiple, high conductances (> or =40 pS), and GABA (> or =10 microM) was able to increase their conductance, up to a maximum of 60 pS. The mean open time of GABA-activated channels in control cells expressing alphabetagamma receptors alone was 2.3 +/- 0.1 ms for the main 30-pS channel and shorter for the subconductance state (20 pS, 0.8 +/- 0.1 ms). Similar values were measured for the 30- and 20-pS channels active in patches from cells co-expressing GABARAP. However higher conductance channels (> or =40 pS) remained open longer, irrespective of whether GABA or GABA plus diazepam activated them. Plotting mean open times against mean conductances revealed a linear relationship between these two parameters. Since high GABA concentrations increase both the maximum single channel conductance and mean open time of GABA(A) channels co-expressed with GABARAP, trafficking processes must influence ion channel properties. This suggests that the organization of extrasynaptic GABA(A) receptors may provide a range of distinct inhibitory currents in the brain and, further, provide differential drug responses.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Ativação do Canal Iônico , Proteínas de Membrana/fisiologia , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Proteínas Reguladoras de Apoptose , Linhagem Celular , Proteínas do Citoesqueleto/biossíntese , Diazepam/farmacologia , Relação Dose-Resposta a Droga , Eletrofisiologia , Fibroblastos/metabolismo , Moduladores GABAérgicos/farmacologia , Proteínas de Membrana/biossíntese , Camundongos , Proteínas Associadas aos Microtúbulos , Modelos Químicos , Técnicas de Patch-Clamp , Proteínas Recombinantes/químicaRESUMO
A chemically synthesized peptide representing the C-terminal subunit (p13-C) of the p13 protein of GB virus B (GBV-B), the most closely related virus to hepatitis C virus (HCV) showed ion channel activity in artificial lipid bilayers. The channels had a variable conductance and were more permeable to potassium ions than to chloride ions. Amantadine but not hexamethylene amiloride (HMA) inhibited the ion channel function of p13-C in the lipid membranes. However, neither agent was able to inhibit the replication and secretion of GBV-B from virus-infected cultured marmoset hepatocytes, which were harvested from a marmoset that was infected in vivo or inhibit replication after in vitro infection of naive hepatocytes. These data suggest that the GBV-B ion channel, contrary to the data derived from the lipid membranes, is either resistant to amantadine or that virus replication and secretion are independent of ion channel function. As the p7 protein of HCV also has ion channel activity that is apparently resistant to amantadine in vivo, the former possibility is most likely. Ion channels are likely to have an important role in the life cycle of many viruses and compounds that block these channels may prove to be useful antiviral agents.
Assuntos
Amantadina/farmacologia , Antivirais/farmacologia , Vírus GB B/efeitos dos fármacos , Canais Iônicos/efeitos dos fármacos , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Callithrix , Células Cultivadas , Infecções por Flaviviridae/virologia , Vírus GB B/genética , Vírus GB B/metabolismo , Vírus GB B/fisiologia , Hepatite Viral Animal/virologia , Hepatócitos/virologia , Canais Iônicos/química , Canais Iônicos/genética , Canais Iônicos/metabolismo , Bicamadas Lipídicas , Dados de Sequência Molecular , Proteínas Virais/química , Proteínas Virais/metabolismoRESUMO
The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [3H]ryanodine binding to native and purified RyR channels, enhances substate activity in individual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca2+. CLIC-2 (0.2-10 microM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10-100 microM) or sub-activating (100 nM) cytoplasmic Ca2+ concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5'-[beta,gamma-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 microM cytoplasmic Ca2+ than with 100 nM or 10 microM Ca2+. Further, CLIC-2 prevented the usual approximately 50-fold increase in activity when the cytoplasmic Ca2+ concentration was increased from 100 nM to 100 microM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels.
Assuntos
Trifosfato de Adenosina/fisiologia , Sinalização do Cálcio/fisiologia , Canais de Cloreto/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/fisiologia , Animais , Canais de Cloreto/química , Citoplasma/fisiologia , Escherichia coli/metabolismo , Humanos , Potenciais da Membrana , Miocárdio/citologia , Organismos Geneticamente Modificados , Ligação Proteica , Rianodina/metabolismo , Retículo Sarcoplasmático/fisiologia , OvinosRESUMO
We report that the amiloride analogues 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride inhibit, at micromolar concentrations, the replication of human immunodeficiency virus type 1 (HIV-1) in cultured human blood monocyte-derived macrophages. These compounds also inhibit the in vitro activities of the HIV-1 Vpu protein and might represent lead compounds for a new class of anti-HIV-1 drugs.
Assuntos
Amilorida/análogos & derivados , Amilorida/farmacologia , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Macrófagos/virologia , Replicação Viral/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , DNA Viral/biossíntese , DNA Viral/genética , Proteína do Núcleo p24 do HIV/biossíntese , Proteína do Núcleo p24 do HIV/genética , HIV-1/crescimento & desenvolvimento , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Macrófagos/efeitos dos fármacos , RNA Viral/biossíntese , RNA Viral/genética , Proteínas Virais Reguladoras e Acessórias/biossínteseRESUMO
We have examined the biochemical and functional properties of the recently identified, uncharacterised CLIC-2 protein. Sequence alignments showed that CLIC-2 has a high degree of sequence similarity with CLIC-1 and some similarity to the omega class of glutathione transferases (GSTO). A homology model of CLIC-2 based on the crystal structure of CLIC-1 suggests that CLIC-2 belongs to the GST structural family but, unlike the GSTs, CLIC-2 exists as a monomer. It also has an unusual enzyme activity profile. While the CXXC active site motif is conserved between CLIC-2 and the glutaredoxins, no thiol transferase activity was detected. In contrast, low glutathione peroxidase activity was recorded. CLIC-2 was found to be widely distributed in tissues including heart and skeletal muscle. Functional studies showed that CLIC-2 inhibited cardiac ryanodine receptor Ca2+ release channels in lipid bilayers when added to the cytoplasmic side of the channels and inhibited Ca2+ release from cardiac sarcoplasmic reticulum vesicles. The inhibition of RyR channels was reversed by removing CLIC-2 from the solution or by adding an anti-CLIC-2 antibody. The results suggest that one function of CLIC-2 might be to limit Ca2+ release from internal stores in cells.
Assuntos
Bloqueadores dos Canais de Cálcio , Canais de Cloreto/fisiologia , Miocárdio/química , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Sequência Conservada , Citoplasma/metabolismo , Glutationa Transferase , Humanos , Músculo Esquelético/química , Miocárdio/metabolismo , Alinhamento de Sequência , Distribuição TecidualRESUMO
High conductance gamma-aminobutyric acid type A (GABA(A)) channels (>40 picosiemens (pS)) have been reported in some studies on GABA(A) channels in situ but not in others, whereas recombinant GABA(A) channels do not appear to display conductances above 40 pS. Furthermore, the conductance of some native GABA(A) channels can be increased by diazepam or pentobarbital, which are effects not reported for expressed GABA(A) channels. GABARAP, a protein associated with native GABA(A) channels, has been reported to cause clustering of GABA(A) receptors and changes in channel kinetics. We have recorded single channel currents activated by GABA in L929 cells expressing alpha(1), beta(1), and gamma(2S) subunits of human GABA(A) receptors. Channel conductance was never higher than 40 pS and was not significantly increased by diazepam or pentobarbital, although open probability was increased. In contrast, in cells expressing the same three subunits together with GABARAP, channel conductance could be significantly higher than 40 pS, and channel conductance was increased by diazepam and pentobarbital. GABARAP caused clustering of receptors in L929 cells, and we suggest that there may be interactions between subunits of clustered GABA(A) receptors that make them open co-operatively to give high conductance "channels." Recombinant channels may require the influence of GABARAP and perhaps other intracellular proteins to adopt a fuller repertoire of properties of native channels.
Assuntos
Proteínas Associadas aos Microtúbulos/biossíntese , Receptores de GABA-A/química , Receptores de GABA-A/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas Reguladoras de Apoptose , Western Blotting , Linhagem Celular , DNA Complementar/metabolismo , Diazepam/farmacologia , Eletrofisiologia , Fibroblastos/metabolismo , Humanos , Camundongos , Microscopia de Fluorescência , Pentobarbital/farmacologia , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , TransfecçãoRESUMO
Neurons were obtained from the CA1 region of the hippocampus of newborn rats and maintained in culture. Channels were activated by pentobarbitone in cell-attached, inside-out or outside-out patches, normally by applying pentobarbitone in flowing bath solution. Currents were outwardly rectifying and blocked by bicuculline, properties of GABAA channels in these cells. Maximum channel conductance increased as pentobarbitone concentration was increased to 500 microM but conductance then decreased as pentobarbitone concentration was raised further. The best fit of a Hill-type equation to the relationship between maximum channel conductance and pentobarbitone concentration (up to 500 microM) gave an EC50 of 41 microM, a maximum conductance of 36 pS and a Hill coefficient of 1.6. Bicuculline decreased the maximum conductance of the channels activated by pentobarbitone, with an IC50 of 224 microM. Diazepam increased channel conductance, with a maximum effect being obtained with 1 microM diazepam. Diazepam (1 microM) decreased the EC50 of the pentobarbitone effect on channel conductance from 41 microM to 7.2 microM and increased maximum conductance to 72 pS. We conclude that GABAA channel conductance is related to the concentration of the allosteric agonist pentobarbitone.
Assuntos
Moduladores GABAérgicos/farmacologia , Hipocampo/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Neurônios/fisiologia , Pentobarbital/farmacologia , Receptores de GABA-A/fisiologia , Animais , Animais Recém-Nascidos , Bicuculina/farmacologia , Células Cultivadas , Diazepam/farmacologia , Condutividade Elétrica , Antagonistas GABAérgicos/farmacologia , Hipocampo/citologia , Técnicas de Patch-Clamp , Ratos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Channels were activated, in ripped-off patches from rat-cultured hippocampal neurons, by propofol alone, propofol plus 0.5 microM GABA (gamma-aminobutyric acid) or GABA alone. The propofol-activated currents were chloride-selective, showed outward-rectification and were enhanced by 1 microM diazepam. The maximum propofol-activated channel conductance increased with propofol concentration from less than 15 pS (10 microM) to about 60 pS (500 microM) but decreased to 40 pS in 1 mM propofol. Fitting the data from 10 to 500 microM propofol with a Hill-type equation gave a maximum conductance of 64 pS, an EC50 value of 32 microM and a Hill coefficient of 1.1. Addition of 0.5 microM GABA shifted the propofol EC50 value to 10 microM and increased the maximum channel conductance to about 100 pS. The Hill coefficient was 0.8. The maximum channel conductance did not increase further when 1 microM diazepam was added together with a saturating propofol concentration and GABA. The results are compared to effects other drugs have on GABAA channels conductance.
Assuntos
Anestésicos Intravenosos/farmacologia , Propofol/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Anestésicos Intravenosos/administração & dosagem , Animais , Células Cultivadas , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/metabolismo , Diazepam/farmacologia , Relação Dose-Resposta a Droga , Moduladores GABAérgicos/farmacologia , Hipocampo/metabolismo , Neurônios/metabolismo , Propofol/administração & dosagem , Ratos , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
Ross River virus and Barmah Forest virus are Australian arboviruses of the Alphavirus genus. Features of alphavirus infection include an increased permeability of cells to monovalent cations followed by virion budding. Virally encoded ion channels are thought to have a role in these processes. In this paper, the 6K proteins of Ross River virus and Barmah Forest virus are shown to form cation-selective ion channels in planar lipid bilayers. Using a novel purification method, bacterially expressed 6K proteins were inserted into bilayers with a defined orientation (i.e. N-terminal cis, C-terminal trans). Channel activity was reversibly inhibited by antibodies to the N and C termini of 6K protein added to the cis and trans baths, respectively. Channel conductances varied from 40-800 picosiemens, suggesting that the protein is able to form channels with a range of possible oligomerization states.
Assuntos
Alphavirus/metabolismo , Canais Iônicos/química , Sequência de Aminoácidos , Western Blotting , Clonagem Molecular , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Escherichia coli/metabolismo , Glutationa Transferase/metabolismo , Íons/metabolismo , Bicamadas Lipídicas/metabolismo , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Peptídeos/química , Mutação Puntual , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Proteínas Virais/químicaRESUMO
During prolonged depolarization of excitable cells, some voltage-activated, tetrodotoxin-sensitive sodium channels are resistant to inactivation and can continue to open for long periods of time, generating a "persistent" sodium current ( I(NaP)). The amplitude of I(NaP) is small [generally less than 1% of the peak amplitude of the transient sodium current ( I(NaT))], activates at potentials close to the resting membrane potential, and is more sensitive to Na channel blocking drugs than I(NaT). It is thought that persistent Na channels are generated by a change in gating of transient Na channels, possibly because of a change in phosphorylation or protein structure, e.g. loss of the inactivation gate. Drugs that block Na channels can prevent the increase in [Ca(2+)](i) in cardiac cells during hypoxia. Hypoxia increases the amplitude of I(NaP). Paradoxically, NO causes a similar increase in I(NaP) and the effects of both can be inhibited by reducing agents such as dithiothreitol and reduced glutathione. It is proposed that an increased inflow of Na(+) during hypoxia increases [Na(+)](i), which in turn reverses the Na/Ca exchanger so that [Ca(2+)](i) rises. An increase in I(NaP) and [Ca(2+)](i) could cause arrhythmias and irreversible cell damage.
Assuntos
Hipóxia/fisiopatologia , Ativação do Canal Iônico , Isquemia Miocárdica/fisiopatologia , Oxigênio/metabolismo , Canais de Sódio , Sódio/metabolismo , Humanos , Hipóxia-Isquemia Encefálica/fisiopatologia , Potenciais da Membrana , Óxido Nítrico/farmacologia , Cianeto de Sódio/farmacologiaRESUMO
The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels and enhances the process of virion budding and release. Mutagenesis studies have shown that the N-terminal transmembrane domain primarily controls both of these activities. Here we report that the Vpu ion channel is inhibited by the amiloride derivatives 5-(N,N-hexamethylene)amiloride and 5-(N,N-dimethyl)amiloride but not by amiloride itself, nor by amantadine. Hexamethyleneamiloride also inhibits budding of virus-like particles from HeLa cells expressing HIV-1 Gag and Vpu proteins. These results confirm the link between Vpu ion channel activity and the budding process and also suggest that amiloride derivatives might have useful anti-HIV-1 properties.
Assuntos
Amilorida/análogos & derivados , Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Canais Iônicos/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Amilorida/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Fenômenos Biofísicos , Biofísica , DNA Viral/genética , HIV-1/genética , HIV-1/crescimento & desenvolvimento , Células HeLa , Proteínas do Vírus da Imunodeficiência Humana , Humanos , Canais Iônicos/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/fisiologiaRESUMO
Mu-conotoxins are peptide inhibitors of voltage-sensitive sodium channels (VSSCs). Synthetic forms of mu-conotoxins PIIIA and PIIIA-(2-22) were found to inhibit tetrodotoxin (TTX)-sensitive VSSC current but had little effect on TTX-resistant VSSC current in sensory ganglion neurons. In rat brain neurons, these peptides preferentially inhibited the persistent over the transient VSSC current. Radioligand binding assays revealed that PIIIA, PIIIA-(2-22), and mu-conotoxins GIIIB discriminated among TTX-sensitive VSSCs in rat brain, that these and GIIIC discriminated among the corresponding VSSCs in human brain, and GIIIA had low affinity for neuronal VSSCs. (1)H NMR studies found that PIIIA adopts two conformations in solution due to cis/trans isomerization at hydroxyproline 8. The major trans conformation results in a three-dimensional structure that is significantly different from the previously identified conformation of mu-conotoxins GIIIA and GIIIB that selectively target TTX-sensitive muscle VSSCs. Comparison of the structures and activity of PIIIA to muscle-selective mu-conotoxins provides an insight into the structural requirements for inhibition of different TTX-sensitive sodium channels by mu-conotoxins.