RESUMO
Voltage-dependent calcium channels (VDCCs) in sensory neurones are tonically up-regulated via Ras/extracellular signal regulated kinase (ERK) signalling. The presence of putative ERK consensus sites within the intracellular loop linking domains I and II of neuronal N-type (Ca(v)2.2) calcium channels and all four neuronal calcium channel beta subunits (Ca(v)beta), suggests that Ca(v)2.2 and/or Ca(v)betas may be ERK-phosphorylated. Here we report that GST-Ca(v)2.2 I-II loop, and to a lesser extent Ca(v)beta1b-His(6), are substrates for ERK1/2 phosphorylation. Serine to alanine mutation of Ser-409 and/or Ser-447 on GST-Ca(v)2.2 I-II loop significantly reduced phosphorylation. Loss of Ser-447 reduced phosphorylation to a greater extent than mutation of Ser-409. Patch-clamp recordings from wild-type Ca(v)2.2,beta1b,alpha2delta1 versus mutant Ca(v)2.2(S447A) or Ca(v)2.2(S409A) channels revealed that mutation of either site significantly reduced current inhibition by UO126, a MEK (ERK kinase)-specific inhibitor that down-regulates ERK activity. However, no additive effect was observed by mutating both residues together, suggesting some functional redundancy between these sites. Mutation of both Ser-161 and Ser-348 on Ca(v)beta1b did not significantly reduce phosphorylation but did reduce UO126-induced current inhibition. Crucially, co-expression of Ca(v)2.2(S447A) with Ca(v)beta1b(S161,348A) had an additive effect, abolishing the action of UO126 on channel current, an effect not seen when Ca(v)beta1b(S161,348A) was co-expressed with Ca(v)2.2(S409A). Thus, Ser-447 on Ca(v)2.2 and Ser-161 and Ser-348 of Ca(v)beta1b appear to be both necessary and sufficient for ERK-dependent modulation of these channels. Together, our data strongly suggest that modulation of neuronal N-type VDCCs by ERK involves phosphorylation of Ca(v)2.2alpha1 and to a lesser extent possibly also Ca(v)beta subunits.
Assuntos
Canais de Cálcio Tipo N/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Butadienos/farmacologia , Células COS , Canais de Cálcio Tipo L/fisiologia , Canais de Cálcio Tipo N/genética , Chlorocebus aethiops , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Nitrilas/farmacologia , Fosforilação , Mutação Puntual , Ratos , Serina/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Structure prediction methods have been used to establish a domain structure for the voltage-dependent calcium channel beta subunit, beta1b. One domain was identified from homology searches as an SH3 domain, whilst another was shown, using threading algorithms, to be similar to yeast guanylate kinase. This domain structure suggested relatedness to the membrane-associated guanylate kinase protein family, and that the N-terminal domain of the beta subunit might be similar to a PDZ domain. Three-dimensional model structures have been constructed for these three domains. The extents of the domains are consistent with functional properties and mutational assays of the subunit, and provide a basis for understanding its modulatory function.
Assuntos
Canais de Cálcio/química , Modelos Moleculares , Conformação Proteica , Sequência de Aminoácidos , Canais de Cálcio/fisiologia , Dados de Sequência Molecular , Relação Estrutura-AtividadeRESUMO
Inhibition of insulin secretion from rat islets of Langerhans is known to involve at least one pertussis toxin-sensitive guanine-nucleotide binding (G) protein. We have used antisera raised against unique antigenic determinants of different members of the family of pertussis toxin-sensitive G proteins to identify these proteins in rat islets. Antiserum SG1, which recognizes both Gi1 and Gi2, reacted with an islet protein having an approximate Mr of 40,000. Antiserum I1C (Gi1 specific) failed to recognize any islet proteins, suggesting that Gi2 is present in much greater amounts than Gi1. Indeed, Gi1 levels were below the detection limit of a sensitive immunogold/silver-staining method, indicating that it may be absent from the cells of rat islets. Two different antisera were used to identify Go-like G proteins in rat islet homogenates. Both antisera reacted with a protein band which, under appropriate conditions, could be resolved to reveal two separate proteins of Mr 39-40,000. Thus, at least two molecular forms of Go are present in rat islets. Subcellular fractionation indicated that all three G proteins identified in this study (Gi2 and two forms of Go) are localized to islet membranes. No immunoreactivity could be detected in the cytosolic fraction.
Assuntos
Proteínas de Ligação ao GTP/análise , Ilhotas Pancreáticas/química , Sequência de Aminoácidos , Animais , Feminino , Técnicas Imunoenzimáticas , Dados de Sequência Molecular , Toxina Pertussis , Ratos , Ratos Endogâmicos , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The selective beta 2-adrenergic agonist clenbuterol was ineffective as a stimulus for insulin secretion when isolated rat pancreatic islets were incubated with glucose at concentrations between 4 and 20 mM. Inclusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine led to potentiation of glucose-induced insulin secretion, but did not facilitate stimulation by clenbuterol. Furthermore, maintenance of isolated rat islets for up to 3 days in tissue culture also failed to result in the appearance of a secretory response to beta-agonists. By contrast, clenbuterol induced a dose-dependent increase in insulin release from isolated human islets incubated with 20 mM glucose. Clenbuterol did not increase the basal rate of insulin secretion (4 mM glucose) in human islets. Under perifusion conditions, the secretory response of human islets to clenbuterol was rapid, of similar magnitude to that seen under static incubation conditions and could be sustained for at least 30 min. The increase in insulin secretion induced by clenbuterol was inhibited by propranolol, indicating that the response was mediated by activation of beta-receptors. In support of this, a similar enhancement of glucose-induced insulin secretion was elicited by a different beta 2-agonist, salbutamol, in human islets. The results indicate that the B cells of isolated rat islets are unresponsive to beta-agonists, whereas those of human islets are equipped with functional beta-receptors which can directly influence the rate of insulin secretion.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Albuterol/farmacologia , Animais , Clembuterol/farmacologia , Técnicas de Cultura , Glucose/farmacologia , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Propranolol/farmacologia , Ratos , Ratos EndogâmicosRESUMO
1. In rat isolated islets of Langerhans the selective beta 2-adrenoceptor agonist, clenbuterol (1 to 20 microM), significantly increased the level of adenosine 3':5'-cyclic monophosphate (cyclic AMP) within 2 min of incubation. 2. The cyclic AMP response to clenbuterol was inhibited in the presence of the selective beta 2 adrenoceptor antagonist, ICI 118551 (0.1 or 10 microM) but remained unchanged when the beta 1-antagonist, atenolol (0.1 microM) was administered. 3. Despite causing an elevation in cyclic AMP, clenbuterol (up to 20 microM) failed to influence insulin secretion at any glucose concentration tested, even in the presence of a phosphodiesterase inhibitor. 4. By contrast, clenbuterol elicited a dose-dependent rise in the rate of glucagon secretion; the maximal agonist-induced increase in secretion was two fold, a response equivalent to that observed with 20 mM L-arginine. 5. ICI 118551 significantly inhibited the rise in glucagon secretion induced by clenbuterol (up to 20 microM). 6. The results indicate that the rat islet A cell population is equipped with functional beta 2-adrenoceptors which influence glucagon secretion via the second messenger cyclic AMP, but that the B cells are deficient in functional beta-receptors.
Assuntos
Glucagon/metabolismo , Ilhotas Pancreáticas/fisiologia , Receptores Adrenérgicos beta/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Atenolol/farmacologia , Clembuterol/farmacologia , Colforsina/farmacologia , AMP Cíclico/sangue , Técnicas In Vitro , Insulina/sangue , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Propanolaminas/farmacologia , Ratos , Ratos EndogâmicosRESUMO
The molecular determinants for G-protein regulation of neuronal calcium channels remain controversial. We have generated a series of alpha 1B/alpha 1E chimeric channels, since rat brain alpha 1E (rbEII), unlike human alpha 1E, showed no G-protein modulation. The study, carried out in parallel using D2 receptor modulation of calcium currents in Xenopus oocytes of G beta gamma modulation of calcium currents in COS-7 cells, consistently showed an essential role for domain I (from the N terminus to the end of the I-II loop) of the alpha 1B Ca2+ channel in G-protein regulation, with no additional effect of the C terminal of alpha 1B. The I-II loop alone of alpha 1B, or the I-II loop together with the C-terminal tail, was insufficient to confer G-protein modulation of alpha 1E (rbEII). We have further observed that the alpha 1E clone rbEII is truncated at the N-terminus compared to other alpha 1 subunits, and we isolated a PCR product from rat brain equivalent to a longer N-terminal isoform. The long N-terminal alpha 1E, unlike the short form, showed G-protein modulation. Furthermore, the equivalent truncation of alpha 1B (delta N1-55) abolished G-protein modulation of alpha 1B. Thus, we propose that the N terminus of alpha 1B and alpha 1E calcium channels contains essential molecular determinants for membrane-delimited G-protein inhibition, and that other regions, including the I-II loop and the C terminus, do not play a conclusive role alone.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Baclofeno/farmacologia , Sítios de Ligação , Canais de Cálcio/genética , Células Cultivadas , Eletrofisiologia , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Oócitos/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , XenopusRESUMO
Rat islets express a pertussis toxin sensitive G-protein involved in receptor-mediated inhibition of insulin secretion. This has been assumed previously to represent "G(i)" which couples inhibitory receptors to adenylate cyclase. Incubation of islet G-proteins with 32P-NAD and pertussis toxin resulted in the labelling of a band of molecular weight 40,000. This band was very broad and did not allow resolution of individual components. Incubation of the radiolabelled proteins with an anti-G(o) antiserum resulted in specific immunoprecipitation of a 32P-labelled band. These results demonstrate that the complement of pertussis toxin sensitive G-proteins in rat islets includes G(o).
Assuntos
Toxina Adenilato Ciclase , Proteínas de Ligação ao GTP/análise , Ilhotas Pancreáticas/metabolismo , Toxina Pertussis , Fatores de Virulência de Bordetella/metabolismo , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Epinefrina/farmacologia , Proteínas de Ligação ao GTP/imunologia , Dados de Sequência Molecular , Norepinefrina/farmacologia , Testes de Precipitina , Ratos , Ratos Wistar , Somatostatina/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The implementation of high-throughput (HTP) cloning and expression screening in Escherichia coli by 14 laboratories in the Structural Proteomics In Europe (SPINE) consortium is described. Cloning efficiencies of greater than 80% have been achieved for the three non-ligation-based cloning techniques used, namely Gateway, ligation-indendent cloning of PCR products (LIC-PCR) and In-Fusion, with LIC-PCR emerging as the most cost-effective. On average, two constructs have been made for each of the approximately 1700 protein targets selected by SPINE for protein production. Overall, HTP expression screening in E. coli has yielded 32% soluble constructs, with at least one for 70% of the targets. In addition to the implementation of HTP cloning and expression screening, the development of two novel technologies is described, namely library-based screening for soluble constructs and parallel small-scale high-density fermentation.
Assuntos
Clonagem Molecular/métodos , Células Procarióticas/metabolismo , Proteômica/tendências , Sequência de Aminoácidos , Automação , Sequência de Bases , Escherichia coli/metabolismo , Europa (Continente) , Fermentação , Deleção de Genes , Biblioteca Gênica , Vetores Genéticos , Dados de Sequência Molecular , Dobramento de Proteína , Análise de Sequência/instrumentação , Análise de Sequência/métodosRESUMO
A collaborative project between two Structural Proteomics In Europe (SPINE) partner laboratories, York and Oxford, aimed at high-throughput (HTP) structure determination of proteins from Bacillus anthracis, the aetiological agent of anthrax and a biomedically important target, is described. Based upon a target-selection strategy combining ;low-hanging fruit' and more challenging targets, this work has contributed to the body of knowledge of B. anthracis, established and developed HTP cloning and expression technologies and tested HTP pipelines. Both centres developed ligation-independent cloning (LIC) and expression systems, employing custom LIC-PCR, Gateway and In-Fusion technologies, used in combination with parallel protein purification and robotic nanolitre crystallization screening. Overall, 42 structures have been solved by X-ray crystallography, plus two by NMR through collaboration between York and the SPINE partner in Utrecht. Three biologically important protein structures, BA4899, BA1655 and BA3998, involved in tRNA modification, sporulation control and carbohydrate metabolism, respectively, are highlighted. Target analysis by biophysical clustering based on pI and hydropathy has provided useful information for future target-selection strategies. The technological developments and lessons learned from this project are discussed. The success rate of protein expression and structure solution is at least in keeping with that achieved in structural genomics programs.
Assuntos
Bacillus anthracis/genética , Proteômica/métodos , Bacillus cereus/genética , Proteínas de Bactérias , Clonagem Molecular , Biologia Computacional , Cristalização , Cristalografia por Raios X , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Vetores Genéticos , Espectroscopia de Ressonância Magnética , RNA de Transferência/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Robótica , Esporos Bacterianos/genética , SulfurtransferasesRESUMO
Neuronal voltage-dependent calcium channels undergo inhibitory modulation by G-protein activation, generally involving both kinetic slowing and steady-state inhibition. We have shown previously that the beta-subunit of neuronal calcium channels plays an important role in this process, because when it is absent, greater receptor-mediated inhibition is observed (). We therefore hypothesized that the calcium channel beta-subunits normally may occlude G-protein-mediated inhibition. Calcium channel beta-subunits bind to the cytoplasmic loop between transmembrane domains I and II of the alpha1-subunits (). We have examined the hypothesis that this loop is involved in G-protein-mediated inhibition by making chimeras containing the I-II loop of alpha1B or alpha1A inserted into alpha1E (alpha1EBE and alpha1EAE, respectively). This strategy was adopted because alpha1B (the molecular counterpart of N-type channels) and, to a lesser extent, alpha1A (P/Q-type) are G-protein-modulated, whereas this has not been observed to any great extent for alpha1E. Although alpha1B, coexpressed with alpha2-delta and beta1b transiently expressed in COS-7 cells, showed both kinetic slowing and steady-state inhibition when recorded with GTPgammaS in the patch pipette, both of which were reversed with a depolarizing prepulse, the chimera alpha1EBE (and, to a smaller extent, alpha1EAE) showed only kinetic slowing in the presence of GTPgammaS, and this also was reversed by a depolarizing prepulse. These results indicate that the I-II loop may be the molecular substrate of kinetic slowing but that the steady-state inhibition shown by alpha1B may involve a separate site on this calcium channel.
Assuntos
Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Animais , Células COS , Canais de Cálcio/efeitos dos fármacos , Quimera , Estimulação Elétrica , Eletrofisiologia , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Homeostase , Cinética , Coelhos , RatosRESUMO
The rat brain alpha1A calcium channel clone has been expressed in COS-7 cells together with the neuronal accessory subunits beta1b and alpha2-delta. From reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and electrophysiology experiments, we have obtained no evidence that these cells contain any endogenous calcium channels. Transfected cells were identified by co-expression of a cDNA for the reporter Green Fluorescent Protein. From immunocytochemical evidence, a high degree of co-expression was obtained between Green Fluorescent Protein and individual calcium channel subunits. When all three calcium channel subunits (alpha1, alpha2-delta and beta1b) were co-expressed, evidence was obtained that all subunits were present at the cell membrane. Voltage-dependent calcium currents were observed between 24 and 72 h after transfection with the three calcium channel subunits. The current density for the combination alpha1A/alpha2-delta/beta1b was 4.19 +/- 0.69 pA.pF(-1) and the current produced was slowly inactivating. The time constant of inactivation of the maximum I(Ba) was 332 +/- 46 ms (n = 5). The voltage-dependence of activation and steady-state inactivation had voltages of half activation and inactivation of 9.5 +/- 2.5 mV and -30.4 +/- 1.5 mV respectively, and there was little overlap between the two curves. The alpha1A current was completely blocked by 100 microM Cd2+ and was also blocked by omega-conotoxin MVIIC (500 nM). Dose-inhibition curves and analysis of k(on) and k(off) for omega-agatoxin IVA both revealed apparent K(D) values of approximately 11 nM for alpha1A currents, with a k(on) of 7.8 x 10(4) M(-1).s(-1). The results suggest that alpha1A expressed in these cells has some resemblance to the P type component of calcium current observed in native neurons, although it shows a somewhat greater degree of inactivation than native P current, more similar to the Q type current component. It also has an affinity for omega-agatoxin IVA 2-5 fold lower than reported for P current, but approximately 9-fold higher than reported for Q current.
Assuntos
Canais de Cálcio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Encéfalo/metabolismo , Células COS , Cádmio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/biossíntese , Clonagem Molecular , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/biossíntese , Substâncias Macromoleculares , Potenciais da Membrana , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Reação em Cadeia da Polimerase , Biossíntese de Proteínas , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Venenos de Aranha/farmacologia , Transcrição Gênica , Transfecção , ômega-Agatoxina IVARESUMO
1. The alpha1B (N-type) calcium channel shows strong G protein modulation in the presence of G protein activators or Gbetagamma subunits. Using transient expression in COS-7 cells of alpha1B together with the accessory subunits alpha2-delta and beta2a, we have examined the role of endogenous Gbetagamma subunits in the tonic modulation of alpha1B, and compared this with modulation by exogenously expressed Gbetagamma subunits. 2. Prepulse facilitation of control alpha1B/alpha2-delta/beta2a currents was always observed. This suggests the existence of tonic modulation of alpha1B subunits. To determine whether endogenous Gbetagamma is involved in the facilitation observed in control conditions, the betaARK1 Gbetagamma-binding domain (amino acids 495-689) was overexpressed, in order to bind free Gbetagamma subunits. The extent of control prepulse-induced facilitation was significantly reduced, both in terms of current amplitude and the rate of current activation. In agreement with this, GDPbetaS also reduced the control facilitation. 3. Co-expression of the Gbeta1gamma2 subunit, together with the alpha1B/alpha2-delta/beta2a calcium channel combination, resulted in a marked degree of depolarizing prepulse-reversible inhibition of the whole-cell ICa or IBa. Both slowing of current activation and inhibition of the maximum current amplitude were observed, accompanied by a depolarizing shift in the mid-point of the voltage dependence of activation. Activation of endogenous Gbetagamma subunits by dialysis with GTPgammaS produced a smaller degree of prepulse-reversible inhibition. 4. The rate of reinhibition of alpha1B currents by activated G protein, following a depolarizing prepulse, was much faster with Gbeta1gamma2 than for the decay of facilitation in control cells. Furthermore, betaARK1 (495-689) co-expression markedly slowed the control rate of reinhibition, suggesting that the kinetics of reinhibition depend on the concentration of free endogenous or exogenously expressed Gbetagamma in the cells. In contrast, the rate of loss of inhibition during a depolarizing prepulse did not vary significantly between the different conditions examined. 5. These findings indicate that, in this system, the voltage-dependent facilitation of alpha1B that is observed under control conditions occurs as a result of endogenous free Gbetagamma binding to alpha1B.
Assuntos
Canais de Cálcio/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Animais , Fenômenos Biofísicos , Biofísica , Biotransformação/efeitos dos fármacos , Biotransformação/fisiologia , Canais de Cálcio/genética , Linhagem Celular , DNA/biossíntese , DNA/genética , Estimulação Elétrica , Eletrofisiologia , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Imuno-Histoquímica , Cinética , Potenciais da Membrana/fisiologia , Técnicas de Patch-Clamp , Coelhos , TransfecçãoRESUMO
1. The role of the voltage-dependent calcium channel (VDCC) beta-subunit has been examined in cultured rat dorsal root ganglion neurones (DRGs). An antipeptide antibody was raised and this recognized proteins corresponding to beta-subunits in a number of preparations. Immunoreactivity for the VDCC beta-subunit in DRGs was concentrated on the internal side of the plasma membrane but was also present in the cytoplasm. 2. A twenty-six-mer antisense oligonucleotide with homology to all published VDCC beta-subunit sequences was microinjected into individual cells, and maximal depletion of VDCC beta-subunit immunoreactivity was observed after 108 h suggesting a half-life for the turnover of the beta-subunit greater than 50 h. No depletion was obtained with nonsense oligonucleotide. 3. The effect of depletion of VDCC beta-subunit immunoreactivity on calcium channel currents in these cells was a reduction in amplitude of the maximum current of about 47%, and a shift in the voltage dependence of current activation of about +7 mV. These effects are the converse of those observed following co-expression of cloned beta- with alpha 1-subunits in oocytes and other expression systems. 4. The ability of the 1,4-dihydropyridine (DHP) agonist Bay K 8644 to enhance calcium channel currents was greatly reduced following depletion of beta-subunit immunoreactivity. This result is in agreement with the finding in several systems that co-expression of the beta-subunit with alpha 1-subunits results in an increased number of DHP binding sites. 5. These results show that calcium channel beta-subunits form part of native neuronal calcium channels and modify their biophysical and pharmacological properties.
Assuntos
Canais de Cálcio/metabolismo , Neurônios/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Cromatografia de Afinidade , Eletrofisiologia , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Immunoblotting , Imuno-Histoquímica , Ativação do Canal Iônico/fisiologia , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Neurônios/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
1. The beta-subunit has marked effects on the biophysical and pharmacological properties of voltage-dependent calcium channels. In the present study we examined the ability of the GABAB agonist (-) -baclofen to inhibit calcium channel currents in cultured rat dorsal root ganglion neurones following depletion of beta-subunit immunoreactivity, 108-116 h after microinjection of a beta-subunit antisense oligonucleotide. 2.We observed that, although the calcium channel current was markedly reduced in amplitude following beta-subunit depletion, the residual current (comprising both N- and L-type calcium channel currents) showed an enhanced response to application of (-) -baclofen. Therefore, it is possible that there is normally competition between activated G protein G(o) and the calcium channel beta-subunit for binding to the calcium channel alpha 1-subunit; and this competition shifts in favour of the binding of activated G(o) following depletion of the beta-subunit, resulting in increased inhibition. 3. This hypothesis is supported by evidence that an antibody against the calcium channel beta-subunit completely abolishes stimulation of the GTPase activity of G(o) by the dihydropyridine agonist S-(-) -Bay K 8644 in brain membranes. This stimulation of GTPase is thought to result from an interaction of G(o) alpha-subunit (G alpha o) with its calcium channel effector which may operate as a GTPase-activating protein. 4. These data suggest that the calcium channel beta-subunit when complexed with the beta 1-subunit normally inhibits its association with activated G(o). It may function as a GTPase-activating protein to reduce the ability of activated G(o) to associate with the calcium channel, and thus limit the efficacy of agonists such as (-) -baclofen.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Sequência de Aminoácidos , Animais , Animais Recém-Nascidos , Baclofeno/farmacologia , Sequência de Bases , Agonistas dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Células Cultivadas , Eletrofisiologia , GTP Fosfo-Hidrolases/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Imuno-Histoquímica , Dados de Sequência Molecular , Neurônios/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , RatosRESUMO
We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.
Assuntos
Canais de Cálcio Tipo N , Canais de Cálcio/genética , Proteínas de Ligação ao GTP/metabolismo , Neurônios/química , Animais , Química Encefálica/fisiologia , Células COS , Canais de Cálcio/química , Canais de Cálcio Tipo R , Proteínas de Transporte de Cátions , Bovinos , Agonistas de Dopamina/farmacologia , Proteínas de Ligação ao GTP/antagonistas & inibidores , Expressão Gênica/fisiologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Isomerismo , Cinética , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estrutura Terciária de Proteína , Quimpirol/farmacologia , Coelhos , Ratos , Homologia de Sequência de Aminoácidos , Tionucleotídeos/farmacologiaRESUMO
beta-Subunits of voltage-dependent Ca(2+) channels regulate both their expression and biophysical properties. We have injected a range of concentrations of beta3-cDNA into Xenopus oocytes, with a fixed concentration of alpha1B (Ca(V)2.2) cDNA, and have quantified the corresponding linear increase of beta3 protein. The concentration dependence of a number of beta3-dependent processes has been studied. First, the dependence of the a1B maximum conductance on beta3-protein occurs with a midpoint around the endogenous concentration of beta3 (approximately 17 nM). This may represent the interaction of the beta-subunit, responsible for trafficking, with the I-II linker of the nascent channel. Second, the effect of beta3-subunits on the voltage dependence of steady-state inactivation provides evidence for two channel populations, interpreted as representing alpha1B without or with a beta3-subunit, bound with a lower affinity of 120 nM. Third, the effect of beta3 on the facilitation rate of G-protein-modulated alpha1B currents during a depolarizing prepulse to +100 mV provides evidence for the same two populations, with the rapid facilitation rate being attributed to Gbetagamma dissociation from the beta-subunit-bound alpha1B channels. The data are discussed in terms of two hypotheses, either binding of two beta-subunits to the alpha1B channel or a state-dependent alteration in affinity of the channel for the beta-subunit.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Ativação do Canal Iônico , Animais , Canais de Cálcio/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Agonistas de Dopamina/farmacologia , Condutividade Elétrica , Expressão Gênica , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana , Mutação , Oligonucleotídeos Antissenso/genética , Oócitos/metabolismo , Ligação Proteica , Subunidades Proteicas , Quimpirol/farmacologia , Coelhos , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Xenopus laevisRESUMO
The plasma membrane expression of the rat brain calcium channel subunits alpha1A, alpha2-delta and the beta subunits beta1b, beta2a, beta3b and beta4 was examined by transient expression in COS-7 cells. Neither alpha1A nor alpha2-delta localized to the plasma membrane, either alone or when coexpressed. However, coexpression of alpha1A or alpha2-delta/alpha1A with any of the beta subunits caused alpha1A and alpha2 to be targetted to the plasma membrane. The alpha1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native alpha1A channels in dorsal root ganglion neurons and for alpha1A expressed with a beta subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of alpha1A. Immunostaining for alpha1A was obtained in depolarized non-permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a beta subunit. In contrast, beta1b and beta2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target alpha1A to the membrane since beta3 and beta4 alone showed no differential localization, but did direct the translocation of alpha1A to the plasma membrane, suggesting a chaperone role for the beta subunits.
Assuntos
Canais de Cálcio/fisiologia , Gânglios Espinais/fisiologia , Neurônios/fisiologia , Animais , Anticorpos/farmacologia , Especificidade de Anticorpos , Encéfalo/fisiologia , Células COS , Canais de Cálcio/biossíntese , Canais de Cálcio/imunologia , Membrana Celular/fisiologia , Células Cultivadas , Eletrofisiologia , Substâncias Macromoleculares , Potenciais da Membrana , Ratos , Proteínas Recombinantes de Fusão/biossíntese , TransfecçãoRESUMO
The properties of the rat brain alpha1E Ca2+ channel subunit and its modulation by accessory rat brain alpha2-delta and beta1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether alpha1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca2+ channel and to examine its pharmacology in detail. alpha1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric alpha1E/alpha2-delta/beta1b channels were largely dictated by the presence of the beta1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. alpha1E/alpha2-delta/beta1b channels did not appear to be regulated by Ca2+-induced inactivation. alpha1E was shown to exhibit a unique pharmacological profile. omega-Agatoxin IVA blocked the current in a dose-dependent manner with an IC50 of approximately 50 nM and a maximum inhibition of about 80%, whilst omega-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 micro;M) produced an inhibition of 51 +/- 7%, whereas the DHP agonist S-(-)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the alpha1E Ca2+ channel subunit; we propose that rat brain alpha1E forms a novel Ca2+ channel with properties more similar to a subtype of LVA than HVA Ca2+ current.
Assuntos
Encéfalo/fisiologia , Canais de Cálcio/fisiologia , Animais , Encéfalo/citologia , Linhagem Celular , Reação em Cadeia da Polimerase , RatosRESUMO
1. High voltage activated (HVA) Ca2+ channels are composed of a pore-forming alpha 1 subunit and the accessory beta and alpha2-delta subunits. However, the subunit composition of low voltage activated (LVA), or T-type, Ca2+ channels has yet to be elucidated. We have examined whether native calcium channels in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, which express predominantly LVA currents when undifferentiated, are modulated by overexpression of accessory calcium channel subunits. 2. Endogenous alpha 1A, B, C, C, and E, and low levels of beta and alpha 2-delta subunit protein were demonstrated in undifferentiated NG108-15 cells. 3. The alpha 2-delta, beta 2a or beta 1b accessory subunits were overexpressed by transfection of the cDNAs into these cells, and the effect examined on the endogenous Ca2+ channel currents. Heterologous expression, particularly of alpha 2-delta but also of beta 2a subunits clearly affected the profile of these currents. Both subunits induced a sustained component in the currents evoked by depolarizing voltages above -30 mV, and alpha 2-delta additionally caused a depolarization in the voltage dependence of current activation, suggesting that it also affected the native T-type currents. In contrast, beta 1b overexpression had no effect on the endogenous Ca2+ currents, despite immunocytochemical evidence for its expression in the transfected cells. 4 These results suggest that in NG108-15 cells, overexpression of the Ca2+ channel accessory subunits alpha 2-delta and beta 2a induce a sustained component of HVA current, and alpha 2-delta also influences the voltage dependence of activation of the LVA current. It is possible that native T-type alpha 1 subunits are not associated with beta subunits.