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1.
J Physiol ; 515 ( Pt 3): 685-94, 1999 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-10066897

RESUMEN

1. Voltage-dependent calcium channels (VDCCs) show a highly non-uniform distribution in many cell types, including neurons and other polarized secretory cells. We have examined whether this can be mimicked in a polarized epithelial cell line (Madin-Darby canine kidney), which has been used extensively to study the targeting of proteins. 2. We expressed the VDCC alpha1A, alpha1B or alpha1C subunits either alone or in combination with accessory subunits alpha2-delta and the different beta subunits, and examined their localization immunocytochemically. An alpha1 subunit was only targeted to the plasma membrane if co-expressed with the accessory subunits. 3. The combination alpha1C/alpha2-delta and all beta subunits was always localized predominantly to the basolateral membrane. It has been suggested that this is equivalent to somatodendritic targeting in neurons. 4. In contrast, the alpha1B subunit was expressed at the apical membrane with all the accessory subunit combinations, by 24 h after microinjection. This membrane destination shows some parallels with axonal targeting in neurons. 5. The alpha1A subunit was consistently observed at the apical membrane in the combinations alpha1A/alpha2-delta/beta1b or beta4. In contrast, when co-expressed with alpha2-delta/beta2a, alpha1A was clearly targeted to the basolateral membrane. 6. In conclusion, the VDCC alpha1 subunit appears to be the primary determinant for targeting the VDCC complex, but the beta subunit can modify this destination, particularly for alpha1A.


Asunto(s)
Canales de Calcio Tipo N , Canales de Calcio/fisiología , Animales , Células COS , Canales de Calcio/química , Canales de Calcio/genética , Línea Celular , Membrana Celular/fisiología , ADN Complementario , Perros , Células Epiteliales , Regulación de la Expresión Génica , Riñón , Sustancias Macromoleculares , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección
2.
Eur J Neurosci ; 9(4): 739-48, 1997 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9153580

RESUMEN

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.


Asunto(s)
Canales de Calcio/fisiología , Proteínas del Tejido Nervioso/fisiología , Animales , Encéfalo/metabolismo , Células COS , Cadmio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/biosíntesis , Clonación Molecular , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/biosíntesis , Sustancias Macromoleculares , Potenciales de la Membrana , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Venenos de Araña/farmacología , Transcripción Genética , Transfección , omega-Agatoxina IVA
3.
Diabetologia ; 45(2): 242-52, 2002 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-11935156

RESUMEN

AIMS/HYPOTHESIS: The neurotransmitters glutamate and gamma-aminobutyric acid (GABA) could participate in the regulation of the endocrine functions of islets of Langerhans. We investigated the role of the metabotropic glutamate (mGluRs) and GABA(B) (GABA(B)Rs) receptors in this process. METHODS: We studied the expression of mGluRs and GABA(B)Rs in rat and human islets of Langerhans and in pancreatic alpha-cell and beta-cell lines using RT-PCR and immunoblot analysis. Effects of mGluR and GABA(B) R agonists on insulin secretion were determined by radioimmunoassays and enzyme-linked immunoadsorbent assays (ELISAs). RESULTS: We detected mGluR3 and mGluR5 (but not mGluR1, 6 and 7) mRNAs in all of the samples examined. Trace amount of mGluR2 was found in MIN6 beta cells; mGluR4 was identified in rat islets; and mGluR8 expression was detected in rat islets, RINm5F and MIN6 cells. GABA(B)R1 a/b and 2 mRNAs were identified in islets of Langerhans and MIN6 cells. The expression of mGluR3, mGluR5, GABA(B)R1 a/b and GABA(B)R2 proteins was confirmed using specific antibodies. Group I (mGluR1/5) and group II (mGluR2/3) specific mGluR agonists increased the release of insulin in the presence of 3 to 10 mmol/l or 3 to 25 mmol/l glucose, respectively, whereas a group III (mGluR4/6-8) specific agonist inhibited insulin release at high (10-25 mmol/l) glucose concentrations. Baclofen, a GABA(B)R agonist, also inhibited the release of insulin but only in the presence of 25 mmol/l glucose. CONCLUSION/INTERPRETATION: These data suggest that mGluRs and GABA(B)Rs play a role in the regulation of the endocrine pancreas with mechanisms probably involving direct activation or inhibition of voltage dependent Ca(2+)-channels, cAMP generation and G-protein-mediated modulation of K(ATP) channels.


Asunto(s)
Glucosa/farmacología , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Receptores de GABA-B/fisiología , Receptores de Glutamato Metabotrópico/fisiología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/fisiología , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/fisiología , Cartilla de ADN , Humanos , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Modelos Biológicos , Ratas , Ratas Wistar , Receptor del Glutamato Metabotropico 5 , Receptores de GABA-B/genética , Receptores de Glutamato Metabotrópico/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
4.
J Physiol ; 509 ( Pt 1): 15-27, 1998 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-9547377

RESUMEN

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.


Asunto(s)
Canales de Calcio/fisiología , Proteínas de Unión al GTP/fisiología , Animales , Fenómenos Biofísicos , Biofisica , Biotransformación/efectos de los fármacos , Biotransformación/fisiología , Canales de Calcio/genética , Línea Celular , ADN/biosíntesis , ADN/genética , Estimulación Eléctrica , Electrofisiología , Proteínas de Unión al GTP/biosíntesis , Proteínas de Unión al GTP/genética , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Inmunohistoquímica , Cinética , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Conejos , Transfección
5.
Eur J Neurosci ; 9(4): 749-59, 1997 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-9153581

RESUMEN

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.


Asunto(s)
Canales de Calcio/fisiología , Ganglios Espinales/fisiología , Neuronas/fisiología , Animales , Anticuerpos/farmacología , Especificidad de Anticuerpos , Encéfalo/fisiología , Células COS , Canales de Calcio/biosíntesis , Canales de Calcio/inmunología , Membrana Celular/fisiología , Células Cultivadas , Electrofisiología , Sustancias Macromoleculares , Potenciales de la Membrana , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Transfección
6.
Eur J Neurosci ; 12(3): 894-902, 2000 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-10762319

RESUMEN

Voltage-dependent calcium channels consist of a pore-forming transmembrane alpha1-subunit, which is known to associate with a number of accessory subunits, including alpha2-delta- and beta-subunits. The beta-subunits, of which four have been identified (beta1-4), are intracellular proteins that have marked effects on calcium channel trafficking and function. In a previous study, we observed that the beta1b-subunit showed selective plasma membrane association when expressed alone in COS7 cells, whereas beta3 and beta4 did not. In this study, we have examined the basis for this, and have identified, by making chimeric beta-subunits, that the C-terminal region, which shows most diversity between beta-subunits, of beta1b is responsible for its plasma membrane association. Furthermore we have identified, by deletion mutations, an 11-amino acid motif present in the C terminus of beta1b but not in beta3 (amino acids 547-556 of beta1b, WEEEEDYEEE), which when deleted, reduces membrane association of beta1b. Future research aims to identify what is binding to this sequence in beta1b to promote membrane association of this calcium channel subunit. It is possible that such membrane association is important for the selective localization or clustering of particular calcium channels with which beta1b is associated.


Asunto(s)
Canales de Calcio/genética , Animales , Células COS , Canales de Calcio/biosíntesis , Membrana Celular/fisiología , Núcleo Celular/fisiología , Núcleo Celular/ultraestructura , Quimera/genética , Chlorocebus aethiops , ADN/biosíntesis , ADN/genética , Perros , Electrofisiología , Eliminación de Gen , Inmunohistoquímica , Riñón/citología , Mutación/genética , Mutación/fisiología , Oocitos/metabolismo , Transfección/genética , Xenopus
7.
J Physiol ; 510 ( Pt 2): 347-60, 1998 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-9705988

RESUMEN

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.


Asunto(s)
Canales de Calcio/biosíntesis , Canales de Calcio/metabolismo , Animales , Neoplasias Encefálicas/metabolismo , Canales de Calcio/genética , Estimulación Eléctrica , Electrofisiología , Glioma/metabolismo , Células Híbridas , Inmunohistoquímica , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Ratones , Neuroblastoma/metabolismo , Técnicas de Placa-Clamp , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección
8.
J Physiol ; 502 ( Pt 2): 307-19, 1997 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-9263912

RESUMEN

1. The presence of calcium channel alpha 1D subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea-pig cardiac myocytes was demonstrated using the reverse transcriptase-polymerase chain reaction. 2. An antipeptide antibody targeted at a region of the voltage-dependent calcium channel alpha 1D subunit C-terminal to the pore-forming SS1-SS2 loop in domain IV (amino acids 1417-1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+. 3. Incubation of cells under depolarizing conditions for 2-4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.05) for cardiac myocytes when compared with controls. 4. S-(-)-Bay K 8644 (1 microM) enhanced calcium channel currents in DRGs by 75 +/- 19% (n = 5) in neurones incubated under depolarizing conditions with antibody that had been preabsorbed with its immunizing peptide (100 micrograms ml-1). This was significantly (P < 0.05) larger than the enhancement by S-(-)-Bay K 8644 that was seen with cells incubated under identical conditions but with antibody alone, which was 15 +/- 4% (n = 5). 5. These results demonstrate the presence of calcium channel alpha 1D subunits in rat DRG neurones and guinea-pig cardiac myocytes. They also show that amino acids 1417-1434 of the alpha 1D subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calcium channel current and reduces the ability of S-(-)-Bay K 8644 to enhance this current, indicating that it is an L-type current that is attenuated.


Asunto(s)
Canales de Calcio/fisiología , Ganglios Espinales/fisiología , Corazón/fisiología , Neuronas/fisiología , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Anticuerpos/farmacología , Secuencia de Bases , Sitios de Unión de Anticuerpos , Canales de Calcio/biosíntesis , Canales de Calcio/química , Células Cultivadas , Cartilla de ADN , Electrofisiología , Epítopos , Cobayas , Ventrículos Cardíacos , Inmunohistoquímica , Masculino , Potenciales de la Membrana , Microscopía Confocal , Datos de Secuencia Molecular , Miocardio/citología , Neuronas/citología , Reacción en Cadena de la Polimerasa/métodos , Ratas , Ratas Sprague-Dawley
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