RESUMO
Multiple membrane organelles require cholesterol for proper function within cells. The Niemann-Pick type C (NPC) proteins export cholesterol from endosomes to other membrane compartments, including the endoplasmic reticulum (ER), plasma membrane (PM), trans-Golgi network (TGN), and mitochondria, to meet their cholesterol requirements. Defects in NPC cause malfunctions in multiple membrane organelles and lead to an incurable neurological disorder. Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), a resident enzyme in the ER, converts cholesterol to cholesteryl esters for storage. In mutant NPC cells, cholesterol storage still occurs in an NPC-independent manner. Here we report the interesting finding that in a mutant Npc1 mouse (Npc1nmf), Acat1 gene (Soat1) knockout delayed the onset of weight loss, motor impairment, and Purkinje neuron death. It also improved hepatosplenic pathology and prolonged lifespan by 34%. In mutant NPC1 fibroblasts, ACAT1 blockade (A1B) increased cholesterol content associated with TGN-rich membranes and mitochondria, while decreased cholesterol content associated with late endosomes. A1B also restored proper localization of syntaxin 6 and golgin 97 (key proteins in membrane trafficking at TGN) and improved the levels of cathepsin D (a key protease in lysosome and requires Golgi/endosome transport for maturation) and ABCA1 (a key protein controlling cholesterol release at PM). This work supports the hypothesis that diverting cholesterol from storage can benefit multiple diseases that involve cholesterol deficiencies in cell membranes.
Assuntos
Longevidade , Doença de Niemann-Pick Tipo C , Acetil-CoA C-Acetiltransferase , Doença de Alzheimer , Animais , Colesterol , Ésteres do Colesterol , Modelos Animais de Doenças , Endossomos/genética , Camundongos , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Esterol O-AciltransferaseRESUMO
Niemann-Pick type C (NP-C) disease is a fatal genetic lipidosis for which there is no Food and Drug Administration (FDA)-approved therapy. Vorinostat, an FDA-approved inhibitor of histone deacetylases, ameliorates lysosomal lipid accumulation in cultured NP-C patient fibroblasts. To assess the therapeutic potential of histone deacetylase inhibition, we pursued these in vitro observations in two murine models of NP-C disease. Npc1nmf164 mice, which express a missense mutation in the Npc1 gene, were treated intraperitoneally, from weaning, with the maximum tolerated dose of vorinostat (150 mg/kg, 5 days/week). Disease progression was measured via gene expression, liver function and pathology, serum and tissue lipid levels, body weight, and life span. Transcriptome analyses of treated livers indicated multiple changes consistent with reversal of liver dysfunction that typifies NP-C disease. Significant improvements in liver pathology and function were achieved by this treatment regimen; however, NPC1 protein maturation and levels, disease progression, weight loss, and animal morbidity were not detectably altered. Vorinostat concentrations were >200 µm in the plasma compartment of treated animals but were almost 100-fold lower in brain tissue. Apolipoprotein B metabolism and the expression of key components of lipid homeostasis in primary hepatocytes from null (Npc1-/-) and missense (Npc1nmf164 ) mutant mice were altered by vorinostat treatment, consistent with a response by these cells independent of the status of the Npc1 locus. These results suggest that HDAC inhibitors have utility to treat visceral NP-C disease. However, it is clear that improved blood-brain barrier penetration will be required to alleviate the neurological symptoms of human NP-C disease.
Assuntos
Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Fígado/efeitos dos fármacos , Fígado/fisiopatologia , Doença de Niemann-Pick Tipo C/tratamento farmacológico , Doença de Niemann-Pick Tipo C/genética , Proteínas/genética , Animais , Apolipoproteínas B/metabolismo , Células Cultivadas , Colesterol/genética , Colesterol/metabolismo , Modelos Animais de Doenças , Inibidores de Histona Desacetilases/farmacocinética , Homeostase/efeitos dos fármacos , Humanos , Ácidos Hidroxâmicos/farmacocinética , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Mutação de Sentido Incorreto , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/patologia , Doença de Niemann-Pick Tipo C/fisiopatologia , Proteínas/metabolismo , Transcriptoma/efeitos dos fármacos , VorinostatRESUMO
It is important for nurses today and for those joining the workforce in the future to have familiarity and training with respect to interprofessional research, evidence-based practice, and quality improvement. In an effort to address this need, we describe a 10-week summer research program that immerses undergraduate nursing students in a broad spectrum of clinical and translational research projects as part of their exposure to advanced nursing roles. In doing so, the program increases the ability of the students to participate in research, effectively interact with academic medical center researchers, and incorporate elements of evidence-based practice into future nursing interventions. Their mentors are nurses practicing in roles as nurse researcher, advanced practice nurses involved in evidence-based practice or quality improvement, and clinical trials research nurses. Each student is matched with 3 of these mentors and involved in 3 different projects. Through this exposure, the students benefit from observing multiple nursing roles, taking an active role in research-related activities participating in interdisciplinary learning experiences. Overall, the program provides benefits to the students, who demonstrate measured improvement with respect to the program objectives, and to their mentors and each of the participating organizations.
Assuntos
Bolsas de Estudo , Pesquisa em Enfermagem , Estudantes de Enfermagem , Escolha da Profissão , Bacharelado em Enfermagem , Enfermagem Baseada em Evidências/métodos , Humanos , MentoresRESUMO
We have identified a point mutation in Npc1 that creates a novel mouse model (Npc1(nmf164)) of Niemann-Pick type C1 (NPC) disease: a single nucleotide change (A to G at cDNA bp 3163) that results in an aspartate to glycine change at position 1005 (D1005G). This change is in the cysteine-rich luminal loop of the NPC1 protein and is highly similar to commonly occurring human mutations. Genetic and molecular biological analyses, including sequencing the Npc1(spm) allele and identifying a truncating mutation, confirm that the mutation in Npc1(nmf164) mice is distinct from those in other existing mouse models of NPC disease (Npc1(nih), Npc1(spm)). Analyses of lifespan, body and spleen weight, gait and other motor activities, as well as acoustic startle responses all reveal a more slowly developing phenotype in Npc1(nmf164) mutant mice than in mice with the null mutations (Npc1(nih), Npc1(spm)). Although Npc1 mRNA levels appear relatively normal, Npc1(nmf164) brain and liver display dramatic reductions in Npc1 protein, as well as abnormal cholesterol metabolism and altered glycolipid expression. Furthermore, histological analyses of liver, spleen, hippocampus, cortex and cerebellum reveal abnormal cholesterol accumulation, glial activation and Purkinje cell loss at a slower rate than in the Npc1(nih) mouse model. Magnetic resonance imaging studies also reveal significantly less demyelination/dysmyelination than in the null alleles. Thus, although prior mouse models may correspond to the severe infantile onset forms of NPC disease, Npc1(nmf164) mice offer many advantages as a model for the late-onset, more slowly progressing forms of NPC disease that comprise the large majority of human cases.
Assuntos
Proteínas de Transporte/genética , Modelos Animais de Doenças , Glicoproteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/genética , Mutação Puntual/genética , Idade de Início , Alelos , Animais , Astrócitos/patologia , Encéfalo/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Análise Mutacional de DNA , Progressão da Doença , Estresse do Retículo Endoplasmático , Gangliosídeos/metabolismo , Homozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Metabolismo dos Lipídeos , Pulmão/citologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/metabolismo , Camundongos , Microglia/patologia , Bainha de Mielina , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Doença de Niemann-Pick Tipo C/fisiopatologia , Fenótipo , Deficiências na Proteostase , Células de Purkinje/patologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Reflexo de Sobressalto , Taxa de SobrevidaRESUMO
Niemann-Pick type C (NPC) disease is an inherited lysosomal storage disease and caused by mutations in Npc1 or Npc2, which mediate cooperatively the egress of cholesterol from lysosomes. The disease entails progressive neurodegeneration, whose cause is poorly understood. Here, we report that Npc1 is distributed in distinct layers of the mouse retina and that its deficiency causes striking retinal degeneration in 2-month-old mice with signs of age-related maculopathies. This includes impaired visual function, accumulation of lipofuscin in the retinal pigment epithelium layer, degeneration of photoreceptor outer segments, disruption of synaptic layers and an increase in autophagy markers in the ganglion cell layer. Moreover, the lack of Npc1 results in the upregulation of proteins that mediate cellular cholesterol release in the retina. Our findings suggest that Npc1 is required for normal retinal function and that its absence may serve as model to study age-related degeneration of the retina.
Assuntos
Degeneração Macular/patologia , Proteínas/metabolismo , Retina/patologia , Animais , Autofagia , Colesterol/metabolismo , Eletrorretinografia , Peptídeos e Proteínas de Sinalização Intracelular , Degeneração Macular/genética , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Degeneração Neural/metabolismo , Degeneração Neural/patologia , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Fagossomos/metabolismo , Fagossomos/ultraestrutura , Proteínas/genética , Retina/citologia , Retina/metabolismoRESUMO
Epitope tags have been increasingly used to understand ion channel subunit assembly and interaction, trafficking, subcellular localization, and function in living cells. In particular, epitope tags have proven extremely useful for analyses of closely related, highly homologous channel subunits in endogenous cell contexts in vitro and in vivo, where multiple channel isoforms may be expressed. However, as the variety of epitope tags that have been used has expanded, and the use of tagged channel subunits has become increasingly sophisticated and widespread, there has also been an increase in the number of examples highlighting the potential problems associated with the use of epitope tags for ion channel studies. Described here are some of the epitope tags that have been used to study ion channel subunits, including the HA, FLAG, myc, His6, and green fluorescent protein (GFP) epitopes, as well as some of the applications and avenues of research in which they have proven advantageous. Potential pitfalls and caveats associated with the use of these epitope tags are also discussed, with an emphasis on the need to include careful characterization of epitope-tagged channel subunits as part of their construction. Finally, potential avenues for future investigation and the development of this approach are considered.
Assuntos
Epitopos/genética , Epitopos/metabolismo , Canais Iônicos/metabolismo , Animais , Mapeamento de Epitopos , Previsões , Proteínas de Fluorescência Verde/metabolismo , Hemaglutininas/metabolismo , Histidina/metabolismo , Humanos , Imuno-Histoquímica , Canais Iônicos/genética , Canais Iônicos/fisiologia , Oligopeptídeos , Peptídeos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismoRESUMO
Niemann-Pick Type C (NP-C) disease, caused by mutations in either human NPC1 (hNPC1) or human NPC2 (hNPC2), is characterized by the accumulation of unesterified cholesterol in late endosomes. Although it is known that the NP-C proteins are targeted to late endosomal/lysosomal compartments, their delivery mechanisms have not been fully elucidated. To identify mechanisms regulating NP-C protein localization, we used Saccharomyces cerevisiae, which expresses functional homologs of both NP-C proteins - scNcr1p and scNpc2p. Targeting of scNcr1p to the vacuole was perturbed in AP-3-deficient yeast cells, whereas the delivery of scNpc2p was affected by deficiencies in either AP-3 or GGA. We focused on the role of the AP-3 pathway in the targeting of the mammalian NP-C proteins. We found that, although mouse NPC1 (mNPC1) and hNPC2 co-localize with AP-3 to a similar extent in fibroblasts, hNPC2 preferentially co-localizes with AP-1. Importantly, the targeting of both mammalian NPC1 and NPC2 is dependent on AP-3. Moreover, and consistent with the NP-C proteins playing a role in cholesterol metabolism, AP-3-deficient cells have reduced levels of cholesterol. These results provide information about how the NP-C proteins are targeted to their sites of action and illustrate the possibility that defective sorting of the NP-C proteins along the endocytic route can alter cellular cholesterol.
Assuntos
Complexo 3 de Proteínas Adaptadoras/metabolismo , Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Glicoproteínas/metabolismo , Proteínas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Colesterol/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteína C1 de Niemann-Pick , Doenças de Niemann-Pick/metabolismo , Transporte Proteico , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Proteínas de Transporte VesicularRESUMO
Cerebellar Purkinje neurons in vivo exhibit high frequency and multi-spike action potentials with transient (INaT), resurgent (INaR) and persistent (INaP) Na+ currents arising from voltage-gated Na+ channels, which play important roles in shaping the action potentials and electrical activity of these cells. However, little is known about Na+ channel expression in cultured Purkinje neurons despite the use of in vitro approaches to study these cells. Therefore, GFP-expressing Purkinje neurons isolated from transgenic mice were analysed after four weeks in culture, when, coincident with distinct axonal and dendritic morphologies, cultured Purkinje neurons exhibited dendrite-specific MAP2 expression characteristic of polarized neurons. In cell-attached patch clamp recordings, Na+ currents occurred at significantly higher frequencies and amplitudes in patches from the soma and axon than from dendrites, similar to the polarized distribution observed in vivo. INaT, INaR and INaP Na+ currents with properties similar to those observed in acutely isolated Purkinje neurons were detected in nucleated outside-out patches from cultured Purkinje cells. RT-PCR analysis detected Nav1.1, Nav1.2 and Nav1.6, but not Nav1.3, Nav1.4, Nav 1.5 or Nav1.8 Na+ channel alpha subunit gene expression in cultured Purkinje neurons, as observed in vivo. Together, the results indicate that key aspects of Na+ channel expression in mature Purkinje neurons in vivo occur in vitro.
Assuntos
Células de Purkinje/metabolismo , Canais de Sódio/metabolismo , Animais , Axônios/metabolismo , Células Cultivadas , Cerebelo/citologia , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Embrião de Mamíferos , Feminino , Expressão Gênica/fisiologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Técnicas de Patch-Clamp/métodos , Gravidez , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Canais de Sódio/classificação , Canais de Sódio/genéticaRESUMO
OBJECTIVE: We have identified a large kindred with multiple endocrine neoplasia 2A (MEN 2A) due to a mutation at RET codon 609 that results in a cysteine to serine substitution, a mutation previously identified in only one case in the literature. We characterized the clinical phenotype of the kindred and the biochemical mechanism of this new mutation. PATIENTS AND DESIGN: The index case, a 42-year-old woman, presented with pheochromocytoma. We screened 29 family members for the presence of the mutation. Of the 15 mutation-positive family members, 11 agreed to undergo further evaluation by physical examination, calcium and pentagastrin-stimulated calcitonin levels, measurement of urinary metanephrines, adrenal imaging and serum calcium levels. Biochemical characterization of the mutation was by transient transfection of human neuroblastoma cells and Western blot analysis. RESULTS: This kindred demonstrated an inheritance pattern consistent with autosomal dominant pheochromocytoma. Strikingly, no clinically evident case of medullary thyroid cancer (MTC) was observed among mutation-positive family members. Thyroidectomy in six cases revealed C-cell hyperplasia in all and microscopic MTC in two cases. Transfection experiments using human neuroblastoma cells showed that the mutant RET, unlike the wild-type receptor, is constitutively phosphorylated in the absence of ligand, and thus resembles other previously characterized MEN 2A mutations. CONCLUSIONS: The identification of a new mutation causing a MEN 2A phenotype that features pheochromocytoma and the surprising absence of clinically apparent MTC has significant implications for carriers of this mutation and provides further insights into the genotype-phenotype correlation in MEN 2A.
Assuntos
Carcinoma Medular/genética , Neoplasia Endócrina Múltipla Tipo 2a/diagnóstico , Feocromocitoma/genética , Mutação Puntual , Proteínas Proto-Oncogênicas c-ret/genética , Neoplasias da Glândula Tireoide/genética , Adolescente , Adulto , Calcitonina/sangue , Cálcio , Carcinoma Medular/metabolismo , Catecolaminas/urina , Criança , Códon , Feminino , Humanos , Masculino , Metanefrina/urina , Pessoa de Meia-Idade , Neoplasia Endócrina Múltipla Tipo 2a/genética , Linhagem , Pentagastrina , Feocromocitoma/metabolismo , Polimorfismo de Fragmento de Restrição , Análise de Sequência de DNA , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/metabolismo , Ácido Vanilmandélico/urinaRESUMO
Niemann-Pick Type C (NPC) disease is an autosomal recessive disorder caused by mutations in either the NPC1 or HE1 genes. Hallmarks of this presently incurable disease include abnormal intracellular accumulation of cholesterol and glycosphingolipids, progressive neuropathology and neurodegeneration, and premature death. There have been increased efforts to understand the effects of NPC disease on neurons of the brain, in part due to the recent development of improved research tools and reagents, and in part due to the rapidly growing appreciation of the importance of cholesterol and lipoproteins in the brain during neuronal development, function, and degeneration. Here, we highlight fundamental aspects of neurons that appear to be affected by NPC disease, including their morphology, metabolism, intracellular transport, electrical signaling, and response to environmental factors, and suggest other potentially important areas for future investigation. This provides a framework for acquiring additional insight to this disorder and shaping new therapeutic approaches to NPC disease.
Assuntos
Neurônios/metabolismo , Neurônios/patologia , Doenças de Niemann-Pick/fisiopatologia , Animais , Transporte Biológico , Colesterol/metabolismo , Previsões , Genes Recessivos , Glicoesfingolipídeos/metabolismo , Humanos , Mutação , Fatores de Crescimento Neural/metabolismo , Fatores de Crescimento Neural/farmacologia , Neuroglia/metabolismo , Neurônios/efeitos dos fármacos , Doenças de Niemann-Pick/genéticaRESUMO
Voltage-gated K+ channels play important roles in shaping the characteristics of action potentials and electrical activity. In a previous study, we isolated cDNAs encoding several distinct K+ channel isoforms, including a novel isoform (XKv1.10) expressed in Xenopus laevis spinal cord neurons and myocytes. Here, we report the biophysical characterization of XKv1.10 expressed in transiently transfected HEK293 cells. Whole cell patch clamp recordings revealed a voltage-gated, rapidly activating and inactivating K+ current. Interestingly, the rate of inactivation of XKv1.10 channels showed apparent voltage dependence, with time constants between 77.7-213.3 ms. The predicted protein sequence of XKv1.10 does not appear to encode an N-terminal inactivating "ball and chain" domain, and instead these channels may inactivate via a C/P-type mechanism. Consistent with this, either increasing the external concentration of K+ or external application of tetraethylammonium caused a decrease in the rate of inactivation. Pharmacologically, XKv1.10 K+ channels were sensitive to 4-aminopyridine and tetraethylammonium with apparent IC50 values of 68.5 microM and 17.1 mM, respectively. When simulated action potentials were used as a voltage command, XKv1.10 was similar to XKv1.4 in that it carried more repolarizing current during the action potential than XKv1.2. However, while XKv1.4 was active during the interspike interval, XKv1.10 and XKv1.2 were not. Overall, the data suggest that XKv1.10 channels make a unique contribution to the developmental maturation of electrical signaling in Xenopus laevis.
Assuntos
Regulação da Expressão Gênica/fisiologia , Potenciais da Membrana/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular , Clonagem Molecular/métodos , Relação Dose-Resposta a Droga , Estimulação Elétrica , Embrião de Mamíferos , Embrião não Mamífero , Humanos , Rim , Canal de Potássio Kv1.2 , Canal de Potássio Kv1.4 , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/fisiologia , Pirimidinas/farmacologia , Superfamília Shaker de Canais de Potássio , Tetraetilamônio/farmacologia , Fatores de Tempo , Transfecção/métodos , Xenopus , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Xenopus laevis/fisiologiaRESUMO
Voltage-gated sodium (Na(+)) channels typically contain a pore-forming alpha subunit and one or two auxiliary beta subunits. Although initial characterization of known alpha and beta subunits has been facilitated by expression in heterologous cells, to understand fully the differences between individual subunits and the functional consequences of selective subunit expression, there is a need to acutely manipulate expression in cells that endogenously express Na(+) channels. To this end, we have constructed a recombinant adenovirus containing a cDNA for a mouse Na(+) channel beta1 subunit with a yellow fluorescent protein fused to its C-terminus (Ad-beta1-EYFP), and with fluorescence microscopy detected beta1-EYFP expression in primary cerebellar neurons and Chinese hamster ovary (CHO) cells upon transduction with this adenovirus, including expression in the plasma membrane. Consistent with this, patch clamp recordings confirmed that Na(+) currents in CHO cells expressing mouse Na(v)1.4 alpha subunits were appropriately modified by the viral-mediated expression of beta1-EYFP subunits. The results demonstrate that adenoviral-mediated gene delivery can be used effectively to express epitope-tagged Na(+) channel subunits with properties similar to wild-type subunits, and suggest that Ad-beta1-EYFP will be a useful reagent for investigating Na(+) channels in a variety of excitable cell types, including neurons.
Assuntos
Neurônios/fisiologia , Canais de Sódio/fisiologia , Transdução Genética , Adenoviridae/genética , Animais , Proteínas de Bactérias , Células CHO , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Cricetinae , Cricetulus , Imuno-Histoquímica , Proteínas Luminescentes , Potenciais da Membrana/fisiologia , Camundongos , Microscopia de Fluorescência , Neurônios/ultraestrutura , Neurônios/virologia , Técnicas de Patch-Clamp , Transdução Genética/métodos , TransfecçãoRESUMO
To understand the molecular basis of nervous system function in the leech, Hirudo medicinalis, we have isolated four novel cDNAs encoding putative voltage-gated sodium (Na) channel alpha subunits, and have analyzed the expression of these genes in individual neurons of known function. To begin, degenerate oligonucleotide primers were used in combination with pre-existing cDNA libraries and reverse transcriptase-coupled polymerase chain reactions (RT-PCR). The putative leech Na channel cDNAs (LeNas) exhibit a higher degree of sequence homology to Na channel genes in other species than to voltage-gated calcium or potassium channel genes, including those expressed in leech. All LeNa cDNAs contain sequences corresponding to regions of functional importance in Na channel alpha subunits, including the "S4 region" involved in activation, the "pore loops" responsible for ion selectivity, and the "inactivation loop" between the third and fourth domains, though the latter lacks the highly conserved "IFM" motif critical for mammalian Na channel inactivation. Sequences corresponding to important determinants of tetrodotoxin sensitivity are found in some, but not all, LeNa cDNAs, consistent with prior electrophysiological evidence of Na channel heterogeneity in the leech with respect to this toxin. Subsequently, two different sets of isoform-specific primers and methods of RT-PCR, including a sensitive, fluorescence-based "real time" RT-PCR, were used to analyze LeNa isoform expression in functionally distinct neurons. The results from both approaches were consistent, and not only demonstrated that individual neurons often express more than one LeNa isoform, but also revealed cell-specific patterns of Na channel isoform expression in the leech nervous system.
Assuntos
Expressão Gênica , Sanguessugas/genética , Neurônios/fisiologia , Análise de Sequência de Proteína , Canais de Sódio/genética , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/análise , Ativação do Canal Iônico/genética , Dados de Sequência Molecular , Isoformas de Proteínas/genética , Subunidades Proteicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Canais de Sódio/isolamento & purificação , Especificidade da EspécieRESUMO
BCtheta is a proteolytically nicked and biotinylated derivative of a cholesterol binding protein perfringolysin O (theta-toxin), and has been used to detect cholesterol-rich domains at the plasma membrane (PM). Here we show that by modifying the cell fixation condition, BCtheta can also be used to detect cholesterol-rich domains intracellularly. When cells were processed for PM cholesterol staining, the difference in BCtheta signals between the CT43 (CT) cell, a mutant Chinese hamster ovary cell line lacking the Niemann-Pick type C1 (NPC1) protein, and its parental cell 25RA (RA) was minimal. However, when cells were fixed with 4% paraformaldehyde, they became permeable to BCtheta. Under this condition, BCtheta mainly stained cholesterol-rich domains inside the cells, with the signal being much stronger in CT cells than in RA cells. The sensitivity of BCtheta staining was superior to that of filipin staining. The staining of cholesterol-rich domain(s) inside RA cells was sensitive to beta-cyclodextrin treatment, while most of the staining inside CT cells was relatively resistant to cyclodextrin treatment. Clear differences in intracellular BCtheta staining were also seen between the normal and mutant NPC1 fibroblasts of human or mouse origin. Thus, BCtheta is a powerful tool for visually monitoring cholesterol-rich domains inside normal and NPC cells.