RESUMEN
To explore whether CaMKII-dependent phosphorylation events mediate reperfusion arrhythmias, Langendorff perfused hearts were submitted to global ischemia/reperfusion. Epicardial monophasic or transmembrane action potentials and contractility were recorded. In rat hearts, reperfusion significantly increased the number of premature beats (PBs) relative to pre-ischemic values. This arrhythmic pattern was associated with a significant increase in CaMKII-dependent phosphorylation of Ser2814 on Ca(2+)-release channels (RyR2) and Thr17 on phospholamban (PLN) at the sarcoplasmic reticulum (SR). These phenomena could be prevented by the CaMKII-inhibitor KN-93. In transgenic mice with targeted inhibition of CaMKII at the SR membranes (SR-AIP), PBs were significantly decreased from 31±6 to 5±1 beats/3min with a virtually complete disappearance of early-afterdepolarizations (EADs). In mice with genetic mutation of the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), PBs decreased by 51.0±14.7%. In contrast, the number of PBs upon reperfusion did not change in transgenic mice with ablation of both PLN phosphorylation sites (PLN-DM). The experiments in SR-AIP mice, in which the CaMKII inhibitor peptide is anchored in the SR membrane but also inhibits CaMKII regulation of L-type Ca(2+) channels, indicated a critical role of CaMKII-dependent phosphorylation of SR proteins and/or L-type Ca(2+) channels in reperfusion arrhythmias. The experiments in RyR2-S2814A further indicate that up to 60% of PBs related to CaMKII are dependent on the phosphorylation of RyR2-Ser2814 site and could be ascribed to delayed-afterdepolarizations (DADs). Moreover, phosphorylation of PLN-Thr17 and L-type Ca(2+) channels might contribute to reperfusion-induced PBs, by increasing SR Ca(2+) content and Ca(2+) influx.
Asunto(s)
Arritmias Cardíacas/enzimología , Arritmias Cardíacas/etiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Daño por Reperfusión Miocárdica/complicaciones , Daño por Reperfusión Miocárdica/enzimología , Transducción de Señal , Potenciales de Acción , Sustitución de Aminoácidos , Animales , Arritmias Cardíacas/genética , Arritmias Cardíacas/prevención & control , Bencilaminas/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Corazón/efectos de los fármacos , Corazón/fisiopatología , Masculino , Ratones , Ratones Transgénicos , Mutación , Daño por Reperfusión Miocárdica/genética , Fosforilación/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Wistar , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Sulfonamidas/farmacologíaRESUMEN
Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.
Asunto(s)
Acidosis/complicaciones , Arritmias Cardíacas/etiología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Miocitos Cardíacos/enzimología , Retículo Sarcoplasmático/metabolismo , Acidosis/enzimología , Acidosis/fisiopatología , Potenciales de Acción , Animales , Arritmias Cardíacas/enzimología , Arritmias Cardíacas/fisiopatología , Bencilaminas/farmacología , Proteínas de Unión al Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Dantroleno/farmacología , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Concentración de Iones de Hidrógeno , Masculino , Ratones , Ratones Transgénicos , Miocitos Cardíacos/efectos de los fármacos , Péptidos/genética , Péptidos/metabolismo , Fosforilación , Ratas , Ratas Wistar , Rianodina/farmacología , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático/efectos de los fármacos , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/antagonistas & inhibidores , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Sulfonamidas/farmacología , Tapsigargina/farmacología , Factores de Tiempo , Función Ventricular Izquierda , Presión VentricularRESUMEN
CLC-3, a member of the CLC family of chloride channels, mediates function in many cell types in the body. The multifunctional calcium-calmodulin-dependent protein kinase II (CaMKII) has been shown to activate recombinant CLC-3 stably expressed in tsA cells, a human embryonic kidney cell line derivative, and natively expressed channel protein in a human colonic tumour cell line T84. We examined the CaMKII-dependent regulation of CLC-3 in a smooth muscle cell model as well as in the human colonic tumour cell line, HT29, using whole-cell voltage clamp. In CLC-3-expressing cells, we observed the activation of a Cl(-) conductance following intracellular introduction of the isolated autonomous CaMKII into the voltage-clamped cell via the patch pipette. The CaMKII-dependent Cl(-) conductance was not observed following exposure of the cells to 1 microm autocamtide inhibitory peptide (AIP), a selective inhibitor of CaMKII. Arterial smooth muscle cells express a robust CaMKII-activated Cl(-) conductance; however, CLC-3(-/-) cells did not. The N-terminus of CLC-3, which contains a CaMKII consensus sequence, was phosphorylated by CaMKII in vitro, and mutation of the serine at position 109 (S109A) abolished the CaMKII-dependent Cl(-) conductance, indicating that this residue is important in the gating of CLC-3 at the plasma membrane.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Activación del Canal Iónico/fisiología , Animales , Aorta/citología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Membrana Celular/metabolismo , Canales de Cloruro/química , Canales de Cloruro/genética , Células HT29 , Humanos , Técnicas In Vitro , Cinética , Ratones , Ratones Mutantes , Músculo Liso Vascular/citología , Músculo Liso Vascular/fisiología , Mutagénesis Sitio-Dirigida , Fosforilación , Estructura Terciaria de Proteína , TransfecciónRESUMEN
Annexin V is an intracellular protein that lacks a hydrophobic signal peptide. However, there are several studies reporting the extracellular presence of annexin V. In this study, we designed transgenes of annexin V with or without an attached secretory signal peptide and investigated the secretion of the transgene products in COS-7 cells. The signal peptide, targeted annexin V to the endoplasmic reticulum (ER), the Golgi and culture media of transfected cells. In contrast, without the signal peptide, annexin V was present only in the cytoplasm and was not detected in the medium. To confirm our results we also evaluated the presence of extracellular annexin V in two cultured cell lines: BeWo, a choriocarcinoma cell model of placental trophoblasts, and human umbilical vein endothelial cells (HUVEC). Our results showed that annexin V was immunolocalized on the surfaces of both cells but could not be detected in the culture medium of either cell type. Our results suggest that the secretion of annexin V required the recombinant addition of a hydrophobic signal peptide and that the limited quantities of endogenous cell surface annexin V on BeWo and HUVEC cells is most likely derived from adjacent damaged cells.
Asunto(s)
Anexina A5/metabolismo , Señales de Clasificación de Proteína/fisiología , Animales , Aniones , Anexina A5/análisis , Anexina A5/genética , Células COS , Membrana Celular/química , Permeabilidad de la Membrana Celular , Células Cultivadas , Coriocarcinoma/química , Coriocarcinoma/metabolismo , Medios de Cultivo Condicionados/química , Endotelio Vascular/química , Endotelio Vascular/metabolismo , Femenino , Humanos , Fosfolípidos/análisis , Embarazo , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/análisis , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Venas Umbilicales , Neoplasias Uterinas/química , Neoplasias Uterinas/metabolismoRESUMEN
Site-directed mutagenesis, electron microscopy, and X-ray crystallography were used to probe the structural basis of annexin IV-induced membrane aggregation and the inhibition of this property by protein kinase C phosphorylation. Site-directed mutants that either mimic (Thr6Asp, T6D) or prevent (Thr6Ala, T6A) phosphorylation of threonine 6 were produced for these studies and compared with wild-type annexin IV. In vitro assays showed that unmodified wild-type annexin IV and the T6A mutant, but not PKC-phosphorylated wild-type or the T6D mutant, promote vesicle aggregation. Electron crystallographic data of wild-type and T6D annexin IV revealed that, similar to annexin V, the annexin IV proteins form 2D trimer-based ordered arrays on phospholipid monolayers. Cryo-electron microscopic images of junctions formed between lipid vesicles in the presence of wild-type annexin IV indicated a separation distance corresponding to the thickness of two layers of membrane-bound annexin IV. In this orientation, a single layer of WT annexin IV, attached to the outer leaflet of one vesicle, would undergo face-to-face self-association with the annexin layer of a second vesicle. The 2.0-A resolution crystal structure of the T6D mutant showed that the mutation causes release of the N-terminal tail from the protein core. This change would preclude the face-to-face annexin self-association required to aggregate vesicles. The data suggest that reversible complex formation through phosphorylation and dephosphorylation could occur in vivo and play a role in the regulation of vesicle trafficking following changes in physiological states.
Asunto(s)
Anexina A4/genética , Anexina A4/metabolismo , Liposomas/química , Liposomas/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alanina/genética , Animales , Anexina A4/química , Anexina A4/ultraestructura , Sitios de Unión/genética , Bovinos , Microscopía por Crioelectrón , Cristalización , Cristalografía por Rayos X/métodos , Fosfatidilcolinas/química , Fosfatidilserinas/química , Fosforilación , Proteína Quinasa C/metabolismo , Ratas , Proteínas Recombinantes/ultraestructura , Treonina/genéticaRESUMEN
The multifunctional calcium/calmodulin-dependent protein kinase II, CaMKII, has been shown to regulate chloride movement and cellular function in both excitable and non-excitable cells. We show that the plasma membrane expression of a member of the ClC family of Cl(-) channels, human CLC-3 (hCLC-3), a 90-kDa protein, is regulated by CaMKII. We cloned the full-length hCLC-3 gene from the human colonic tumor cell line T84, previously shown to express a CaMKII-activated Cl(-) conductance (I(Cl,CaMKII)), and transfected this gene into the mammalian epithelial cell line tsA, which lacks endogenous expression of I(Cl,CaMKII). Biotinylation experiments demonstrated plasma membrane expression of hCLC-3 in the stably transfected cells. In whole cell patch clamp experiments, autonomously active CaMKII was introduced into tsA cells stably transfected with hCLC-3 via the patch pipette. Cells transfected with the hCLC-3 gene showed a 22-fold increase in current density over cells expressing the vector alone. Kinase-dependent current expression was abolished in the presence of the autocamtide-2-related inhibitory peptide, a specific inhibitor of CaMKII. A mutation of glycine 280 to glutamic acid in the conserved motif in the putative pore region of the channel changed anion selectivity from I(-) > Cl(-) to Cl(-) > I(-). These results indicate that hCLC-3 encodes a Cl(-) channel that is regulated by CaMKII-dependent phosphorylation.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Canales de Cloruro/metabolismo , Secuencia de Aminoácidos , Canales de Cloruro/química , Canales de Cloruro/genética , Clonación Molecular , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecciónRESUMEN
We have studied the regulation of Ca(2+)-dependent chloride (Cl(Ca)) channels in a human pancreatoma epithelial cell line (CFPAC-1), which does not express functional cAMP-dependent cystic fibrosis transmembrane conductance regulator chloride channels. In cell-free patches from these cells, physiological Ca(2+) concentrations activated a single class of 1-picosiemens Cl(-)-selective channels. The same channels were also stimulated by a purified type II calmodulin-dependent protein kinase (CaMKII), and in cell-attached patches by purinergic agonists. In whole-cell recordings, both Ca(2+)- and CaMKII-dependent mechanisms contributed to chloride channel stimulation by Ca(2+), but the CaMKII-dependent pathway was selectively inhibited by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P(4)). This inhibitory effect of Ins(3,4,5,6)P(4) on Cl(Ca) channel stimulation by CaMKII was reduced by raising [Ca(2+)] and prevented by inhibition of protein phosphatase activity with 100 nm okadaic acid. These data provide a new context for understanding the physiological relevance of Ins(3,4,5,6)P(4) in the longer term regulation of Ca(2+)-dependent Cl(-) fluxes in epithelial cells.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Calcio/metabolismo , Canales de Cloruro/metabolismo , Regulación de la Expresión Génica , Fosfatos de Inositol/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Membrana Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Electrofisiología , Células Epiteliales/metabolismo , Humanos , Ionóforos/farmacología , Cinética , Modelos Biológicos , Ácido Ocadaico/farmacología , Técnicas de Placa-Clamp , Isoformas de Proteínas , Células Tumorales Cultivadas , Regulación hacia Arriba , Uridina Trifosfato/metabolismoRESUMEN
BACKGROUND: Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Annexin V contains four such domains. Each of these domains has a highly conserved arginine (R). METHODS: To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. RESULTS: Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation. R43E and R115E exhibited much greater susceptibility to proteolytic digestion than the wild type. While Ca(2+)-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 microM Ca2+), R43E and R115E exhibited a 6- and 2-fold decrease in phospholipid affinity, respectively. Consistent with the different phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. CONCLUSIONS: Our data suggest that mutation of these highly conserved arginine residues in each of the four canonical domains of annexin have differential effects on the phospholipid binding, tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation, R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of annexin V by establishing hydrogen bonds and ionic bridges.
Asunto(s)
Anexina A5/genética , Arginina/genética , Secuencia Conservada , Secuencia de Aminoácidos , Animales , Anexina A5/metabolismo , Mutagénesis Sitio-Dirigida , Ratas , Relación Estructura-ActividadRESUMEN
Hexagonal phase (H(II))-preferring lipids such as phosphatidate, cardiolipin, and phosphatidylserine form nonbilayer molecular arrangements in lipid bilayers. While their presence in biological membranes has not been established, in vitro studies suggest that alterations in membrane properties modify their function. In this study, antiphospholipid monoclonal antibodies were developed against nonbilayer structures. One of the monoclonal antibodies identifies nonplanar surfaces in liposomes and in membranes of cultured cells. These results are the first evidence that natural membranes maintain a fragile balance between bilayer and nonbilayer lipid arrangements. Therefore, these antibodies can be used to evaluate the role of H(II)-preferring lipids in the modulation of membrane activities. Our studies demonstrated that nonplanar surfaces are highly immunogenic. Although these structures are normally transient, their formation can be stabilized by temperature variations, drugs, antibiotics, apolar peptides, and divalent cations. Our studies demonstrated that abnormal exposure of nonbilayer arrangements may induce autoimmune responses as found in the antiphospholipid syndrome.
Asunto(s)
Antígenos de Superficie/química , Antígenos de Superficie/inmunología , Liposomas/inmunología , Fosfolípidos/química , Fosfolípidos/inmunología , Animales , Anticuerpos/inmunología , Especificidad de Anticuerpos , RatonesRESUMEN
OBJECTIVES: Recurrent fetal loss can be a consequence of placental thrombosis, frequently occurring in autoimmune disorders such as antiphospholipid syndrome. A potent anticoagulant, annexin V, is abundant in placental tissues. We investigated the role of annexin V in maintaining fetal viability. STUDY DESIGN: Sites of annexin V activity in placenta were found and neutralized, and the physiologic consequences on fetal development were evaluated. To find extracellular binding sites for annexin V on placental membrane, 2 approaches were taken. An epitope-tagged recombinant annexin V was infused into pregnant BALB/c mice. Endogenous annexin V was evaluated by immunohistochemical techniques. To define a role for annexin V during pregnancy, annexin V was neutralized by tail-vein infusion of affinity-purified anti-annexin V antibodies immediately before mating, 16 hours before the vaginal plugs were observed. Fetal viability, number, and size were evaluated at days 11 or 15 after conception. RESULTS: Endogenous annexin V is enriched along the apical surfaces of trophoblasts. Animals infused with epitope-tagged annexin V had confirmed presence of extracellular binding sites for annexin V exclusively along these surfaces. In mice infused with anti-annexin V antibodies, various degrees of fetal absorption were observed. Thrombosis and necrosis were present in the fetal component of placentas from partially absorbed embryos. Focal necrosis and fibrosis were present in the decidua of placentas from embryos that were significantly smaller than the normal embryos in the same uterus. CONCLUSIONS: Apical surfaces of syncytiotrophoblasts in the placenta possess annexin V binding sites. The binding of annexin V to these coagulation-promoting surfaces is crucial for the maintenance of blood flow through the placenta and consequently for fetal viability. Infusion of anti-annexin V antibodies decreased the availability of annexin V to bind to the trophoblast surfaces and caused placental thrombosis, necrosis, and fetal loss. Our study suggests that anti-annexin V autoantibodies may contribute to recurrent pregnancy failure resulting from placental thrombosis, as found in patients with certain autoimmune diseases.
Asunto(s)
Aborto Espontáneo/etiología , Anexina A5/fisiología , Desarrollo Embrionario y Fetal/fisiología , Placentación , Animales , Anexina A5/inmunología , Anexina A5/farmacología , Autoanticuerpos/inmunología , Desarrollo Embrionario y Fetal/efectos de los fármacos , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos BALB C , Placenta/efectos de los fármacos , Placenta/patología , EmbarazoRESUMEN
1. We have studied the regulation of whole-cell chloride current in T84 colonic epithelial cells by inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5,6)P4). New information was obtained using (a) microcystin and okadaic acid to inhibit serine/threonine protein phosphatases, and (b) a novel functional tetrakisphosphate analogue, 1, 2-bisdeoxy-1,2-bisfluoro-Ins(3,4,5,6)P4 (i.e. F2-Ins(3,4,5,6)P4). 2. Calmodulin-dependent protein kinase II (CaMKII) increased chloride current 20-fold. This current (ICl,CaMK) continued for 7 +/- 1.2 min before its deactivation, or running down, by approximately 60 %. This run-down was prevented by okadaic acid, whereupon ICl,CaMK remained near its maximum value for >= 14.3 +/- 0.6 min. 3. F2-Ins(3, 4,5,6)P4 inhibited ICl,CaMK (IC50 = 100 microM) stereo-specifically, since its enantiomer, F2-Ins(1,4,5,6)P4 had no effect at >= 500 microM. Dose-response data (Hill coefficient = 1.3) showed that F2-Ins(3,4,5,6)P4 imitated only the non-co-operative phase of inhibition by Ins(3,4,5,6)P4, and not the co-operative phase. 4. Ins(3,4,5,6)P4 was prevented from blocking ICl,CaMK by okadaic acid (IC50 = 1.5 nM) and microcystin (IC50 = 0.15 nM); these data lead to the novel conclusion that, in situ, protein phosphatase activity is essential for Ins(3,4,5,6)P4 to function. The IC50 values indicate that more than one species of phosphatase was required. One of these may be PP1, since F2-Ins(3,4,5,6)P4-dependent current blocking was inhibited by okadaic acid and microcystin with IC50 values of 70 nM and 0.15 nM, respectively.
Asunto(s)
Calcio/fisiología , Comunicación Celular/fisiología , Canales de Cloruro/fisiología , Colon/fisiología , Fosfatos de Inositol/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Línea Celular , Colon/citología , Colon/metabolismo , Estimulación Eléctrica , Electrofisiología , Inhibidores Enzimáticos/farmacología , Células Epiteliales/metabolismo , Células Epiteliales/fisiología , Humanos , Potenciales de la Membrana/fisiología , Ácido Ocadaico/farmacología , Técnicas de Placa-Clamp , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , EstereoisomerismoRESUMEN
Calmodulin (CaM) acts as a primary mediator of calcium signaling by interacting with target proteins. We have previously shown that nuclear CaM is critical for cell cycle progression using a transgene containing four repeats of a CaM inhibitor peptide and nuclear targeting signals (J. Wang et al., J. Biol. Chem. 270 (1995) 30245 30248; Biochim. Biophys. Acta 1313 (1996) 223-228). To evaluate the role of CaM in the nucleus specifically during S phase of the cell cycle, a motif which stabilizes the mRNA only during S phase was included in the transgene. The CaM inhibitor mRNA transcript contains a self-annealing stem-loop derived from histone H2B at the 3' end. This structure provides stability of the mRNA only during S phase, thereby restricting CaM inhibitor expression to S phase. The inhibitor accumulates in the nucleus, particularly in the nucleoli. Flow cytometric analysis demonstrated that the CaM inhibitor is expressed in S and G2. Transfected cells show growth inhibition and a reduction in DNA synthesis. The CaM inhibitor peptide is a versatile reagent that allows spatial as well as temporal dissection of calmodulin function.
Asunto(s)
Calmodulina/antagonistas & inhibidores , Núcleo Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Quinasa de Cadena Ligera de Miosina/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Células COS , Ciclo Celular/efectos de los fármacos , ADN/biosíntesis , Células HeLa , Histonas/genética , Humanos , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Quinasa de Cadena Ligera de Miosina/genética , ARN Mensajero/genética , Conejos , Fase S , Factores de Tiempo , TransfecciónRESUMEN
The stunning sensations produced by electric fish, particularly the electric eel, Electrophorus electricus, have fascinated scientists for centuries. Within the last 50 years, however, electric cells of Electrophorus have provided a unique model system that is both specialized and appropriate for the study of excitable cell membrane electrophysiology and biochemistry. Electric tissue generates whole animal electrical discharges by means of membrane potentials that are remarkably similar to those of mammalian neurons, myocytes and secretory cells. Electrocytes express ion channels, ATPases and signal transduction proteins common to these other excitable cells. Action potentials of electrocytes represent the specialized end function of electric tissue whereas other excitable cells use membrane potential changes to trigger sophisticated cellular processes, such as myofilament cross-bridging for contraction, or exocytosis for secretion. Because electric tissue lacks these functions and the proteins associated with them, it provides a highly specialized membrane model system. This review examines the basic mechanisms involved in the generation of the electrical discharge of the electric eel and the membrane proteins involved. The valuable contributions that electric tissue continues to make toward the understanding of excitable cell physiology and biochemistry are summarized, particularly those studies using electrocytes as a model system for the study of the regulation of membrane excitability by second messengers and signal transduction pathways.
Asunto(s)
Órgano Eléctrico/fisiología , Electrophorus/fisiología , Animales , Membrana Celular/fisiología , Electrophorus/anatomía & histología , Electrofisiología , Proteínas de la Membrana/fisiologíaRESUMEN
Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a model system for the study of excitable membrane biochemistry and electrophysiology. Membrane receptors, ion channels, and ATPases utilized by electrocytes are conserved in mammalian neurons and myocytes. In this study, we show that Ca2+ predominates as the major mediator of electric tissue phosphorylation relative to cyclic AMP and cyclic GMP-induced phosphorylation. Mastoparan, a calmodulin inhibitor peptide, and a peptide corresponding to the pseudosubstrate region of mammalian calmodulin-dependent protein kinase II (CaMKII (281-302)) attenuated Ca(2+)-dependent phosphorylation in a dose-dependent manner. These experiments demonstrated that calmodulin-dependent protein kinase II activity predominates in electric tissue. The Electrophorus kinase was purified by a novel affinity chromatography procedure utilizing Ca2+/calmodulin-dependent binding to the CaMKII (281-302) peptide coupled to Sepharose. The purified 51 kDa calmodulin-dependent protein kinase II demonstrated extensive autophosphorylation and exhibited a 3- to 4-fold increase in Ca(2+)-independent activity following autophosphorylation. Immunofluorescent localization experiments demonstrated calmodulin to be abundant in electrocytes, particularly subjacent to the plasma membrane. Calmodulin-dependent protein kinase II had a punctate distribution indicating that it may be compartmentalized by association with vesicles or the cytoskeleton. As the primary mediator of phosphorylation within electric tissue, CaM kinase II may be critical for the regulation of the specialized electrophysiological function of electrocytes.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Órgano Eléctrico/enzimología , Electrophorus/metabolismo , Sistemas de Mensajero Secundario/fisiología , Animales , Encéfalo/enzimología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Técnica del Anticuerpo Fluorescente , Fosforilación , ConejosRESUMEN
Annexin VI is a member of a Ca(2+)-dependent phospholipid-binding protein family that participates in the transduction of the intracellular Ca2+ signal. We have identified annexin VI as one of the major annexins expressed differentially by sensory neurons of dorsal root ganglia (DRG) and by neurons of spinal cord (SC) of the rat and the mouse. This annexin shows a preferential localization at the plasma membrane of the soma and cellular processes, particularly in motoneurons of the SC. This finding suggests an active role of annexin VI in the Ca(2+)-dependent regulation of plasma membrane functions. To test this possibility, the neuronal function of annexin VI was evaluated by whole cell electrophysiology of mouse embryo SC and DRG neurons. An antibody was developed that has the property of neutralizing annexin VI-phospholipid interactions. The intracellular perfusion of individual neurons in culture, either from SC or DRG, with monospecific affinity-purified anti-annexin VI antibodies resulted in an increase in the magnitude of the K+ current and in an increase in the Ca2+ current in sensory neurons. Our results suggest that the endogenous annexin VI regulates the Ca2+ conductance, which indirectly modifies Ca(2+)-dependent ionic conductances in SC and DRG neurons.
Asunto(s)
Anexina A6/metabolismo , Calcio/metabolismo , Ganglios Espinales/metabolismo , Neuronas/metabolismo , Potasio/metabolismo , Transducción de Señal , Médula Espinal/metabolismo , Animales , Anexina A6/análisis , Células Cultivadas , Femenino , Transporte Iónico , Ratones , Embarazo , Ratas , Ratas Sprague-DawleyRESUMEN
Calmodulin (CaM) is a major intracellular calcium binding protein which has been implicated in the regulation of cell proliferation. Previous studies using chemically synthesized CaM antagonists and anti-sense RNA indicated that CaM is important for initiation of DNA synthesis and cell cycle progression. However, these methods reduce total intracellular CaM and globally interfering with all the CaM-dependent processes. In order to explore the function of nuclear CaM during the cell cycle, a CaM inhibitor peptide was targeted to the nucleus of intact mammalian cells. Cell progression through S-phase was assessed by incorporation of the thymidine analogue, BrdU. Cells were transfected for 48 h with either the CaM inhibitor peptide gene or the control plasmid prior to analysis. Approx. 70% of the control cells incorporated BrdU. In striking contrast, double immunofluorescent labeling demonstrated that none of the cells expressing the CaM inhibitor peptide entered S-phase. This result indicates that neutralization of nuclear CaM by targeted expression of a CaM inhibitor peptide blocks DNA synthesis and cell cycle progression.
Asunto(s)
Calmodulina/fisiología , Ciclo Celular/fisiología , Núcleo Celular/fisiología , ADN/biosíntesis , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Células COS , Calmodulina/antagonistas & inhibidores , Núcleo Celular/química , Expresión Génica , Datos de Secuencia Molecular , Músculo Esquelético/enzimología , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/genética , Péptidos/farmacología , Conejos , Proteínas Recombinantes de Fusión/análisis , TransfecciónRESUMEN
Calmodulin (CaM) is a major intracellular Ca2+ mediator protein involved in cell growth and differentiation. To evaluate calmodulin function in lung, it was necessary to construct a gene that encodes a high-affinity calmodulin binding peptide, since chemically synthesized calmodulin inhibitors lack binding and targeting specificity. This calmodulin inhibitor peptide gene was targeted to type II epithelial cells in transgenic mice using the human surfactant protein C promoter. Neutralization of calmodulin function in progenitor type II epithelial pneumocytes alters epithelial cell growth and differentiation, which prevents branching morphogenesis of the bronchial tree. Newborn transgenic animals have undeveloped lungs. This study indicates that type II lung epithelial cells require functional CaM for proliferation and development. The targeting of specific inhibitor peptides to a single lung cell type is an approach to evaluate the role of calmodulin, the ubiquitous calcium-dependent regulator protein, in lung development and disease.
Asunto(s)
Calmodulina/fisiología , Pulmón/embriología , Animales , Secuencia de Bases , Calmodulina/antagonistas & inhibidores , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario y Fetal , Femenino , Humanos , Ratones , Ratones Transgénicos/genética , Sondas Moleculares/genética , Datos de Secuencia Molecular , Alveolos Pulmonares/citología , Alveolos Pulmonares/embriología , Alveolos Pulmonares/metabolismo , Células Madre/metabolismoRESUMEN
The mechanism by which inositol 3,4,5,6-tetrakisphosphate (Ins(3,4,5, 6)P4) regulates chloride (Cl-) secretion was evaluated in the colonic epithelial cell line T84 using whole cell voltage clamp techniques. Our studies focused on the calcium-dependent chloride conductance (gClCa) that was activated either by mobilizing intracellular calcium (Cai) stores with thapsigargin or by introduction of the autonomous, autophosphorylated calmodulin-dependent protein kinase II (CaMKII) into the cell via the patch pipette. Basal concentrations of Ins(3,4,5,6)P4 (1 microM) present in the pipette solution had no significant effect on Cl- current; however, as the concentration of the polyphosphate was increased there was a corresponding reduction in anion current, with near complete inhibition at 8-10 microM Ins(3,4,5,6)P4. Corresponding levels are found in cells after sustained receptor-dependent activation of phospholipase C. The Ins(3,4,5, 6)P4-induced inhibition of gClCa was isomer specific; neither Ins(1, 3,4,5)P4, Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, nor Ins(1,3,4,5,6)P5 induced current inhibition at concentrations of up to 100 microM. Annexin IV also plays an inhibitory role in modulating gClCa in T84 cells. When 2 microM annexin IV was present in the pipette solution, a concentration that by itself has no effect on gClCa, the potency of Ins(3,4,5,6)P4 was approximately doubled. The combination of Ins(3,4,5,6)P4 and annexin IV did not alter the in vitro activity of CaMKII. These data demonstrate that Ins(3,4,5,6)P4 is an additional cellular signal that participates in the control of salt and fluid secretion, pH balance, osmoregulation, and other physiological activities that depend upon gClCa activation. Ins(3,4,5,6)P4 metabolism and action should also be taken into account when designing treatment strategies for cystic fibrosis.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Canales de Cloruro/fisiología , Cloruros/metabolismo , Fosfatos de Inositol/farmacología , Mucosa Intestinal/fisiología , Calcimicina/farmacología , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Línea Celular , Canales de Cloruro/efectos de los fármacos , Colon , Inhibidores Enzimáticos/farmacología , Humanos , Fosfatos de Inositol/síntesis química , Fosfatos de Inositol/química , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Potenciales de la Membrana/efectos de los fármacos , Estructura Molecular , Terpenos/farmacología , TapsigarginaRESUMEN
The annexins are a family of Ca(2+)-dependent phospholipid-binding proteins. In the present study, the spatial expression patterns of annexins I-VI were evaluated in the rat dorsal root ganglia (DRG) and spinal cord (SC) by using indirect immunofluorescence. Annexin I is expressed in small sensory neurons of the DRG, by most neurons of the SC, and by ependymal cells lining the central canal. Annexin II is expressed by most sensory neurons of the DRG but is primarily expressed in the SC by glial cells. Annexin III is expressed by most sensory neurons, regardless of size, by endothelial cells lining the blood vessels, and by the perineurium. In the SC, annexin III is primarily expressed by astrocytes. In the DRG and the SC, annexin IV is primarily expressed by glial cells and at lower levels by neurons. In the DRG, annexin V is expressed in relatively high concentrations in small sensory neurons in contrast to the SC, where it is expressed mainly by ependymal cells and by small-diameter axons located in the superficial laminae of the dorsal horn areas. Annexin VI is differentially expressed by sensory neurons of the DRG, being more concentrated in small neurons. In the SC, annexin VI has the most striking distribution. It is concentrated subjacent to the plasma membrane of motor neurons and their processes. The differential localization pattern of annexins in cells of the SC and DRG could reflect their individual biological roles in Ca(2+)-signal transduction within the central nervous system.
Asunto(s)
Anexinas/biosíntesis , Ganglios Espinales/química , Ratas Sprague-Dawley/fisiología , Médula Espinal/química , Animales , Anexina A1/análisis , Anexina A1/biosíntesis , Anexina A2/análisis , Anexina A2/biosíntesis , Anexina A3/análisis , Anexina A3/biosíntesis , Anexina A4/análisis , Anexina A4/biosíntesis , Anexina A5/análisis , Anexina A5/biosíntesis , Anexina A6/análisis , Anexina A6/biosíntesis , Anexinas/análisis , Anexinas/inmunología , Especificidad de Anticuerpos , Electroforesis en Gel de Poliacrilamida , Técnica del Anticuerpo Fluorescente Indirecta , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Neuronas Motoras/química , Neuroglía/química , Neuronas Aferentes/química , Conejos , Ratas , Ovinos , Especificidad de la Especie , Médula Espinal/citología , Médula Espinal/metabolismo , PorcinosRESUMEN
Annexin VI is a member of a family of Ca(2+)-dependent phospholipid-binding proteins that is expressed in many tissues, including the heart. It is a regulator of membrane-associated events, including the skeletal muscle ryanodine-sensitive Ca2+ release channel and the cardiac Na+/Ca2+ exchanger. The potential roles of annexin VI in Ca2+ signaling in cardiac myocytes were evaluated by targeting its overexpression to the hearts of transgenic mice. Expression of full-length human annexin VI cDNA was targeted to the heart using the alpha-myosin heavy chain gene promoter (Subramaniam, A., W. K. Jones, J. Gulick, S. Wert, J. Neumann, and J. Robbins. J. Biol. Chem. 266: 24613-24620, 1991). Five transgenic lines exhibited at least 10-fold overexpression of annexin VI protein in both atria and ventricles. Pathological evaluation indicated mice overexpressing annexin VI had enlarged dilated hearts, acute diffuse myocarditis, lymphocytic infiltration, moderate to severe fibrosis throughout the heart, and mild fibrosis around the pulmonary veins of the lungs. Contractile mechanics of cardiomyocytes isolated from hearts of transgenic animals showed frequency-dependent reduced percent shortening and decreased rates of contraction and relaxation compared with control animals. Cardiomyocytes isolated from transgenic animals had lower basal levels of intracellular free Ca2+ and a reduced rise in free Ca2+ following depolarization. After stimulation, intracellular free Ca2+ returned to basal levels faster in transgenic cells than in cells from control animals. These data demonstrate that the overexpression of annexin VI in the heart disrupts normal Ca2+ homeostasis and suggests that this dysfunction may be due to annexin VI regulation of pumps and/or exchangers in the membranes of cardiomyocytes.