RESUMEN
Voltage-gated Ca2+ channels are key transducers of cellular excitability and participate in several crucial physiological responses. In vertebrates, 10 Ca2+ channel genes, grouped in 3 families (CaV1, CaV2 and CaV3), have been described and characterized. Insects possess only one member of each family. These genes have been isolated in a limited number of species and very few have been characterized although, in addition to their crucial role, they may represent a collateral target for neurotoxic insecticides. We have isolated the 3 genes coding for the 3 Ca2+ channels expressed in Apis mellifera. This work provides the first detailed characterization of the honeybee T-type CaV3 Ca2+ channel and demonstrates the low toxicity of inhibiting this channel. Comparing Ca2+ currents recorded in bee neurons and myocytes with Ca2+ currents recorded in Xenopus oocytes expressing the honeybee CaV3 gene suggests native expression in bee muscle cells only. High-voltage activated Ca2+ channels could be recorded in the somata of different cultured bee neurons. These functional data were confirmed by in situ hybridization, immunolocalization and in vivo analysis of the effects of a CaV3 inhibitor. The biophysical and pharmacological characterization and the tissue distribution of CaV3 suggest a role in honeybee muscle function.
Asunto(s)
Abejas/efectos de los fármacos , Abejas/fisiología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/metabolismo , Locomoción/efectos de los fármacos , Animales , Canales de Calcio Tipo T/genética , Expresión Génica , Mibefradil/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Bulbo Olfatorio/efectos de los fármacos , Bulbo Olfatorio/fisiología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , XenopusRESUMEN
The administration of autologous (recipient-derived) tolerogenic dendritic cells (ATDCs) is under clinical evaluation. However, the molecular mechanisms by which these cells prolong graft survival in a donor-specific manner is unknown. Here, we tested mouse ATDCs for their therapeutic potential in a skin transplantation model. ATDC injection in combination with anti-CD3 treatment induced the accumulation of CD8(+) CD11c(+) T cells and significantly prolonged allograft survival. TMEM176B is an intracellular protein expressed in ATDCs and initially identified in allograft tolerance. We show that Tmem176b(-/-) ATDCs completely failed to trigger both phenomena but recovered their effect when loaded with donor peptides before injection. These results strongly suggested that ATDCs require TMEM176B to cross-present antigens in a tolerogenic fashion. In agreement with this, Tmem176b(-/-) ATDCs specifically failed to cross-present male antigens or ovalbumin to CD8(+) T cells. Finally, we observed that a Tmem176b-dependent cation current controls phagosomal pH, a critical parameter in cross-presentation. Thus, ATDCs require TMEM176B to cross-present donor antigens to induce donor-specific CD8(+) CD11c(+) T cells with regulatory properties and prolong graft survival.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Presentación de Antígeno/inmunología , Complejo CD3/inmunología , Células Dendríticas/inmunología , Supervivencia de Injerto/fisiología , Proteínas de la Membrana/fisiología , Trasplante de Piel , Aloinjertos , Animales , Linfocitos T CD8-positivos/inmunología , Reactividad Cruzada , Electrofisiología , Endocitosis/fisiología , Femenino , Citometría de Flujo , Tolerancia Inmunológica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis/fisiologíaRESUMEN
RGK (Rad-Gem-Rem) GTPases have been described as potent negative regulators of the Ca(2+) influx via high-threshold voltage-activated Ca(2+) channels. Recent work, mostly performed on Ca(V)1.2 Ca(2+) channels, has highlighted the crucial role played by the channel auxiliary Ca(V)beta subunits and identified several GTPase and beta-subunit protein domains involved in this regulation. We now extend these conclusions by producing the first complete characterization of the effects of Gem, Rem, and Rem2 on the neuronal Ca(V)2.1 Ca(2+) channels expressed with Ca(V)beta(1) or Ca(V)beta(2) subunits. Current inhibition is limited to a decrease in amplitude with no modification in the voltage dependence or kinetics of the current. We demonstrate that this inhibition can occur for Ca(V)beta constructs with impaired capacity to induce current potentiation, but that it is lost for Ca(V)beta constructs deleted for their beta-interaction domain. The RGK C-terminal last approximately 80 amino acids are sufficient to allow potent current inhibition and in vivo beta-subunit/Gem interaction. Interestingly, although Gem and Gem carboxy-terminus induce a completely different pattern of beta-subunit cellular localization, they both potently inhibit Ca(V)2.1 channels. These data therefore set the status of neuronal Ca(V)2.1 Ca(2+) channel inhibition by RGK GTPases, emphasizing the role of short amino acid sequences of both proteins in beta-subunit binding and channel inhibition and revealing a new mechanism for channel inhibition.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Secuencia de Aminoácidos , Animales , Bario/metabolismo , Secuencia de Bases , Sitios de Unión , Fenómenos Biofísicos , Bloqueadores de los Canales de Calcio/metabolismo , Canales de Calcio Tipo N/química , Señalización del Calcio , Cartilla de ADN/genética , Femenino , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Neuronas/metabolismo , Oocitos/metabolismo , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Calcium influx into cardiac myocytes via voltage-gated Ca channels is a key step in initiating the contractile response. During prolonged depolarizations, toxic Ca(2+) overload is prevented by channel inactivation occurring through two different processes identified by their primary trigger: voltage or intracellular Ca(2+). In physiological situations, cardiac L-type (Ca(V)1.2) Ca(2+) channels inactivate primarily via Ca(2+)-dependent inactivation (CDI), while neuronal P/Q (Ca(V)2.1) Ca(2+) channels use preferentially voltage-dependent inactivation (VDI). In certain situations however, these two types of channels have been shown to be able to inactivate by both processes. From a structural view point, the rearrangement occurring during CDI and VDI is not precisely known, but functional studies have underlined the role played by at least 2 channel sequences: a C-terminal binding site for the Ca(2+) sensor calmodulin, essential for CDI, and the loop connecting domains I and II, essential for VDI. The conserved regulation of VDI and CDI by the auxiliary channel beta subunit strongly suggests that these two mechanisms may use a set of common protein-protein interactions that are influenced by the auxiliary subunit. We will review our current knowledge of these interactions. New data are presented on L-P/Q (Ca(V)1.2/Ca(V)2.1) channel chimera that confirm the role of the I-II loop in VDI and CDI, and reveal some of the essential steps in Ca(2+) channel inactivation.
Asunto(s)
Canales de Calcio Tipo L/fisiología , Calcio/fisiología , Activación del Canal Iónico , Potenciales de la Membrana/fisiología , Modelos Biológicos , Secuencia de Aminoácidos , Animales , Femenino , Técnicas In Vitro , Datos de Secuencia Molecular , Miocitos Cardíacos/fisiología , Oocitos/fisiología , Xenopus laevis/fisiologíaRESUMEN
Starting from a comparative study of different Ca2+ chelators on the G-protein-induced inhibition of the CaV2.1 Ca channels, we demonstrate that BAPTA and DM-nitrophen are able to interact, in a Ca2+- and lipid-dependent manner, with phospholipid monolayers. Critical insertion pressure and sensitivity to charged lipids indicated that insertion in the lipid film may occur in biological membranes as those found on Xenopus oocytes. This novel property is not found for EGTA and EDTA and may participate to the unusual ability of BAPTA-related molecules to chelate Ca2+ ions in the very close vicinity of the plasma membrane, where most of the Ca2+-dependent signalling triggered by voltage-gated Ca2+ currents occurs.
Asunto(s)
Calcio/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/metabolismo , Fosfolípidos/metabolismo , Acetatos/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Quelantes/farmacología , Relación Dosis-Respuesta a Droga , Ácido Egtácico/farmacología , Etilenodiaminas/farmacología , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Microelectrodos , Microinyecciones , Oocitos/efectos de los fármacos , Técnicas de Placa-Clamp , Xenopus laevisRESUMEN
Voltage gated Ca(2+) channel (VGCC) auxiliary beta subunits increase membrane expression of the main pore-forming alpha(1) subunits and finely tune channel activation and inactivation properties. In expression studies, co-expression of beta subunits also reduced neuronal Ca(2+) channel regulation by heterotrimeric G protein. Biochemical studies suggest that VGCC beta subunits and G protein betagamma can compete for overlapping interaction sites on VGCC alpha(1) subunits, suggesting a dynamic association of these subunits with alpha(1). In this work we have analyzed the stability of the alpha(1)/beta association under physiological conditions. Regulation of the alpha(1A) Ca(2+) channel inactivation properties by beta(1b) and beta(2a) subunits had two major effects: a shift in voltage-dependent inactivation (E(in)), and an increase of the non-inactivating current (R(in)). Unexpectedly, large variations in magnitude of the effects were recorded on E(in), when beta(1b) was expressed, and R(in), when beta(2a) was expressed. These variations were not proportional to the current amplitude, and occurred at similar levels of beta subunit expression. beta(2a)-induced variations of R(in) were, however, inversely proportional to the magnitude of G protein block. These data underline the two different mechanisms used by beta(1b) and beta(2a) to regulate channel inactivation, and suggest that the VGCC beta subunit can unbind the alpha1 subunit in physiological situations.
Asunto(s)
Canales de Calcio/metabolismo , Animales , Western Blotting , Canales de Calcio/química , Canales de Calcio/genética , Electrofisiología , Encefalina Ala(2)-MeFe(4)-Gli(5)/metabolismo , Mutación , Oocitos/metabolismo , Unión Proteica , Subunidades de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMEN
Stargazin or [gamma]2, the product of the gene mutated in the stargazer mouse, is a homologue of the [gamma]1 protein, an accessory subunit of the skeletal muscle L-type Ca2+ channel. [gamma]2 is selectively expressed in the brain, and considered to be a putative neuronal Ca2+ channel subunit based mainly on homology to [gamma]1. Two new members of the [gamma] family expressed in the brain have recently been identified: [gamma]3 and [gamma]4. We have co-expressed, in Xenopus oocytes, the human [gamma]2, [gamma]3 and [gamma]4 subunits with the P/Q-type (Ca(V)2.1) Ca2+ channel and different regulatory subunits ([alpha]2-[delta]; [beta]1, [beta]2, [beta]3 or [beta]4). Subcellular distribution of the [gamma] subunits confirmed their membrane localization. Ba2+ currents, recorded using two-electrode voltage clamp, showed that the effects of the [gamma] subunits on the electrophysiological properties of the channel are, most of the time, minor. However, a fraction of the oocytes expressing [beta] subunits displayed an unusual slow-inactivating Ba2+ current. Expression of both [beta] and [gamma] subunits increased the appearance of the slow-inactivating current. Our data support a role for the [gamma] subunit as a brain Ca2+ channel modulatory subunit and suggest that [beta] and [gamma] subunits are involved in a switch between two regulatory modes of the P/Q-type channel inactivation.
Asunto(s)
Canales de Calcio Tipo N , Activación del Canal Iónico/fisiología , Animales , Bario/farmacocinética , Calcio/metabolismo , Canales de Calcio Tipo N/química , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Línea Celular , Femenino , Expresión Génica/fisiología , Humanos , Riñón/citología , Cinética , Oocitos/fisiología , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína , Transfección , Xenopus laevisRESUMEN
The effects of zinc (Zn2+) on excitability and ionic conductances were analysed on RINm5F insulinoma cells under whole-cell and outside-out patch-clamp recording conditions. We found that extracellular application of 10-20 microM Zn2+ induced a reversible abolition of Ca2+ action potential firing, which was accompanied by an hyperpolarisation of the resting membrane potential. Higher concentrations of Zn2+, in the tens to hundreds micromolar range, induced a reversible reduction of voltage-gated Ca2+ and, to a lesser extent, K+ currents. Low-voltage-activated Ca2+ currents were more sensitive to Zn2+ block than high voltage-activated Ca2+ currents. The Zn2+-induced hyperpolarisation arose from a dose-dependent increase in a voltage-independent K+ conductance that was pharmacologically identified as an ATP-sensitive K+ (KATP) conductance. The effect was rapid in onset, readily reversible, voltage independent, and related to intracellular ATP concentration. In the presence of 1 mM intracellular ATP, half-maximal activation of KATP channels was obtained with extracellular application of 1.7 microM Zn2+. Single channel analysis revealed that extracellular Zn2+ increased the KATP channel open-state probability with no change in the single channel conductance. Our data support the hypothesis that Zn2+ binding to KATP protein subunits results in an activation of the channels, therefore regulating the resting membrane potential and decreasing the excitability of RINm5F cells. Taken together, our results suggest that Zn2+ can influence insulin secretion in pancreatic beta-cells through a negative feedback loop, involving both KATP and voltage-gated conductances.
Asunto(s)
Adenosina Trifosfato/fisiología , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/fisiología , Canales de Potasio/fisiología , Zinc/farmacología , Animales , Canales de Calcio/fisiología , Conductividad Eléctrica , Electrofisiología , Espacio Extracelular/metabolismo , Membranas Intracelulares/metabolismo , Iones , Técnicas de Placa-Clamp , Potasio/fisiología , Ratas , Células Tumorales Cultivadas , Zinc/metabolismoRESUMEN
Ca(2+) channel inactivation is a key element in controlling the level of Ca(2+) entry through voltage-gated Ca(2+) channels. Interaction between the pore-forming alpha(1) subunit and the auxiliary beta subunit is known to be a strong modulator of voltage-dependent inactivation. Here, we demonstrate that an N-terminal membrane anchoring site (MAS) of the beta(2a) subunit strongly reduces alpha(1A) (Ca(V)2.1) Ca(2+) channel inactivation. This effect can be mimicked by the addition of a transmembrane segment to the N terminus of the beta(2a) subunit. Inhibition of inactivation by beta(2a) also requires a link between MAS and another important molecular determinant, the beta interaction domain (BID). Our data suggest that mobility of the Ca(2+) channel I-II loop is necessary for channel inactivation. Interaction of this loop with other identified intracellular channel domains may constitute the basis of voltage-dependent inactivation. We thus propose a conceptually novel mechanism for slowing of inactivation by the beta(2a) subunit, in which the immobilization of the channel inactivation gate occurs by means of MAS and BID.
Asunto(s)
Canales de Calcio/metabolismo , Activación del Canal Iónico/fisiología , Subunidades de Proteína , Animales , Bario/farmacología , Sitios de Unión/genética , Canales de Calcio/efectos de los fármacos , Canales de Calcio/genética , Canales de Calcio Tipo P/efectos de los fármacos , Canales de Calcio Tipo P/genética , Canales de Calcio Tipo P/metabolismo , Canales de Calcio Tipo Q/efectos de los fármacos , Canales de Calcio Tipo Q/genética , Canales de Calcio Tipo Q/metabolismo , Línea Celular , Quelantes/farmacología , Electrofisiología , Técnica del Anticuerpo Fluorescente , Glutatión Transferasa/genética , Humanos , Activación del Canal Iónico/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Oocitos/citología , Oocitos/metabolismo , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Transfección , XenopusRESUMEN
Ca2+ channel auxiliary beta subunits have been shown to modulate voltage-dependent inactivation of various types of Ca2+ channels. The beta1 and beta2 subunits, that are differentially expressed with the L-type alpha1 Ca2+ channel subunit in heart, muscle and brain, can specifically modulate the Ca2+-dependent inactivation kinetics. Their expression in Xenopus oocytes with the alpha1C subunit leads, in both cases, to biphasic Ca2+ current decays, the second phase being markedly slowed by expression of the beta2 subunit. Using a series of beta subunit deletion mutants and chimeric constructs of beta1 and beta2 subunits, we show that the inhibitory site located on the amino-terminal region of the beta2a subunit is the major element of this regulation. These results thus suggest that different splice variants of the beta2 subunit can modulate, in a specific way, the Ca2+ entry through L-type Ca2+ channels in different brain or heart regions.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/farmacología , Empalme Alternativo , Animales , Encéfalo/metabolismo , Canales de Calcio Tipo L , Cartilla de ADN/genética , Regulación de la Expresión Génica , Miocardio/metabolismo , Oocitos/metabolismo , Técnicas de Placa-Clamp , Ratas , Proteínas Recombinantes/genética , Eliminación de Secuencia , XenopusRESUMEN
During sustained depolarization, voltage-gated Ca2+ channels progressively undergo a transition to a nonconducting, inactivated state, preventing Ca2+ overload of the cell. This transition can be triggered either by the membrane potential (voltage-dependent inactivation) or by the consecutive entry of Ca2+ (Ca2+-dependent inactivation), depending on the type of Ca2+ channel. These two types of inactivation are suspected to arise from distinct underlying mechanisms, relying on specific molecular sequences of the different pore-forming Ca2+ channel subunits. Here we report that the voltage-dependent inactivation (of the alpha1A Ca2+ channel) and the Ca2+-dependent inactivation (of the alpha1C Ca2+ channel) are similarly influenced by Ca2+ channel beta subunits. The same molecular determinants of the beta subunit, and therefore the same subunit interactions, influence both types of inactivation. These results strongly suggest that the voltage and the Ca2+-dependent transitions leading to channel inactivation use homologous structures of the different alpha1 subunits and occur through the same molecular process. A model of inactivation taking into account these new data is presented.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Activación del Canal Iónico , Secuencia de Bases , Canales de Calcio/genética , Cartilla de ADN , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Ca2+ current potentiation by conditioning depolarization is a general mechanism by which excitable cells can control the level of Ca2+ entry during repetitive depolarizations. Several types of Ca2+ channels are sensitive to conditioning depolarization, however, using clearly distinguishable mechanisms. In the case of L-type Ca2+ channels, prepulse-induced current facilitation can only be recorded when the pore-forming alpha1C subunit is coexpressed with the auxiliary beta1, beta3, or beta4, but not beta2, subunit. These four beta subunits are composed of two conserved domains surrounded by central, N-terminal, and C-terminal variable regions. Using different deleted and chimeric forms of the beta1 and beta2 subunits, we have mapped essential sequences for L-type Ca2+ channel facilitation. A first sequence, located in the second conserved domain of all beta subunits, is responsible for the promotion of current facilitation by the beta subunit. A second sequence of 16 amino acids, located on the N-terminal tail of the beta2 subunit, induces a transferable block of L-type current facilitation. Site-specific mutations reveal the essential inhibitory role played by three positive charges on this segment. The lack of prepulse-induced current facilitation recorded with some truncated forms of the beta2 subunit suggests the existence of an additional inhibitory sequence in the beta2 subunit.
Asunto(s)
Canales de Calcio/fisiología , Proteínas del Tejido Nervioso/fisiología , Plasticidad Neuronal , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Canales de Calcio/química , Canales de Calcio/genética , Canales de Calcio Tipo L , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Oocitos/metabolismo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Conformación Proteica , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , XenopusRESUMEN
We have expressed the alpha 1 A calcium channel subunit alone, and in combination with different beta subunits, and investigated the effect of the external Ba2+ concentration on the voltage dependence of activation. Increasing the external Ba2+ concentration from 2.5 to 40 mM induced in all cases a depolarising shift of the potential for half-activation. The magnitude of this shift however, was different depending on whether the alpha 1 A subunit was expressed alone or with a beta subunit. Consistently, calculated external surface-charge density and potential were larger when a beta subunit was expressed. These results suggest that expression of an auxiliary subunit can influence calcium channel gating by modifying the sensitivity of the voltage sensor to the membrane potential profile.
Asunto(s)
Canales de Calcio/fisiología , Potenciales de la Membrana , Animales , Bario/farmacología , Canales de Calcio/genética , Conductividad Eléctrica , Electroquímica , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Oocitos/metabolismo , Proteínas Recombinantes , XenopusRESUMEN
Xenopus oocytes have been injected with different combinations of expression plasmids carrying the rat brain alpha 1A and different beta (beta 1-4) Ca2+ channel subunit cDNAs. Whole-cell Ba2+ and Ca2+ currents were recorded up to seven days after injection. Intra-oocyte injection of BAPTA allowed us to record uncontaminated Ba2+, Sr2+ currents. The alpha 1A calcium channel showed relative current amplitudes according to the sequence: IBa2+ > ISr2+ > ICa2+. The ratio ICa2+/IBa2+ was significantly larger when compared to the class C L-type Ca2+ channel (alpha 1C). However, currents flowing through alpha 1A and alpha (1C) subunits saturate for similar Ba2+ concentrations and display the anomalous mole fraction effect in the presence of mixtures of Ba2+ and Ca2+ ions in the external medium. In oocytes expressing the alpha 1A Ca2+ channel subunit, switching from extracellular Ba2+ to Ca2+ also induced a depolarising shift of current-to-voltage relation and the steady-state inactivation curve, and increased the time-to-peak of the current. Inactivation kinetics were poorly affected. Changes in gating and voltage-dependence of activation, but not in the voltage-dependent inactivation, were independent from the coexpressed beta subunit (except with the beta 4 subunit). Our data constitute strong evidence for the existence of differences in intra-pore Ca2+ binding sites between the alpha 1C and alpha 1A subunits, and emphasise the influence of the charge carrier on the modulation of alpha 1A properties by the beta subunits.
Asunto(s)
Bario/metabolismo , Canales de Calcio/fisiología , Calcio/metabolismo , Estroncio/metabolismo , Animales , Sitios de Unión , Encéfalo/metabolismo , Calcio/farmacología , Canales de Calcio/química , Electrofisiología , Expresión Génica/genética , Activación del Canal Iónico/fisiología , Cinética , Microinyecciones , Oocitos , Técnicas de Placa-Clamp , Plásmidos , Ratas , XenopusRESUMEN
We have expressed muscle embryonic (alpha beta gamma delta) and adult (alpha beta delta epsilon) nicotinic acetylcholine receptors in Xenopus Laevis oocytes and measured their current reversal potentials in the presence of extracellular Na+, Ca2+, Sr2+ or Ba2+ ions. The ionic permeability ratio PCa2+/PNa+ was increased about 3 fold by the change in the subunit composition of the nicotinic acetylcholine receptor (replacement of the gamma by the epsilon subunit). A similar increase was also found when permeability to Ba2+ and Sr2+ ions was studies. Comparison of the nicotinic Ca2+ currents recorded from oocytes injected with embryonic or adult receptor subunit combinations also showed that the Ca2+ influx was significantly increased by expressing the epsilon subunit. This increase was estimated to change the contribution of the Ca2+ current to the total net inward current from 0.8% (in the case of the alpha beta gamma delta receptor) to 2.5% (in the case of the alpha beta delta epsilon receptor). Taken together, these results suggest that important modifications in the acetylcholine-mediated Ca2+ influx occurred during muscle innervation and underline the role of the nicotinic receptor in the developmental regulation of Ca2+ influx.
Asunto(s)
Calcio/metabolismo , Canales Iónicos/fisiología , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacología , Secuencia de Aminoácidos , Animales , Permeabilidad de la Membrana Celular , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Electrofisiología , Activación del Canal Iónico/fisiología , Metales Alcalinotérreos/farmacología , Ratones , Datos de Secuencia Molecular , Músculos/química , Músculos/embriología , Oocitos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Sodio/farmacología , Xenopus laevisRESUMEN
The class A Ca2+ channel alpha 1 subunit (alpha 1A) was expressed in Xenopus oocytes alone or in combination with the beta 1b, beta 2a, beta 3, or beta 4 subunit. Analysis of voltage-dependent activation and inactivation in the presence of 1.8 mM external Ca2+ showed an hyperpolarising shift of both relations when compared to similar recordings performed in the presence of 40 mM Ba2+. These shifts, which differed for activation and inactivation, were strongly modulated by the nature of the coexpressed beta subunit. On the other hand, for each combination, the kinetics of inactivation were similar in 1.8 mM Ca2+ and 40 mM Ba2+ (for example co-expression of the beta 2a subunit reduced inactivation using either 40 mM Ba2+ or 1.8 mM Ca2+). Thus, modulation of channel properties by the beta subunit is different in physiological Ca2+ or high Ba2+ concentrations. These results must be taken into consideration to extrapolate the role of the beta subunit in native cells.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Animales , Bario/metabolismo , Ácido Egtácico/análogos & derivados , Electrofisiología , Femenino , Indicadores y Reactivos , Cinética , Plásmidos/metabolismo , XenopusRESUMEN
Voltage-dependent facilitation of L-type Ca2+ channels is an important regulatory mechanism by which excitable cells modulate Ca2+ entry during a train of action potentials. Expression of the alpha1 and beta subunits of the alpha1C Ca2+ channel is necessary and sufficient to reproduce this kind of facilitation in Xenopus oocytes. Here we show that, by expressing the alpha1C together with different beta subunits in oocytes, the beta1, beta3 and beta4, but not the beta2 subunits are permissive for Ca2+ channel facilitation. The poor facilitation observed in rat ventricular cells, together with the presence of the beta2 subunit mRNA, suggest that beta2 may be the beta subunit associated with functional cardiac L-type Ca2+ channels.