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1.
BMC Neurosci ; 12: 16, 2011 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-21272290

RESUMO

BACKGROUND: The Na+,K+-ATPase plays an important role for ion homeostasis in virtually all mammalian cells, including neurons. Despite this, there is as yet little known about the isoform specific distribution in neurons. RESULTS: With help of superresolving stimulated emission depletion microscopy the spatial distribution of Na+,K+-ATPase in dendritic spines of cultured striatum neurons have been dissected. The found compartmentalized distribution provides a strong evidence for the confinement of neuronal Na+,K+-ATPase (α3 isoform) in the postsynaptic region of the spine. CONCLUSIONS: A compartmentalized distribution may have implications for the generation of local sodium gradients within the spine and for the structural and functional interaction between the sodium pump and other synaptic proteins. Superresolution microscopy has thus opened up a new perspective to elucidate the nature of the physiological function, regulation and signaling role of Na+,K+-ATPase from its topological distribution in dendritic spines.


Assuntos
Espinhas Dendríticas/enzimologia , Nanotecnologia/métodos , ATPase Trocadora de Sódio-Potássio/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Espinhas Dendríticas/ultraestrutura , Interpretação de Imagem Assistida por Computador/instrumentação , Interpretação de Imagem Assistida por Computador/métodos , Isoenzimas/metabolismo , Isoenzimas/ultraestrutura , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Dados de Sequência Molecular , Ratos , Ratos Sprague-Dawley , ATPase Trocadora de Sódio-Potássio/ultraestrutura
2.
Microsc Res Tech ; 75(2): 220-8, 2012 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21809413

RESUMO

Protein localization in dendritic spines is the focus of intense investigations within neuroscience. Applications of super-resolution microscopy to dissect nanoscale protein distributions, as shown in this work with dual-color STED, generate spatial correlation coefficients having quite small values. This means that colocalization analysis to some extent looses part of its correlative impact. In this study we thus introduced nearest neighbor analysis to quantify the spatial relations between two important proteins in neurons, the dopamine D1 receptor and Na(+),K(+)-ATPase. The analysis gave new information on how dense the D1 receptor and Na(+),K(+)-ATPase constituting nanoclusters are located both with respect to the homogenous (self to same) and the heterogeneous (same to other) topology. The STED dissected nanoscale topologies provide evidence for both a joint as well as a separated confinement of the D1 receptor and the Na(+),K(+)-ATPase in the postsynaptic areas of dendritic spines. This confined topology may have implications for generation of local sodium gradients and for structural and functional interactions modulating slow synaptic transmission processes.


Assuntos
Espinhas Dendríticas/química , Microscopia de Fluorescência/instrumentação , Receptores de Dopamina D1/química , ATPase Trocadora de Sódio-Potássio/química , Animais , Células Cultivadas , Interpretação de Imagem Assistida por Computador/instrumentação , Interpretação de Imagem Assistida por Computador/métodos , Imuno-Histoquímica , Imunoprecipitação , Microscopia Eletrônica de Transmissão e Varredura , Microscopia de Fluorescência/métodos , Nanotecnologia/métodos , Neurônios/química , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Dopamina D1/análise , ATPase Trocadora de Sódio-Potássio/análise
3.
J Biol Chem ; 283(17): 11461-8, 2008 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-18303017

RESUMO

Na,K-ATPase and inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) can form a signaling microdomain that in the presence of ouabain triggers highly regular calcium oscillations. Downstream effects include NF-kappaB activation. Here we report that ankyrin B (Ank-B), expressed in most mammalian cells, plays a pivotal role in the function of the Na,K-ATPase/IP3R signaling microdomain. In studies performed on a monkey kidney cell line, we show that Ank-B co-precipitates with both Na,K-ATPase and IP3R. We identify the N terminus tail of the Na,K-ATPase catalytic subunit and the N-terminal portion 1-604 of the IP3R as novel binding sites for Ank-B. Knockdown of Ank-B with small interfering RNA reduced the expression of Ank-B to 15-30%. This down-regulation of Ank-B attenuated the interaction between Na,K-ATPase and IP3R, reduced the number of cells responding to pm doses of ouabain with calcium oscillations, altered the calcium oscillatory pattern, and abolished the ouabain effect on NF-kappaB. In contrast, Ank-B down-regulation had no effect on the ion transporting function of Na,K-ATPase and no effect on the distribution and apparent mobility of Na,K-ATPase in the plasma membrane.


Assuntos
Anquirinas/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microdomínios da Membrana/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Anquirinas/metabolismo , Células COS , Membrana Celular/enzimologia , Chlorocebus aethiops , Regulação para Baixo , Humanos , Ouabaína/farmacologia , Conformação Proteica , Proteínas Recombinantes/química
4.
Am J Physiol Renal Physiol ; 295(4): F1110-6, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18701624

RESUMO

Sodium excretion is bidirectionally regulated by dopamine, acting on D1-like receptors (D1R) and angiotensin II, acting on AT1 receptors (AT1R). Since sodium excretion has to be regulated with great precision within a short frame of time, we tested the short-term effects of agonist binding on the function of the reciprocal receptor within the D1R-AT1R complex in renal proximal tubule cells. Exposure of rat renal proximal tubule cells to a D1 agonist was found to result in a rapid partial internalization of AT1R and complete abolishment of AT1R signaling. Similarly, exposure of rat proximal tubule cells and renal tissue to angiotensin II resulted in a rapid partial internalization of D1R and abolishment of D1R signaling. D1R and AT1R were, by use of coimmunoprecipitation studies and glutathione-S-transferase pull-down assays, shown to be partners in a multiprotein complex. Na+-K+-ATPase, the target for both receptors, was included in this complex, and a region in the COOH-terminal tail of D1R (residues 397-416) was found to interact with both AT1R and Na+-K+-ATPase. Results indicate that AT1R and D1R function as a unit of opposites, which should provide a highly versatile and sensitive system for short-term regulation of sodium excretion.


Assuntos
Sinalização do Cálcio/fisiologia , Túbulos Renais Proximais/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores de Dopamina D1/metabolismo , Sódio/metabolismo , Angiotensina II/farmacologia , Animais , Benzazepinas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Masculino , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptor Cross-Talk/fisiologia , Receptor Tipo 1 de Angiotensina/agonistas , Receptor Tipo 1 de Angiotensina/química , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Vasoconstritores/farmacologia
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