RESUMEN
Na(+)/Ca(2+)-K(+) exchangers (NCKX; gene family SLC24) are plasma membrane Ca(2+) transporters that mediate the extrusion of one Ca(2+) ion and one K(+) ion in exchange for four Na(+) ions. NCKX is modeled to have two sets of five transmembrane segments separated by a large cytosolic loop; within each set of transmembrane segments are regions of internal symmetry termed alpha(1) and alpha(2) repeats. The central residues that are important for Ca(2+) and K(+) liganding and transport have been identified in NCKX2, and they comprise three central acidic residues, Glu(188) in alpha(1) and Asp(548) and Asp(575) in alpha(2), as well as Ser/Thr residues one-helical turn away from these residues. In this study, we have scanned through more than 100 single-residue substitutions of NCKX2 for shifts in Na(+) affinity using a fluorescence assay to monitor changes in free Ca(2+) in HEK293 cells treated with gramicidin to control intracellular Na(+). We have identified 31 residues that, when substituted, result in shifts in Na(+) affinity, either toward higher or lower K(m) values when compared with wild type NCKX2 (K(m) for Na(+) 58 mm). These residues include the central acidic residues Glu(188), Asp(548), and Asp(575), and their neighboring residues in alpha(1) and alpha(2), in addition to a number of newly investigated residues in transmembrane segment 3. Our results relate the identification of residues important for Na(+) transport in this study to those previously identified as important in the counter-transport of Ca(2+) and K(+), lending support to the alternating access model of transmembrane transport.
Asunto(s)
Intercambiador de Sodio-Calcio/metabolismo , Sodio/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Humanos , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Aminoácido , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/genéticaRESUMEN
BACKGROUND: Smoothelin-like 1 (SMTNL1, also known as CHASM) plays a role in promoting relaxation as well as adaptive responses to exercise, pregnancy and sexual development in smooth and skeletal muscle. Investigations of Smtnl1 transcriptional regulation are still lacking. Thus, in this study, we identify and characterize key regulatory elements of the mouse Smtnl1 gene. RESULTS: We mapped the key regulatory elements of the Smtnl1 promoter region: the transcriptional start site (TSS) lays -44 bp from the translational start codon and a TATA-box motif at -75 bp was conserved amongst all mammalian Smtnl1 promoters investigated. The Smtnl1 proximal promoter enhances expression up to 8-fold in smooth muscle cells and a second activating region lays 500 bp further upstream. Two repressing motifs were present (-118 to -218 bp and -1637 to -1869 bp). The proximal promoter is highly conserved in mammals and contains a mirror repeat sequence. In silico analysis suggests many transcription factors (notably MyoD) could potentially bind within the Smtnl1 proximal promoter sequence. CONCLUSION: Smtnl1 transcript was identified in all smooth muscle tissues examined to date, albeit at much lower levels than found in skeletal muscle. It is unlikely that multiple SMTNL1 isoforms exist since a single Smtnl1 transcription start site was identified in both skeletal and intestinal smooth muscle. Promoter studies suggest restrictive control of Smtnl1 expression in non-muscle cells.
Asunto(s)
Proteínas Musculares/genética , Músculo Liso/metabolismo , Fosfoproteínas/genética , Regiones Promotoras Genéticas , Animales , Ratones , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Reacción en Cadena de la Polimerasa , Factores de Transcripción/metabolismo , Sitio de Iniciación de la TranscripciónRESUMEN
Polyphosphate (polyP) consists of tens to hundreds of phosphates, linked by ATP-like high-energy bonds. Although polyP is present in mammalian mitochondria, its physiological roles there are obscure. Here, we examine the involvement of polyP in mitochondrial energy metabolism and ion transport. We constructed a vector to express a mitochondrially targeted polyphosphatase, along with a GFP fluorescent tag. Specific reduction of mitochondrial polyP, by polyphosphatase expression, significantly modulates mitochondrial bioenergetics, as indicated by the reduction of inner membrane potential and increased NADH levels. Furthermore, reduction of polyP levels increases mitochondrial capacity to accumulate calcium and reduces the likelihood of the calcium-induced mitochondrial permeability transition, a central event in many types of necrotic cell death. This confers protection against cell death, including that induced by beta-amyloid peptide, a pathogenic agent in Alzheimer's disease. These results demonstrate a crucial role played by polyP in mitochondrial function of mammalian cells.
Asunto(s)
Ácido Anhídrido Hidrolasas/metabolismo , Calcio/metabolismo , Muerte Celular , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Línea Celular , Metabolismo Energético , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Transporte Iónico , Potenciales de la Membrana , Mitocondrias/enzimología , NAD/metabolismoRESUMEN
AIMS: Inflammatory bowel disease (IBD)-associated precancerous lesions may be adenomatous or non-adenomatous with various histomorphologies. We aim to validate the newly proposed classification, to explore the neoplastic nature of the non-adenomatous lesions and to elucidate the molecular mechanisms underlying the different histomorphologies. METHODS: 44 background precursor lesions identified in 53 cases of surgically resected IBD-associated colorectal and ileal carcinomas were reviewed for the histomorphological features (classified into adenomatous, mucinous, sessile serrated adenoma (SSA)-like, traditional serrated adenoma-like, differentiated, eosinophilic and serrated not otherwise specified (NOS)) and analysed for a key panel of colonic cancer-related molecular markers. RESULTS: Approximately 60% of the lesions were adenomatous, of which some had mixed serrated, mucinous or eosinophilic changes. The remaining non-adenomatous lesions, including all other types except SSA-like type, mostly showed mixed features and focal adenomatous dysplasia. KRAS mutation and p53 mutant-type expression were found in about half cases across all types, while PIK3CA mutation only in some of adenomatous and eosinophilic lesions and MLH1/PMS2 loss in a subset of adenomatous, mucinous and eosinophilic but not in differentiated and serrated lesions. SAT-B2 or PTEN loss and IMP3 overexpression were seen in a small subset of lesions. No BRAF, NRAS or EGFR gene mutation was detected in any type. Certain molecular-morphological correlations were demonstrated; however, no single or combined molecular alteration(s) was specific to any particular morphological type. CONCLUSIONS: IBD-associated precancerous lesions are heterogeneous both histologically and molecularly. True colitis-associated adenomatous lesions are unlikely conventional adenomas. Non-adenomatous lesions without frank cytologic dysplasia should also be regarded as neoplastic.
Asunto(s)
Adenoma/patología , Neoplasias Colorrectales/patología , Enfermedades Inflamatorias del Intestino/patología , Lesiones Precancerosas/patología , Adenoma/genética , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias Colorrectales/genética , Femenino , Tracto Gastrointestinal/patología , Marcadores Genéticos/genética , Humanos , Enfermedades Inflamatorias del Intestino/genética , Masculino , Persona de Mediana Edad , Lesiones Precancerosas/genética , Estudios RetrospectivosRESUMEN
Ca(2+) transport by the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) is sensitive to monovalent cations. Possible K(+) binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca(2+) transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca(2+) transport correlated in most cases with their direct effects on SERCA. Choline(+), however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca(2+) transport. Of the monovalent cations tested, only Cs(+) significantly affected the Hill coefficient of Ca(2+) transport (n(H)). An increase in n(H) from approximately 2 in K(+) to approximately 3 in Cs(+) was seen in all of the forms of SERCA examined. The effects of Cs(+) on the maximum velocity of Ca(2+) uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K(+) binding sites of the different forms of the protein.
Asunto(s)
Señalización del Calcio/efectos de los fármacos , Cationes Monovalentes/farmacología , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Línea Celular , Cesio/farmacología , Colina/farmacología , Perros , Corazón/efectos de los fármacos , Humanos , Técnicas In Vitro , Cinética , Datos de Secuencia Molecular , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Conejos , Retículo Sarcoplasmático/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/química , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
NCKX5 is a bidirectional K+ -dependent Na+ -Ca2+ exchanger, which belongs to the SLC24A gene family. In particular, the A111T mutation of NCKX5 has been associated with reduced pigmentation in European populations. In contrast to other NCKX isoforms, which function in the plasma membrane (PM), NCKX5 has been shown to localize either in the trans-Golgi network (TGN) or in melanosomes. Moreover, sequences responsible for retaining its intracellular localization are unknown. This study addresses two major questions: (i) clarification of intracellular location of NCKX5 and (ii) identification of sequences that retain NCKX5 inside the cell. We designed a set of cDNA constructs representing NCKX5 loop deletion mutants and NCKX2-NCKX5 chimeras to address these two questions after expression in pigmented MNT1 cells. Our results show that NCKX5 is not a PM resident and is exclusively located in the TGN. Moreover, the large cytoplasmic loop is the determinant for retaining NCKX5 in the TGN.
Asunto(s)
Pigmentación , Potasio/farmacología , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/metabolismo , Secuencia de Aminoácidos , Animales , Autoantígenos/metabolismo , Calcio/metabolismo , Recuento de Células , Células HEK293 , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Mutación/genética , Pigmentación/efectos de los fármacos , Estructura Secundaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Relación Estructura-Actividad , Pez Cebra , Red trans-Golgi/efectos de los fármacos , Red trans-Golgi/metabolismoRESUMEN
Light-dependent changes in cytoplasmic free Ca(2+) are much faster in the outer segment of cone than rod photoreceptors in the vertebrate retina. In the limit, this rate is determined by the activity of an electrogenic Na(+)/Ca(2+) exchanger located in the outer segment plasma membrane. We investigate the functional properties of the exchanger activity in intact, single cone photoreceptors isolated from striped bass retina. Exchanger function is characterized through analysis both of the electrogenic exchanger current and cytoplasmic free Ca(2+) measured with optical probes. The exchanger in cones is K(+) dependent and operates both in forward and reverse modes. In the reverse mode, the K(+) dependence of the exchanger is described by binding to a single site with K(1/2) about 3.6 mM. From the retina of the fish we cloned exchanger molecules bassNCKX1 and bassNCKX2. BassNCKX1 is a single class of molecules, homologous to exchangers previously cloned from mammalian rods. BassNCKX2 exists in four splice variants that differ from each other by small sequence differences in the single, large cytoplasmic loop characteristic of these molecules. We used RT-PCR (reverse transcriptase polymerase chain reaction) of individual cells to identify the exchanger molecule specifically expressed in bass single and twin cone photoreceptors. Each and every one of the four bassNCKX2 splice variants is expressed in both single and twin cones indistinguishably. BassNCKX1 is not expressed in cones and, by exclusion, it is likely to be an exchanger expressed in rods.
Asunto(s)
Células Fotorreceptoras Retinianas Conos/fisiología , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/fisiología , Secuencia de Aminoácidos , Animales , Lubina , Calcio , Clonación Molecular , ADN/genética , Matemática , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambiador de Sodio-Calcio/análisis , Visión Ocular/genética , Visión Ocular/fisiologíaRESUMEN
NCKX5 is an ion exchanger expressed mostly in pigment cells; however, the functional role for this protein in melanogenesis is not clear. A variant allele of SLC24A5, the gene encoding NCKX5, has been shown to correlate with lighter skin pigmentation in humans, indicating a key role for SLC24A5 in determining human skin colour. SLC24A5 expression has been found to be elevated in melanoma. Knockdown analyses have shown SLC24A5 to be important for pigmentation, but to date the function of this ion exchanger in melanogenesis has not been fully established. Our data suggest NCKX5 may have an alternative activity that is key to its role in the regulation of pigmentation. Here Xenopus laevis is employed as an in vivo model system to further investigate the function of NCKX5 in pigmentation. SLC24A5 is expressed in the melanophores as they differentiate from the neural crest and develop in the RPE of the eye. Morpholino knockdown and rescue experiments were designed to elucidate key residues and regions of the NCKX5 protein. Unilateral morpholino injection at the 2 cell stage resulted in a reduction of pigmentation in the eye and epidermis of one lateral side of the tadpole. Xenopus and human SLC24A5 can rescue the morpholino effects. Further rescue experiments including the use of ion exchange inactive SLC24A5 constructs raise the possibility that full ion exchanger function of NCKX5 may not be required for rescue of pigmentation.
Asunto(s)
Pigmentación de la Piel/genética , Intercambiador de Sodio-Calcio/genética , Proteínas de Xenopus/genética , Xenopus laevis/genética , Animales , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Morfolinos/farmacología , Mutación/genética , Fenotipo , Pigmentación de la Piel/efectos de los fármacos , Intercambiador de Sodio-Calcio/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologíaRESUMEN
The Na+/Ca2+ -K+ exchanger (NCKX) utilizes the inward Na+ gradient and the outward K+ gradient to promote Ca2+ extrusion from cells. Here, we have characterized a second NCKX from Drosophila. Based on its chromosomal location (X chromosome) we have named it Ncxk-x. Three splice variants were isolated with three distinct N-terminal sequences. NCKX-X differs from NCKX proteins described so far in other species by lacking an N-terminal signal peptide. Heterologous expression of the respective cDNA's resulted in NCKX-X protein expression and K+ -dependent Na+/Ca2+ exchange activity for two of the three splice variants. Transcript localization of Nckx-x was investigated and compared with that previously described by us for Nckx30C.
Asunto(s)
Drosophila/metabolismo , Intercambiador de Sodio-Calcio/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Drosophila/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Potasio/metabolismo , Estructura Secundaria de Proteína , Sodio/metabolismo , Intercambiador de Sodio-Calcio/química , Intercambiador de Sodio-Calcio/genéticaRESUMEN
Transcription factors play important roles in development and homeostasis. We have completed an embryonic stem cell-based neural differentiation screen, which was carried out with a view to isolating early regulators of neurogenesis. Fifty eight of the expressed sequence tags isolated from this screen represent known transcription factors or sequences containing transcription factor motifs. We have determined the full-length sequence of a novel mouse zinc finger-containing gene (ZFEND; also known as Mus musculus zinc finger protein 358 (Zfp358)) that was identified from this screen. ZFEND has 87% nucleotide and 86% amino acid identity to a previously identified human cDNA, FLJ10390, which is moderately similar to zinc finger protein 135. Northern blotting and RPAs demonstrate highest expression of ZFEND during mid-late mouse embryogenesis. Expression is also observed in several adult tissues with highest expression in heart, brain, and liver. Whole-mount in situ hybridization studies reveal apparent ubiquitous expression of ZFEND during mid-gestation stages (embryonic days 11.5, 12.5), while sections of whole-mount embryos reveal much higher expression levels in the neural folds during neural tube closure and at the boundary between the forelimb buds and the body wall. Bioinformatic analysis maps ZFEND to mouse chromosome 8pter, while FLJ10390 resides on 19p13.3-p13.2, a gene-rich region to which a number of disorders have been mapped. More precise mapping indicates that the involvement of FLJ10390 in atherogenic lipoprotein phenotype, familial febrile convulsions 2, and psoriasis susceptibility cannot be ruled out.
Asunto(s)
Proteínas Portadoras/genética , Mapeo Cromosómico/métodos , Proteínas de la Membrana/genética , Factores de Transcripción/genética , Dedos de Zinc/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Biología Computacional , ADN Complementario/química , ADN Complementario/genética , Embrión de Mamíferos/citología , Embrión de Mamíferos/efectos de los fármacos , Embrión de Mamíferos/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Genes/genética , Hibridación in Situ , Ratones , Datos de Secuencia Molecular , Filogenia , ARN/genética , ARN/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Tretinoina/farmacologíaRESUMEN
PURPOSE: To study the possible involvement of the rod (SLC24A1) and cone (SLC24A2) Na-Ca+K exchanger (NCKX) genes in retinal diseases. METHODS: DNA was collected from unrelated patients with retinal disease, mainly from North America. A human genomic library was screened with the cone NCKX cDNA, and hybridizing clones were sequenced to determine the genomic organization of the SLC24A2 gene. The single-strand conformation polymorphism (SSCP) technique and direct sequencing were used to screen the patients' DNA for mutations in SLC24A1 and SLC24A2. The effect of selected missense changes on protein function was tested by measuring potassium-dependent Na-Ca exchange of the mutant proteins expressed in insect cells. RESULTS: Twenty-seven novel sequence changes were found in the rod NCKX gene, 21 of which are unlikely to be pathogenic, because they did not cosegregate with the disease or did not affect conserved regions of the protein. Of the remaining six, two were frameshift mutations found in one patient each. If translated, these alleles would encode nonfunctional proteins. Three of the six possibly pathogenic mutations were missense changes located in conserved regions, and their protein functions were assayed. Only one (Ile992Thr) had a significantly low level of exchanger function, but it was found in two unrelated patients who were heterozygotes with different retinal diseases, and this mutation could not be unequivocally associated with either disease. The last of the six changes is likely to create a new splice acceptor site. The genomic organization of the cone NCKX gene was determined, and it contained 11 exons with a few splice variants. Fifteen novel sequence changes were identified in the cone exchanger gene in patients with a cone dysfunction or degeneration. Only three of these sequence changes, all missense changes found in heterozygous patients, were considered possibly pathogenic. Functional analysis showed only a slight reduction in the activity of the corresponding mutant proteins. CONCLUSIONS: Although variant alleles of the rod and cone NCKX genes were found, none could be definitively associated with a specific retinal disease. The human phenotype associated with mutant exchanger alleles remains unknown.
Asunto(s)
Mutación , Células Fotorreceptoras de Vertebrados/metabolismo , Enfermedades de la Retina/genética , Intercambiador de Sodio-Calcio/genética , Alelos , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Bovinos , Análisis Mutacional de ADN , ADN Complementario/análisis , Delfines , Proteínas de Transporte de Ácidos Grasos , Femenino , Expresión Génica , Biblioteca Genómica , Humanos , Masculino , Datos de Secuencia Molecular , Linaje , Polimorfismo Conformacional Retorcido-Simple , Ratas , Enfermedades de la Retina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Ca(2+) extrusion driven by both the inward Na(+) gradient as well as the outward K(+) gradient is essential for visual transduction in retinal rod and cone photoreceptors because it removes Ca(2+) that enters photoreceptors via the cGMP-gated and light-sensitive channels. We have cloned rod and cone Na/Ca-K exchanger (NCKX) cDNAs from several species, and we have cloned NCKX cDNAs from lower organisms that lack vertebrate-type vision. Although in situ NCKX physiology has only been documented for vertebrate photoreceptors, it is now clear that NCKX gene products have a much broader distribution pattern. Here, we review some of the structural and functional features that have emerged from our studies on different members of the NCKX gene family.
Asunto(s)
Enfermedades de la Retina/genética , Células Fotorreceptoras Retinianas Bastones/fisiología , Intercambiador de Sodio-Calcio/genética , Animales , Transporte Biológico , Caenorhabditis elegans , Calcio/metabolismo , Bovinos , Pollos , Clonación Molecular , Delfines , Humanos , Cinética , Familia de Multigenes , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Segmento Externo de la Célula en Bastón/fisiología , Rubidio/farmacocinética , Intercambiador de Sodio-Calcio/químicaRESUMEN
Polyhydroxybutyrate (PHB) is a biological polymer which belongs to the class of polyesters and is ubiquitously present in all living organisms. Mammalian mitochondrial membranes contain PHB consisting of up to 120 hydroxybutyrate residues. Roles played by PHB in mammalian mitochondria remain obscure. It was previously demonstrated that PHB of the size similar to one found in mitochondria mediates calcium transport in lipid bilayer membranes. We hypothesized that the presence of PHB in mitochondrial membrane might play a significant role in mitochondrial calcium transport. To test this, we investigated how the induction of PHB hydrolysis affects mitochondrial calcium transport. Mitochondrial PHB was altered enzymatically by targeted expression of bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. The expression of PhaZ7 induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential in HepG2 but not in U87 and HeLa cells. Furthermore, it significantly inhibited mitochondrial calcium uptake in intact HepG2, U87 and HeLa cells stimulated by the ATP or by the application of increased concentrations of calcium to the digitonin permeabilized cells. Calcium uptake in PhaZ7 expressing cells was restored by mimicking calcium uniporter properties with natural electrogenic calcium ionophore - ferutinin. We propose that PHB is a previously unrecognized important component of the mitochondrial calcium uptake system.
Asunto(s)
Calcio/metabolismo , Hidroxibutiratos/metabolismo , Mitocondrias/metabolismo , Poliésteres/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/metabolismo , Línea Celular Tumoral , Supervivencia Celular , Femenino , Células HeLa , Células Hep G2 , Humanos , Potencial de la Membrana Mitocondrial/fisiología , Prohibitinas , TransfecciónRESUMEN
Heat shock proteins (Hsps) are a set of molecular chaperones involved in cellular repair. They provide protective mechanisms that allow cells to survive potentially lethal insults, In response to a conditioning stress their expression is increased. Here we examined the connection between Hsps and Aß(42), the amyloid peptide involved in the pathological sequence of Alzheimer's disease (AD). Extracellular Aß(42) associates with neuronal cells and is a major constituent of senile plaques, one of the hallmarks of AD. Although Hsps are generally thought to prevent accumulation of misfolded proteins, there is a lack of mechanistic evidence that heat shock chaperones directly modulate Aß(42) toxicity. In this study we show that neither extracellular Aß(42) nor Aß(42/)PrP(C) trigger the heat shock response in neurons. To address the influence of the neuroprotective heat shock response on cellular Aß(42), Western analysis of Aß(42) was performed following external Aß(42) application. Five hours after a conditioning heat shock, Aß(42) association with CAD cells was increased compared to control neurons. However, at forty-eight hours following heat shock Aß(42) levels were reduced compared to that found for control cells. Moreover, transient transfection of the stress induced Hsp40, decreased CAD levels of Aß(42). In contrast to CAD cells, hippocampal neurons transfected with Hsp40 retained Aß(42) indicating that Hsp40 modulation of Aß(42) proteostasis is cell specific. Mutation of the conserved HPD motif within Hsp40 significantly reduced the Hsp40-mediated Aß(42) increase in hippocampal cultures indicating the importance of this motif in regulating cellular Aß(42). Our data reveal a biochemical link between Hsp40 expression and Aß(42) proteostasis that is cell specific. Therefore, increasing Hsp40 therapeutically with the intention of interfering with the pathogenic cascade leading to neurodegeneration in AD should be pursued with caution.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Espacio Extracelular/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Fragmentos de Péptidos/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Bovinos , Línea Celular Tumoral , Demencia/metabolismo , Demencia/patología , Regulación de la Expresión Génica , Hipocampo/citología , Humanos , Cinética , Ratones , Priones/metabolismo , RatasRESUMEN
A non-synonymous single nucleotide polymorphism in the human SLC24A5 gene is associated with natural human skin color variation. Multiple sequence alignments predict that this gene encodes a member of the potassium-dependent sodium-calcium exchanger family denoted NCKX5. In cultured human epidermal melanocytes we show using affinity-purified antisera that native human NCKX5 runs as a triplet of approximately 43 kDa on SDS-PAGE and is partially localized to the trans-Golgi network. Removal of the NCKX5 protein through small interfering RNA-mediated knockdown disrupts melanogenesis in human and murine melanocytes, causing a significant reduction in melanin pigment production. Using a heterologous expression system, we confirm for the first time that NCKX5 possesses the predicted exchanger activity. Site-directed mutagenesis of NCKX5 and NCKX2 in this system reveals that the non-synonymous single nucleotide polymorphism in SLC24A5 alters a residue that is important for NCKX5 and NCKX2 activity. We suggest that NCKX5 directly regulates human epidermal melanogenesis and natural skin color through its intracellular potassium-dependent exchanger activity.
Asunto(s)
Antiportadores/metabolismo , Aparato de Golgi/metabolismo , Melaninas/biosíntesis , Melanocitos/metabolismo , Pigmentación/fisiología , Polimorfismo de Nucleótido Simple , Animales , Antiportadores/genética , Calcio/metabolismo , Línea Celular Tumoral , Aparato de Golgi/genética , Humanos , Transporte Iónico/fisiología , Masculino , Melaninas/genética , Melanocitos/citología , Ratones , Potasio/metabolismo , Sodio/metabolismo , Intercambiador de Sodio-Calcio/genética , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
S100A11 is a member of the S100 family of EF-hand Ca(2+)-binding proteins, which is expressed in smooth muscle and other tissues. Ca(2+) binding to S100A11 induces a conformational change that exposes a hydrophobic surface for interaction with target proteins. Affinity chromatography with immobilized S100A11 was used to isolate a 70-kDa protein from smooth muscle that bound to S100A11 in a Ca(2+)-dependent manner and was identified by mass spectrometry as annexin A6. Direct Ca(2+)-dependent interaction between S100A11 and annexin A6 was confirmed by affinity chromatography of the purified bacterially expressed proteins, by gel overlay of annexin A6 with purified S100A11, by chemical cross-linking, and by coprecipitation of S100A11 with annexin A6 bound to liposomes. The expression of S100A11 and annexin A6 in the same cell type was verified by RT-PCR and immunocytochemistry of isolated vascular smooth muscle cells. The site of binding of S100A11 on annexin A6 was investigated by partial tryptic digestion and deletion mutagenesis. The unique NH(2) terminal head region of annexin A6 was not required for S100A11 binding, but binding sites were identified in both NH(2)- and COOH-terminal halves of the molecule. We hypothesize that an agonist-induced increase in cytosolic free [Ca(2+)] leads to formation of a complex of S100A11 and annexin A6, which forms a physical connection between the plasma membrane and the cytoskeleton, or plays a role in the formation of signaling complexes at the level of the sarcolemma.
Asunto(s)
Anexina A6/metabolismo , Calcio/metabolismo , Músculo Liso/metabolismo , Fosfolípidos/metabolismo , Proteínas S100/metabolismo , Secuencia de Aminoácidos , Animales , Anexina A6/química , Anexina A6/genética , Pollos , Inmunohistoquímica , Técnicas In Vitro , Liposomas , Masculino , Datos de Secuencia Molecular , Músculo Liso Vascular/metabolismo , Fosfolípidos/química , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/química , Proteínas S100/química , Proteínas S100/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
L-type, voltage-gated Ca2+ channels (CaL) play critical roles in brain and muscle cell excitability. Here we show that currents through heterologously expressed neuronal and smooth muscle CaL channel isoforms are acutely potentiated following alpha5beta1 integrin activation. Only the alpha1C pore-forming channel subunit is critical for this process. Truncation and site-directed mutagenesis strategies reveal that regulation of Cav1.2 by alpha5beta1 integrin requires phosphorylation of alpha1C C-terminal residues Ser1901 and Tyr2122. These sites are known to be phosphorylated by protein kinase A (PKA) and c-Src, respectively, and are conserved between rat neuronal (Cav1.2c) and smooth muscle (Cav1.2b) isoforms. Kinase assays are consistent with phosphorylation of these two residues by PKA and c-Src. Following alpha5beta1 integrin activation, native CaL channels in rat arteriolar smooth muscle exhibit potentiation that is completely blocked by combined PKA and Src inhibition. Our results demonstrate that integrin-ECM interactions are a common mechanism for the acute regulation of CaL channels in brain and muscle. These findings are consistent with the growing recognition of the importance of integrin-channel interactions in cellular responses to injury and the acute control of synaptic and blood vessel function.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Integrina alfa5beta1/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Encéfalo/metabolismo , Proteína Tirosina Quinasa CSK , Datos de Secuencia Molecular , Músculos/metabolismo , Miocitos del Músculo Liso/metabolismo , Neuronas/metabolismo , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Porcinos , Familia-src QuinasasRESUMEN
Plasma membrane Na+/Ca2+-exchangers play a predominant role in Ca2+ extrusion in brain. Neurons express several different Na+/Ca2+-exchangers belonging to both the K+-independent NCX family and the K+-dependent NCKX family. The unique contributions of each of these proteins to neuronal Ca2+ homeostasis and/or physiology remain largely unexplored. To address this question, we generated mice in which the gene encoding the abundant neuronal K+ -dependent Na+/Ca2+-exchanger protein, NCKX2, was knocked out. Analysis of these animals revealed a significant reduction in Ca2+ flux in cortical neurons, a profound loss of long term potentiation and an increase in long term depression at hippocampal Schaffer/CA1 synapses, and clear deficits in specific tests of motor learning and spatial working memory. Surprisingly, there was no obvious loss of photoreceptor function in cones, where expression of the NCKX2 protein had been reported previously. These data emphasize the critical and non-redundant role of NCKX2 in the local control of neuronal [Ca2+] that is essential for the development of synaptic plasticity associated with learning and memory.
Asunto(s)
Aprendizaje/fisiología , Memoria/fisiología , Intercambiador de Sodio-Calcio/fisiología , Animales , Animales Recién Nacidos , Calcio/metabolismo , Diferenciación Celular/genética , Células Cultivadas , Hipocampo/fisiología , Ratones , Ratones Noqueados , Neuronas/citología , Neuronas/fisiología , Técnicas de Placa-Clamp , Células Fotorreceptoras Retinianas Conos/citología , Células Fotorreceptoras Retinianas Conos/fisiología , Intercambiador de Sodio-Calcio/genética , Sinapsis/fisiologíaRESUMEN
Kinetics and voltage dependence of inactivation of a prokaryotic voltage-gated sodium channel (NaChBac) were investigated in an effort to understand its molecular mechanism. NaChBac inactivation kinetics show strong, bell-shaped voltage dependence with characteristic time constants ranging from approximately 50 ms at depolarized voltages to a maximum of approximately 100 s at the inactivation midpoint. Activation and inactivation parameters for four different covalently linked tandem dimer or tandem tetramer constructs were indistinguishable from those of the wild-type channel. Point mutations in the outer part of the pore revealed an important influence of the S195 residue on the process of inactivation. For two mutants (S195D and S195E), the maximal and minimal rates of inactivation observed were increased by approximately 2.5-fold, and the midpoint of the steady-state inactivation curve was shifted approximately 20 mV in the hyperpolarizing direction, compared to the wild-type channel. Our data suggest that pore vestibule structure is an important determinant of NaChBac inactivation, whereas the inactivation mechanism is independent of the number of free cytoplasmic N- and C-termini in the functional channel. In these respects, NaChBac inactivation resembles C-type or slow inactivation modes observed in other voltage-gated K and Na channels.
Asunto(s)
Citoplasma/metabolismo , Canales de Sodio/química , Secuencia de Aminoácidos , Bacterias/enzimología , Fenómenos Biofísicos , Biofisica , Western Blotting , Cadmio/química , Cadmio/farmacología , Línea Celular , Citoplasma/química , Dimerización , Electrofisiología , Epítopos/química , Colorantes Fluorescentes/farmacología , Humanos , Cinética , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , Mutación , Plásmidos/metabolismo , Mutación Puntual , Estructura Terciaria de Proteína , Sodio/química , Factores de TiempoRESUMEN
The Na+/Ca2+-K+ exchanger (NCKX) gene products are polytopic membrane proteins that utilize the existing cellular Na+ and K+ gradients to extrude cytoplasmic Ca2+. NCKX proteins are made up of two clusters of hydrophobic segments, both thought to consist of five putative membrane-spanning alpha-helices, and separated by a large cytoplasmic loop. The two most conserved regions within the NCKX sequence are known as the alpha1 and alpha2 repeats, and are found within the first and second set of transmembrane domains, respectively. The alpha repeats have previously been shown to contain residues critical for transport function. Here we used site-directed disulfide mapping to report that the alpha repeats are found in close proximity in three-dimensional space, bringing together key functional NCKX residues, e.g., the two critical acidic residues, Glu188 and Asp548. Glu188Cys in the alpha1 repeat could form a disulfide cross-link with Asp548Cys in the alpha2 repeat. Surprisingly, cysteine substitutions of Ser185 in the alpha1 repeat could form disulfide cross-links with cysteine substitutions of three residues in the alpha2 repeat (Ser545, Asp548, and Ser552), thought to cover close to two full turns of an alpha helix, implying an area of increased flexibility. Using the same method, Asp575, a residue critical for the K+ dependence of NCKX, was shown to be in the proximity of Ser185 and Glu188, consistent with its role in enabling K+ to bind to a single Ca2+ and K+ binding pocket.