RESUMEN
The H(+)-ATP synthase is a reversible engine of mitochondria that synthesizes or hydrolyzes ATP upon changes in cell physiology. ATP synthase dysfunction is involved in the onset and progression of diverse human pathologies. During ischemia, the ATP hydrolytic activity of the enzyme is inhibited by the ATPase inhibitory factor 1 (IF1). The expression of IF1 in human tissues and its participation in the development of human pathology are unknown. Here, we have developed monoclonal antibodies against human IF1 and determined its expression in paired normal and tumor biopsies of human carcinomas. We show that the relative mitochondrial content of IF1 increases significantly in carcinomas, suggesting the participation of IF1 in oncogenesis. The expression of IF1 varies significantly in cancer cell lines. To investigate the functional activity of IF1 in cancer, we have manipulated its cellular content. Overexpression of IF1 or of its pH-insensitive H49K mutant in cells that express low levels of IF1 triggers the up-regulation of aerobic glycolysis and the inhibition of oxidative phosphorylation with concurrent mitochondrial hyperpolarization. Treatment of the cells with the H(+)-ATP synthase inhibitor oligomycin mimicked the effects of IF1 overexpression. Conversely, small interfering RNA-mediated silencing of IF1 in cells that express high levels of IF1 promotes the down-regulation of aerobic glycolysis and the increase in oxidative phosphorylation. Overall, these findings support that the mitochondrial content of IF1 controls the activity of oxidative phosphorylation mediating the shift of cancer cells to an enhanced aerobic glycolysis, thus supporting an oncogenic role for the de-regulated expression of IF1 in cancer.
Asunto(s)
ATPasas de Translocación de Protón Mitocondriales/metabolismo , Neoplasias/metabolismo , Proteínas/metabolismo , Animales , Western Blotting , Línea Celular , Línea Celular Tumoral , Glucólisis/efectos de los fármacos , Glucólisis/genética , Células HeLa , Células Hep G2 , Humanos , Técnicas In Vitro , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Microscopía Fluorescente , Mitocondrias/metabolismo , ATPasas de Translocación de Protón Mitocondriales/antagonistas & inhibidores , Mutación , Oligomicinas/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Proteínas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/fisiología , Ratas , Proteína Inhibidora ATPasaRESUMEN
Nicotinic acetylcholine receptors (nAChRs) transmit the agonist signal to the channel gate through a number of extracellular domains. We have previously shown that particular details of the process of coupling binding to gating could be quantitative and qualitatively different in muscle and neuronal type nAChRs. We have extended previous studies on homomeric alpha7 nAChRs to heteromeric alpha3beta4 nAChRs, by mutating residues located at loops 2 and 7, and M2-M3 linker of both alpha3 and beta4 subunits which, in order to monitor surface expression, were modified to bind alpha-bungarotoxin, and expressed in Xenopus oocytes. We show that, in general, mutations in these domains of both alpha3 and beta4 subunits affect the gating function, although the effects are slightly larger if they are inserted in the alpha3 subunit. However, the involvement of a previously reported intrasubunit interaction in coupling (Gln48-Ile130) seems to be restricted to the beta4 subunit. We also show that mutations at these domains, particularly loop 2 of the alpha3 subunit, change the pharmacological profile of alpha3beta4 nAChRs, decreasing nicotine's and increasing cytisine's effectiveness relative to acetylcholine. It is concluded that, unlike muscle nAChRs, the non-alpha subunits play a relevant role in the coupling process of neuronal alpha3beta4 nAChRs.
Asunto(s)
Membrana Celular/química , Activación del Canal Iónico/genética , Receptores Nicotínicos/química , Acetilcolina/metabolismo , Acetilcolina/farmacología , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Femenino , Humanos , Activación del Canal Iónico/efectos de los fármacos , Mutación/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Oocitos , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/química , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/genética , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/genética , Xenopus laevisRESUMEN
Recently, the inevitable metabolic reprogramming experienced by cancer cells as a result of the onset of cellular proliferation has been added to the list of hallmarks of the cancer cell phenotype. Proliferation is bound to the synchronous fluctuation of cycles of an increased glycolysis concurrent with a restrained oxidative phosphorylation. Mitochondria are key players in the metabolic cycling experienced during proliferation because of their essential roles in the transduction of biological energy and in defining the life-death fate of the cell. These two activities are molecularly and functionally integrated and are both targets of commonly altered cancer genes. Moreover, energetic metabolism of the cancer cell also affords a target to develop new therapies because the activity of mitochondria has an unquestionable tumor suppressor function. In this review, we summarize most of these findings paying special attention to the opportunity that translation of energetic metabolism into the clinics could afford for the management of cancer patients. More specifically, we emphasize the role that mitochondrial beta-F1-ATPase has as a marker for the prognosis of different cancer patients as well as in predicting the tumor response to therapy.
Asunto(s)
Proliferación Celular , Genes Supresores de Tumor , Mitocondrias/metabolismo , Mitocondrias/patología , Neoplasias/patología , ATPasas de Translocación de Protón/genética , Metabolismo Energético , Humanos , Neoplasias/metabolismo , Fosforilación OxidativaRESUMEN
We studied the role of the alpha-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in alpha7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the alpha-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of alpha7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (alpha3beta4 and alpha4beta2) and 5-HT(3A) receptors also abolished their expression at the membrane. We conclude that the N-terminal alpha-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.
Asunto(s)
Receptores Nicotínicos/química , Receptores Nicotínicos/fisiología , Animales , Bungarotoxinas/metabolismo , Bovinos , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/genética , Leucina/genética , Modelos Moleculares , Mutagénesis/fisiología , Mutación/genética , Neuroblastoma , Oocitos , Prolina/genética , Estructura Secundaria de Proteína/genética , Estructura Secundaria de Proteína/fisiología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/genética , Receptores de Serotonina 5-HT3/genética , Transfección/métodos , Xenopus , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Binding of agonists to nicotinic acetylcholine receptors (nAChR) is coupled to channel opening through local rearrangements of different domains of the protein. Recent structural data suggest that two of these regions could be the loop 5 (L5) and the beta-strand beta6', both forming the inner part of the N-terminal domain. Amino acids in these domains were mutated in alpha7 nAChRs, and expression levels and functional responses of mutant receptors were measured. Mutations located at the putative apex of L5, Asp97 and Glu98, and also at Phe100, gave receptors with smaller currents, showing qualitative differences with respect to muscle nAChRs. In contrast, mutations in the beta-strand beta6' (at Phe124 and Lys125) showed increased functional responses. Mutations affected equally the responses to acetylcholine and dimethylphenylpiperazinium, except in Phe100 where the latter was sevenfold less effective than in wild-type. Currents in mutants decayed with almost the same kinetics, ruling out large effects on desensitization. Analysis of double mutants demonstrated a functional coupling among the three electrically charged amino acids Asp97, Glu98, and Lys125, and also between Phe100 and Phe124. The results are compatible with the involvement of functional interactions between L5 and beta-strand beta6' during nAChR activation.
Asunto(s)
Activación del Canal Iónico/fisiología , Receptores Nicotínicos/química , Receptores Nicotínicos/fisiología , Alanina/genética , Sustitución de Aminoácidos , Animales , Bungarotoxinas/farmacocinética , Bovinos , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Mutación/fisiología , Agonistas Nicotínicos/farmacocinética , Agonistas Nicotínicos/farmacología , Oocitos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Binding of agonists to nicotinic acetylcholine receptors results in channel opening. Previously, we have shown that several charged residues at three different domains of the alpha7 nicotinic receptor are involved in coupling binding and gating, probably through a network of electrostatic interactions. This network, however, could also be integrated by other residues. To test this hypothesis, non-charged amino acids were mutated and expression levels and electrophysiological responses of mutant receptors were determined. Mutants at positions Asn47 and Gln48 (loop 2), Ile130, Trp134, and Gln140 (loop 7), and Thr264 (M2-M3 linker) showed poor or null functional responses, despite significant membrane expression. By contrast, mutants F137A and S265A exhibited a gain of function effect. In all cases, changes in dose-response relationships were small, EC(50) values being between threefold smaller and fivefold larger, arguing against large modifications of agonist binding. Peak currents decayed at the same rate in all receptors except two, excluding large effects on desensitization. Thus, the observed changes could be mostly caused by alterations of the gating characteristics. Moreover, analysis of double mutants showed an interconnection between some residues in these domains, especially Gln48 with Ile130, suggesting a potential coupling between agonist binding and channel gating through these amino acids.
Asunto(s)
Aminoácidos/fisiología , Activación del Canal Iónico/fisiología , Agonistas Nicotínicos/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacología , Sustitución de Aminoácidos , Animales , Bungarotoxinas/metabolismo , Bovinos , Yoduro de Dimetilfenilpiperazina/farmacología , Relación Dosis-Respuesta a Droga , Electrofisiología , Activación del Canal Iónico/genética , Mutagénesis , Oocitos , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
We studied the role of the cytoplasmic regions adjacent to the M3 and M4 transmembrane segments of alpha7 nicotinic receptors in the expression of functional channels. For this purpose, a total of 50 amino acids were mutated throughout the mentioned regions. Mutants close to M3, from Arg294 to Leu321, showed slight modifications in the levels of alpha-bungarotoxin binding sites and acetylcholine-evoked currents. Exceptions were mutants located at two clusters (His296 to Pro300 and Ile312 to Trp316), which exhibited low expression levels. In addition, some mutants showed altered functional responses. Many mutants close to M4 showed increased receptor expression, especially the ones located at the hydrophobic face of a putative amphipathic helix. This effect seems to be the consequence of a combination of increased receptor biosynthesis, higher transport efficiency and delayed degradation, such that we postulate that elements in the amphipathic domain strongly influence receptor stability. Finally, some mutants in this region showed altered functional responses: elimination of positively charged residues (Arg424 and Arg426) increased currents, whereas the opposite was observed upon suppression of negatively charged ones (Glu430 and Glu432). These results suggest that the cytoplasmic regions close to M3 and M4 play important structural and functional roles.
Asunto(s)
Aminoácidos/metabolismo , Citoplasma/metabolismo , Expresión Génica/fisiología , Proteínas Mutantes/metabolismo , Receptores Nicotínicos/fisiología , Acetilcolina/farmacología , Animales , Bungarotoxinas/farmacocinética , Citoplasma/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Isótopos de Yodo/farmacocinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Microinyecciones/métodos , Datos de Secuencia Molecular , Oocitos , Técnicas de Placa-Clamp/métodos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína , Ratas , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Relación Estructura-Actividad , Xenopus , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
This study was designed to examine the kinetics of neurotransmitter release using the carbon fiber amperometric technique on cells in slices of mouse adrenal glands superfused with bicarbonate phosphate buffer-based solutions. The exocytotic amperometric response evoked by electrical stimulation was significantly faster than that produced after exogenous application of ACh or K+. Splanchnic nerve-evoked neurotransmitter release was blocked by hexamethonium, indicating the involvement of ACh nicotinic receptors. We discuss the implications of our data for understanding the mechanisms underlying the vesicle fusion process.
Asunto(s)
Médula Suprarrenal/fisiología , Células Cromafines/fisiología , Exocitosis/fisiología , Acetilcolina/farmacología , Médula Suprarrenal/efectos de los fármacos , Animales , Células Cromafines/efectos de los fármacos , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/fisiología , Hexametonio/farmacología , Técnicas In Vitro , Cinética , Ratones , Neurotransmisores/fisiología , Potasio/farmacología , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/fisiologíaRESUMEN
Although the specific interaction between synaptic protein SNAP-25 and the alpha1A subunit of the Cav2.1 channels, which conduct P/Q-type Ca2+ currents, has been confirmed in in vitro-translated proteins and brain membrane studies, the question of how native proteins can establish this association in situ in developing neurons remains to be elucidated. Here we report data regarding this interaction in bovine chromaffin cells natively expressing both proteins. The two carboxyl-terminal splice variants of the alpha1A subunit identified in these cells share a synaptic protein interaction ('synprint') site within the II/III loop segment and are immunodetected by a specific antibody against bovine alpha1A protein. Moreover, both alpha1A isoforms form part of the P/Q-channels-SNARE complexes in situ because they are coimmunoprecipitated from solubilized chromaffin cell membranes by a monoclonal SNAP-25 antibody. The distribution of alpha1A and SNAP-25 was studied in round or transdifferentiated chromaffin cells using confocal microscopy and specific antibodies: the two proteins are colocalized at the cell body membrane in both natural cell types. However, during the first stages of the cell transdifferentiation process, SNAP-25 migrates alone out to the developing growth cone and what will become the nerve endings and varicosities of the mature neurites; alpha1A follows and colocalizes to SNAP-25 in the now mature processes. These observations lead us to propose that the association between SNAP-25 and alpha1A during neuritogenesis might promote not only the efficient coupling of the exocytotic machinery but also the correct insertion of P/Q-type channels at specialized active zones in presynaptic neuronal terminals.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Células Cromafines/citología , Células Cromafines/fisiología , Neuritas/metabolismo , Subunidades de Proteína/metabolismo , Proteína 25 Asociada a Sinaptosomas/metabolismo , Animales , Secuencia de Bases , Northern Blotting/métodos , Western Blotting/métodos , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/genética , Bovinos , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Células Cultivadas , Células Cromafines/clasificación , Células Cromafines/efectos de los fármacos , Dopamina beta-Hidroxilasa/metabolismo , Relación Dosis-Respuesta a Droga , Técnica del Anticuerpo Fluorescente/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoprecipitación/métodos , Ratones , Microscopía Confocal/métodos , Modelos Moleculares , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Potasio/farmacología , Subunidades de Proteína/genética , ARN Mensajero/metabolismo , Conejos , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Alineación de Secuencia/métodos , Proteína 25 Asociada a Sinaptosomas/genéticaRESUMEN
In bovine chromaffin cells fast-superfused with Krebs-HEPES solution containing 1-2 mM Ca2+, 5 s pulses of choline (1-10 mM), elicited catecholamine secretory responses that were only approximately 10% of those evoked by ACh (0.01-0.1 mM). However, in high-Ca2+ solutions (10-20 mM) the size of the choline secretory responses approached those of ACh. The choline responses (10 mM choline in 20 mM Ca2+, 10Cho/20Ca2+) tended to decline upon repetitive pulsing, whereas those of ACh were well maintained. The confocal [Ca2+]c increases evoked by 10Cho/20Ca2+ were similar to those of ACh. Whereas 10Cho/20Ca2+ caused mostly hyperpolarization of chromaffin cells, 0.1ACh/20 Ca2+ caused first depolarization and then hyperpolarization; in regular solutions (2 mM Ca2+), the hyperpolarizing responses did not show up. In Xenopus oocytes injected with mRNA for bovine alpha7 nicotinic receptors (nAChRs), 10Cho/20 Ca2+ fully activated an inward current; in oocytes expressing alpha3beta4, however, the inward current elicited by choline amounted to only 4% of the size of alpha7 current. Our results suggest that choline activates the entry of Ca2+ through alpha7 nAChRs; this leads to a cytosolic concentration of calcium ([Ca2+]c) rise that causes the activation of nearby Ca2+-dependent K+ channels and the hyperpolarization of the chromaffin cell. This response, which could be unmasked provided that cells were stimulated with high-Ca2+ solutions, may be the underlying mechanism through which choline exerts a modulatory effect on the electrical activity of the chromaffin cell and on neurotransmitter release at cholinergic synapses.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Catecolaminas/metabolismo , Colina/farmacología , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Acetilcolina/farmacología , Animales , Atropina/farmacología , Calcio/farmacología , Bovinos , Conductividad Eléctrica , Mecamilamina/farmacología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Potasio/farmacología , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Xenopus laevisRESUMEN
Cyclin-dependent kinase 5 (Cdk5) is a serine/threonine kinase involved in synaptogenesis and brain development, and its enzymatic activity is essential for slow forms of synaptic vesicle endocytosis. Recent work also has implicated Cdk5 in exocytosis and synaptic plasticity. Pharmacological inhibition of Cdk5 modifies secretion in neuroendocrine cells, synaptosomes, and brain slices; however, the specific mechanisms involved remain unclear. Here we demonstrate that dominant-negative inhibition of Cdk5 increases quantal size and broadens the kinetics of individual exocytotic events measured by amperometry in adrenal chromaffin cells. Conversely, Cdk5 overexpression narrows the kinetics of fusion, consistent with an increase in the extent of kiss-and-run exocytosis. Cdk5 inhibition also increases the total charge and current of catecholamine released during the amperometric foot, representing a modification of the conductance of the initial fusion pore connecting the granule and plasma membrane. We suggest that these effects are not attributable to an alteration in catecholamine content of secretory granules and therefore represent an effect on the fusion mechanism itself. Finally, mutational silencing of the Cdk5 phosphorylation site in Munc18, an essential protein of the late stages of vesicle fusion, has identical effects on amperometric spikes as dominant-negative Cdk5 but does not affect the amperometric feet. Cells expressing Munc18 T574A have increased quantal size and broader kinetics of fusion. These results suggest that Cdk5 could, in part, control the kinetics of exocytosis through phosphorylation of Munc18, but Cdk5 also must have Munc18-independent effects that modify fusion pore conductance, which may underlie a role of Cdk5 in synaptic plasticity.
Asunto(s)
Quinasas Ciclina-Dependientes/fisiología , Exocitosis , Fusión de Membrana , Animales , Catecolaminas/metabolismo , Bovinos , Permeabilidad de la Membrana Celular , Células Cultivadas , Células Cromafines/fisiología , Quinasa 5 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , Conductividad Eléctrica , Células HeLa , Humanos , Cinética , Proteínas Munc18 , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células PC12 , Fosforilación , Ratas , Transfección , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMEN
The auxiliary Ca(v)beta subunit is essential for functional expression of high-voltage activated Ca(2+) channels. Here, we describe a lure sequence designed to sequester the Ca(v)beta subunits in transfected bovine chromaffin cells. This sequence is composed of the extracellular and transmembrane domains of the alpha chain of the human CD8, the I-II loop of Ca(v)2.1 subunit, and EGFP. We showed that expressing the CD8-I-II-EGFP sequence in chromaffin cells led to a >50% decrease in overall Ca(2+) current density. Although this decrease involved all the Ca(2+) channel types (L, N, P/Q, R), the proportion of each type supporting the remaining current was altered. A similar effect was observed after transfection when measuring the functional role of Ca(2+) channels in catecholamine release by chromaffin cells: global decrease of release and change of balance between the different channel types supporting it. Possible explanations for this apparent discrepancy are further discussed.
Asunto(s)
Antígenos CD8/metabolismo , Canales de Calcio Tipo N/fisiología , Activación del Canal Iónico/fisiología , Proteínas Luminiscentes/metabolismo , Ingeniería de Proteínas/métodos , Subunidades de Proteína/fisiología , Animales , Antígenos CD8/genética , Canales de Calcio/genética , Canales de Calcio/fisiología , Canales de Calcio Tipo N/genética , Bovinos , Células Cultivadas , Células Cromafines , Regulación de la Expresión Génica/fisiología , Proteínas Fluorescentes Verdes , Humanos , Activación del Canal Iónico/genética , Proteínas Luminiscentes/fisiología , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
The effects of the toxin SXN482 on Ca2+ channel currents (ICa), Na+ currents (INa), and K+ currents (IK) have been studied in bovine adrenal medullary chromaffin cells voltage-clamped at -80 mV. Currents were elicited by depolarising pulses to 0-10 mV (ICa and INa) or to +60 mV (IK). SNX482 blocked ICa in a concentration-dependent manner. The inhibition curve exhibited two phases. The first high-affinity phase comprised 28% of the whole-cell current and exhibited an IC50 of 30.2 nM. The second low-affinity phase comprised over 70% of ICa and had an IC50 of 758.6 nM. Blockade was rapid and fully reversible upon washout of the toxin. Occlusion experiments showed additivity of blockade exerted by nifedipine plus SNX482 (0.3 microM) and by omega-conotoxin GVIA plus SNX482. In contrast, blockade exerted by combined omega-agatoxin IVA plus SNX482 (about 50% of the whole cell) did not show additivity. At 0.3 microM and higher concentrations, SNX482 delayed the inactivation of INa. The time constant (tau) for inactivation of INa in control conditions doubled in the presence of 0.5 microM SNX482. At 0.3 microM, SNX482 did not affect IK. Our data demonstrate that: (i) SNX482 selectively blocks P/Q Ca2+ channels at submicromolar concentrations; (ii) the toxin partially blocks Na+ channels; (iii) SNX482 delays the inactivation of Na+ channels. These results reveal novel properties of SNX482 and cast doubts on the claimed selectivity and specificity of the toxin to block the R-type Ca2+ channel.
Asunto(s)
Canales de Calcio Tipo P/fisiología , Canales de Calcio Tipo Q/fisiología , Células Cromafines/efectos de los fármacos , Canales de Sodio/fisiología , Venenos de Araña/farmacología , Glándulas Suprarrenales/citología , Glándulas Suprarrenales/efectos de los fármacos , Glándulas Suprarrenales/fisiología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Bovinos , Células Cultivadas , Células Cromafines/citología , Células Cromafines/fisiología , Relación Dosis-Respuesta a Droga , Técnicas de Placa-ClampRESUMEN
At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca(2+) concentration, [Ca(2+)](c), depend on at least three efficient regulatory mechanisms: (1) the plasmalemmal Ca(2+) channels; (2) the endoplasmic reticulum (ER); and (3) the mitochondria. High-voltage activated Ca(2+) channels of the L, N, P/Q, and R subtypes are expressed with different densities in various mammalian species; they are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca(2+)](c). Targeted aequorin and confocal microscopy show that Ca(2+) entry through Ca(2+) channels can refill the ER to near millimolar concentrations and causes the release of ER Ca(2+) (CICR). We have also seen that, depending on its degree of filling, the ER may act as a sink or source of Ca(2+) that modulates the release of catecholamine. Targeted aequorins with different Ca(2+) affinities show that mitochondria undergo surprisingly rapid millimolar Ca(2+) transients ([Ca(2+)](M)) upon stimulation of chromaffin cells with ACh, high K(+), or caffeine. Physiological stimuli generate [Ca(2+)](c) microdomains at these functional complexes in which the local subplasmalemmal [Ca(2+)](c) rises abruptly from 0.1 micro M to about 50 micro M. This triggers CICR, mitochondrial Ca(2+) uptake, and exocytosis in nearby secretory active sites. That this is true is shown by the observation that protonophores abolish mitochondrial Ca(2+) uptake and drastically increase catecholamine release by 3- to 5-fold. This increase is likely due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; such transport might be controlled by Ca(2+) redistribution to the cytoskeleton, through CICR and/or mitochondrial Ca(2+) release.
Asunto(s)
Calcio/metabolismo , Células Cromafines/metabolismo , Exocitosis , Animales , Sitios de Unión , Cafeína/farmacología , Catecolaminas/metabolismo , Bovinos , Citoesqueleto , Retículo Endoplásmico/metabolismo , Humanos , Cinética , Modelos Biológicos , Potasio/metabolismo , Potasio/farmacología , Estructura Terciaria de ProteínaAsunto(s)
Células Cromafines/metabolismo , Intercambiador de Sodio-Calcio/fisiología , Tiourea/análogos & derivados , Animales , Catecolaminas/metabolismo , Bovinos , Citosol/metabolismo , Relación Dosis-Respuesta a Droga , Exocitosis , Perfusión , Potasio/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Tiourea/farmacología , Factores de TiempoRESUMEN
Simultaneous recordings of inward whole-cell Ca(2+) channel currents (I(Ca) ) and increments of capacitance as an indication of exocytosis (Delta(Cm)), were performed in voltage-clamped single adrenal chromaffin cells from wild-type and alpha(1A) subunit deficient mice, using the perforated-patch configuration of the patch-clamp technique. Using protocol #1 (one single Ca(2+) channel blocker per cell), to dissect the components of I(Ca), L channels contributed 43%, N channels 35% and P/Q channels 30% to the total I(Ca) of wild-type cells. Using protocol #2 (cumulative sequential addition of 3 microm nifedipine, 1 microm omega-conotoxin GVIA, and 1 microm omega-agatoxin IVA), L, N and P/Q channels contributed 40%, 34% and 14%, respectively, to I(Ca); an R component of around 11% remained. In wild-type mice the changes of Delta(Cm) paralleled those of I(Ca). In alpha(1A) deficient mice the L component of I(Ca) rose to 53% while the P/Q disappeared; the N and R components were similar. In these mice, Delta(Cm) associated to N and R channels did not vary; however, the P/Q component was abolished while the L component increased by 20%. In conclusion, exocytosis was proportional to the relative density of each Ca(2+) channel subtype, L, N, P/Q, R. Ablation of the alpha(1A) gene led to a loss of P/Q channel current and to a compensatory increase of L channel-associated secretion; however, this compensation was not sufficient to maintain the overall exocytotic response, that was diminished by 35% in alpha(1A) -deficient mice. This may be due to altered Ca(2+) homeostasis in these mice, as compared to wild mouse chromaffin cells.
Asunto(s)
Canales de Calcio Tipo N/deficiencia , Canales de Calcio Tipo N/metabolismo , Canales de Calcio/metabolismo , Células Cromafines/metabolismo , Exocitosis/fisiología , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Canales de Calcio Tipo L/metabolismo , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo R/metabolismo , Células Cromafines/efectos de los fármacos , Capacidad Eléctrica , Estimulación Eléctrica , Exocitosis/efectos de los fármacos , Genotipo , Heterocigoto , Homocigoto , Técnicas In Vitro , Ratones , Ratones Noqueados , Nifedipino/farmacología , Técnicas de Placa-Clamp , Subunidades de Proteína , omega-Agatoxina IVA/farmacología , omega-Conotoxina GVIA/farmacologíaRESUMEN
During fast superfusion of bovine chromaffin cells with normal Krebs-HEPES solution containing 2 mM Ca2+, pulses of 100 microM ACh or 100 mM K+ of increasing duration (1-5 s) caused similar exocytosis of about 3-4 microC catecholamine. Depletion of endoplasmic reticulum (ER) Ca2+ by pretreatment with 1 microM thapsigargin, 10 mM caffeine and 10 microM ryanodine more than halved the responses to ACh but did not affect the responses to K+ responses. In these ER Ca2+-depleted cells the protonophore carbonylcyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) (20 microM given during the 5 s preceding each pulse) augmented the responses to ACh responses fourfold for all pulse durations applied (1-5 s) whereas responses to K+ were potentiated twofold with 1 to 2 s pulses but were not affected with longer pulse durations. ACh pulses applied to fura-2-loaded cells evoked increases of bulk cytosolic [Ca2+] ([Ca2+](c)) that were substantially smaller than those elicited by K+ pulses. Confocal microscopy of fluo-3-loaded cells showed that ACh pulses elicited discrete and more localized [Ca2+](c) elevations, whereas K+ pulses produced higher [Ca2+](c) transients that spread out quickly throughout the cytosol. These results suggest that mitochondria sense the increase of local [Ca2+](c) elicited by ACh (that evokes firing of action potentials) much better than that induced by K+ (that produces sustained cell depolarisation). This implies that mitochondria are more sensitive to the local [Ca2+](c) changes resulting from the physiological triggering of action potentials by ACh.