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1.
Adv Exp Med Biol ; 1131: 93-129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646508

RESUMO

Plasma membrane Ca2+ transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca2+ from the cell in order to keep the cytosolic Ca2+ ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca2+ homeostasis they also change the duration and amplitude of the Ca2+ signal and can create Ca2+ gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.


Assuntos
Membrana Celular , ATPases Transportadoras de Cálcio da Membrana Plasmática , Animais , Membrana Celular/enzimologia , Membrana Celular/patologia , Citosol/metabolismo , Homeostase/fisiologia , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
2.
Biochim Biophys Acta ; 1863(6 Pt B): 1351-63, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26707182

RESUMO

Plasma membrane Ca(2+) ATPases (PMCAs) are intimately involved in the control of intracellular Ca(2+) concentration. They reduce Ca(2+) in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca(2+) release from the endoplasmic reticulum Ca(2+) pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca(2+) with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca(2+) homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca(2+) homeostasis of cancer cells. This article is part of a Special Issue entitled: Calcium and Cell Fate. Guest Editors: Jacques Haiech, Claus Heizmann, Joachim Krebs, Thierry Capiod and Olivier Mignen.


Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Membrana Celular/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Homeostase , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética
3.
J Cell Sci ; 127(Pt 1): 72-84, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24198396

RESUMO

Plasma membrane Ca(2+) ATPases (PMCAs, also known as ATP2B1-ATP2B4) are known targets of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], but if and how they control the PtdIns(4,5)P2 pool has not been considered. We demonstrate here that PMCAs protect PtdIns(4,5)P2 in the plasma membrane from hydrolysis by phospholipase C (PLC). Comparison of active and inactive PMCAs indicates that the protection operates by two mechanisms; one requiring active PMCAs, the other not. It appears that the mechanism requiring activity is the removal of the Ca(2+) required for sustained PLC activity, whereas the mechanism not requiring activity is PtdIns(4,5)P2 binding. We show that in PMCA overexpressing cells, PtdIns(4,5)P2 binding can lead to less inositol 1,4,5-triphosphate (InsP3) and diminished Ca(2+) release from intracellular Ca(2+) pools. Inspection of a homology model of PMCA suggests that PMCAs have a conserved cluster of basic residues forming a 'blue collar' at the interface between the membrane core and the cytoplasmic domains. By molecular dynamics simulation, we found that the blue collar forms four binding pockets for the phosphorylated inositol head group of PtdIns(4,5)P2; these pockets bind PtdIns(4,5)P2 strongly and frequently. Our studies suggest that by having the ability to bind PtdIns(4,5)P2, PMCAs can control the accessibility of PtdIns(4,5)P2 for PLC and other PtdIns(4,5)P2-mediated processes.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Sinalização do Cálcio , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Membrana Celular/química , Expressão Gênica , Regulação da Expressão Gênica , Células HeLa , Humanos , Hidrólise , Inositol 1,4,5-Trifosfato/química , Transporte de Íons , Simulação de Dinâmica Molecular , Fosfatidilinositol 4,5-Difosfato/química , Ligação Proteica , Coelhos , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Fosfolipases Tipo C/química , Fosfolipases Tipo C/genética
4.
J Biol Chem ; 287(35): 29664-71, 2012 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-22767601

RESUMO

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.


Assuntos
Calmodulina/química , Membrana Celular/enzimologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Calmodulina/genética , Calmodulina/metabolismo , Membrana Celular/genética , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína
5.
J Biol Chem ; 285(6): 4015-24, 2010 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-19996092

RESUMO

Using solution NMR spectroscopy, we obtained the structure of Ca(2+)-calmodulin (holoCaM) in complex with peptide C28 from the binding domain of the plasma membrane Ca(2+)-ATPase (PMCA) pump isoform 4b. This provides the first atomic resolution insight into the binding mode of holoCaM to the full-length binding domain of PMCA. Structural comparison of the previously determined holoCaM.C20 complex with this holoCaM.C28 complex supports the idea that the initial binding step is represented by (holoCaM.C20) and the final bound complex by (holoCaM.C28). This affirms the existing multi-step kinetic model of PMCA4b activation by CaM. The complex exhibits a new binding motif in which holoCaM is wrapped around helical C28 peptide using two anchoring residues from the peptide at relative positions 18 and 1. The anchors correspond to Phe-1110 and Trp-1093, respectively, in full-length PMCA4b, and the peptide and CaM are oriented in an anti-parallel manner. This is a greater sequence distance between anchors than in any of the known holoCaM complexes with a helical peptide. Analysis of the geometry of holoCaM-peptide binding for the cases where the target peptide adopts an alpha(D)-helix with its anchors buried in the main hydrophobic pockets of the two CaM lobes establishes that only relative sequential positions of 10, 14, 17, and 18 are allowed for the second anchor.


Assuntos
Cálcio/metabolismo , Calmodulina/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Domínios e Motivos de Interação entre Proteínas , Sítios de Ligação , Cálcio/química , Calmodulina/química , Humanos , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Fenilalanina/química , Fenilalanina/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Triptofano/química , Triptofano/metabolismo
6.
J Biol Chem ; 282(35): 25640-8, 2007 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-17595168

RESUMO

The inhibition by the regulatory domain and the interaction with calmodulin (CaM) vary among plasma membrane calcium pump (PMCA) isoforms. To explore these differences, the kinetics of CaM effects on PMCA4a were investigated and compared with those of PMCA4b. The maximal apparent rate constant for CaM activation of PMCA4a was almost twice that for PMCA4b, whereas the rates of activation for both isoforms showed similar dependence on Ca2+. The inactivation of PMCA4a by CaM removal was also faster than for PMCA4b, and Ca2+ showed a much smaller effect (2- versus 30-fold modification). The rate constants of the individual steps that determine the overall rates were obtained from stopped-flow experiments in which binding of TA-CaM was observed by changes in its fluorescence. TA-CaM binds to two conformations of PMCA4a, an "open" conformation with high activity, and a "closed" one with lower activity. Compared with PMCA4b (Penheiter, A. R., Bajzer, Z., Filoteo, A. G., Thorogate, R., Török, K., and Caride, A. J. (2003) Biochemistry 41, 12115-12124), the model for PMCA4a predicts less inhibition in the closed form and a much faster equilibrium between the open and closed forms. Based on the available kinetic parameters, we determined the constants to fit the shape of a Ca2+ signal in PMCA4b-overexpressing Chinese hamster ovary cells. Using the constants for PMCA4a, and allowing small variations in parameters of other systems contributing to a Ca2+ signal, we then simulated the effect of PMCA4a on the shape of a Ca2+ signal in Chinese hamster ovary cells. The results reproduce the published data (Brini, M., Coletto, L., Pierobon, N., Kraev, N., Guerini, D., and Carafoli, E. (2003) J. Biol. Chem. 278, 24500-24508), and thereby demonstrate the importance of altered regulatory kinetics for the different functional properties of PMCA isoforms.


Assuntos
Sinalização do Cálcio/fisiologia , Calmodulina/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Animais , Células CHO , Calmodulina/genética , Linhagem Celular , Cricetinae , Cricetulus , Humanos , Cinética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Ligação Proteica/fisiologia , Conformação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Spodoptera
7.
Ann N Y Acad Sci ; 1099: 226-36, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17446463

RESUMO

Plasma membrane Ca2+ ATPases (PMCAs) are essential components of the cellular toolkit to regulate and fine-tune cytosolic Ca2+ concentrations. Historically, the PMCAs have been assigned a housekeeping role in the maintenance of intracellular Ca2+ homeostasis. More recent work has revealed a perplexing multitude of PMCA isoforms and alternative splice variants, raising questions about their specific role in Ca2+ handling under conditions of varying Ca2+ loads. Studies on the kinetics of individual isoforms, combined with expression and localization studies suggest that PMCAs are optimized to function in Ca2+ regulation according to tissue- and cell-specific demands. Different PMCA isoforms help control slow, tonic Ca2+ signals in some cells and rapid, efficient Ca2+ extrusion in others. Localized Ca2+ handling requires targeting of the pumps to specialized cellular locales, such as the apical membrane of cochlear hair cells or the basolateral membrane of kidney epithelial cells. Recent studies suggest that alternatively spliced regions in the PMCAs are responsible for their unique targeting, membrane localization, and signaling cross-talk. The regulated deployment and retrieval of PMCAs from specific membranes provide a dynamic system for a cell to respond to changing needs of Ca2+ regulation.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Isoenzimas/metabolismo , Sinalização do Cálcio , Membrana Celular/enzimologia , Cinética
8.
Ann N Y Acad Sci ; 1099: 440-50, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17446484

RESUMO

Maintenance of Ca2+ homeostasis is essential for normal cellular function and survival. Recent evidences suggest that Ca2+ is also an important player of apoptosis. We demonstrated that the plasma membrane Ca2+ ATPase (PMCA) isoform 4b, a key element of cellular Ca2+ homeostasis, was cleaved by caspase-3 during the course of apoptosis. This cleavage of PMCA removed the entire regulatory region from the C terminus, leaving behind a 120-kDa catalytic fragment. Since loss of PMCA activity could lead to intracellular Ca2+ overload and consequently necrotic cell death, an important question is whether the apoptotic fragment of PMCA retains full activity or it is inactivated. To address this question, we constructed a C-terminally truncated mutant that corresponded to the caspase-3 fragment of PMCA4b and showed that it was fully and constitutively active. This mutant was targeted properly to the plasma membrane when it was expressed stably or transiently in several different cell lines. We followed truncation of PMCA during apoptosis induced by mitochondrial or receptor-mediated pathways and found that a similar fragment of 120 kDa was formed and remained intact for several hours after treatment. We have also demonstrated that the caspase-3 cleavage site is an important structural element of PMCA and found that the accessibility of the caspase-3 site depended strongly on the conformational state of the protein.


Assuntos
Apoptose , ATPases Transportadoras de Cálcio/metabolismo , Animais , Caspase 3/metabolismo , Linhagem Celular , Membrana Celular/enzimologia , Humanos , Imuno-Histoquímica , Proteínas Recombinantes/metabolismo
9.
Cell Calcium ; 42(6): 590-605, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-17433436

RESUMO

In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.


Assuntos
Diferenciação Celular/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Animais , Transporte Biológico , Células COS , Células CACO-2 , Cálcio/metabolismo , Cálcio/farmacocinética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cercopithecus aethiops , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ácidos Graxos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Células HT29 , Células HeLa , Humanos , Imuno-Histoquímica , Isoenzimas/genética , Isoenzimas/metabolismo , Microssomos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Regulação para Cima/efeitos dos fármacos
10.
Biochem J ; 391(Pt 3): 687-92, 2005 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-16080782

RESUMO

The calmodulin-activated transporter hPMCA4 (human plasma membrane Ca2+-ATPase isoform 4) is a target for cleavage by caspase-3 during apoptosis. We have demonstrated that caspase-3 generates a 120 kDa fragment of this pump which lacks the complete autoinhibitory sequence [Paszty, Verma, Padanyi, Filoteo, Penniston and Enyedi (2002) J. Biol. Chem. 277, 6822-6829]. In the present study we analysed further the characteristics of the fragment of hPMCA4b produced by caspase-3. We did this by overexpressing the caspase-3 cleavage product of hPMCA4b in COS-7 and MDCKII (Madin-Darby canine kidney II) cells. This technique made it possible to clearly define the properties of this fragment, and we showed that it is constitutively active, as it forms a phosphoenzyme intermediate and has high Ca2+ transport activity in the absence of calmodulin. When this fragment of hPMCA4b was stably expressed in MDCKII cell clones, it was targeted without degradation to the basolateral plasma membrane. In summary, our studies emphasize that the caspase-3 cleavage product of hPMCA4b is constitutively active, and that the C-terminus is not required for proper targeting of hPMCA4b to the plasma membrane. Also, for the first time, we have generated cell clones that stably express a constitutively active PMCA.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Caspases/metabolismo , Membrana Celular/metabolismo , Animais , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/química , Calmodulina/metabolismo , Caspase 3 , Linhagem Celular , Membrana Celular/enzimologia , Cercopithecus aethiops , Cães , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , ATPases Transportadoras de Cálcio da Membrana Plasmática , Transporte Proteico , Especificidade por Substrato
11.
N Engl J Med ; 352(15): 1557-64, 2005 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-15829536

RESUMO

Five adult siblings presented with autosomal recessive sensorineural hearing loss: two had high-frequency loss, whereas the other three had severe-to-profound loss affecting all frequencies. Genetic evaluation revealed that a homozygous mutation in CDH23 (which encodes cadherin 23) caused the hearing loss in all five siblings and that a heterozygous, hypofunctional variant (V586M) in plasma-membrane calcium pump PMCA2, which is encoded by ATP2B2, was associated with increased loss in the three severely affected siblings. V586M was detected in two unrelated persons with increased sensorineural hearing loss, in the other caused by a mutation in MYO6 (which encodes myosin VI) in one and by noise exposure, suggesting that this variant may modify the severity of sensorineural hearing loss caused by a variety of factors.


Assuntos
Caderinas/genética , ATPases Transportadoras de Cálcio/genética , Perda Auditiva Neurossensorial/genética , Herança Multifatorial , Mutação de Sentido Incorreto , Adulto , Alelos , Proteínas de Transporte de Cátions , Feminino , Genes Recessivos , Genótipo , Perda Auditiva Neurossensorial/classificação , Heterozigoto , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática , Mutação Puntual , Irmãos
12.
Biochemistry ; 44(6): 2009-20, 2005 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-15697226

RESUMO

The sequence L(1086)RRGQILWFRGLNRIQTQIKVVKAFHSS(1113) (peptide C28) is responsible for calmodulin binding to PMCA4b. In this work, peptides following the above sequence were progressively shortened either at the N-terminus (C28NDelta3, C28NDelta5, or C28NDelta6) or at the C-terminus (C20, C22, C23, and C25). Competitive inhibition of PMCA activity was used to measure apparent dissociation constants of the complexes between calmodulin and C28 or progressively shortened peptides. Additionally, equilibrium titrations were used to measure the apparent dissociation constants of the various peptides with TA-calmodulin by changes in TA-calmodulin fluorescence and Trp fluorescence of the peptides. At the N-terminus, deletion of five residues did not change calmodulin affinity, but deletion of six residues resulted in a 5-fold decrease in affinity. There were no major differences in the time course of TA-CaM binding, but C28NDelta6 exhibited a different time course of Trp fluorescence change. At the C-terminus, deletion of five residues (C23) or more resulted in a net increase in fluorescence of TA-CaM upon binding, while longer peptides (C25 and C28) produced both a transient increase and a net decrease in the fluorescence of TA-CaM. Global regression analysis revealed that binding of TA-CaM to the C23 peptide could be fit by a two-step model, while longer peptides required three-step models for adequate fitting. TA-calmodulin dissociated rapidly from C23, C22, and C20, resulting in a marked increase in apparent K(d). Thus, the sequence I(1091)LWFRGLNRIQTQIKVVKAF(1110) (C25NDelta5) is required to reproduce the calmodulin-binding properties of C28. When F(1110) was replaced by A, the TA-calmodulin association and dissociation kinetics resembled C23 kinetics, but changing V(1107) to A produced a smaller effect, suggesting that F(1110), rather than V(1107), is the main anchor for the N-terminal lobe of calmodulin in PMCA4b.


Assuntos
ATPases Transportadoras de Cálcio/metabolismo , Calmodulina/análogos & derivados , Calmodulina/metabolismo , Fragmentos de Peptídeos/metabolismo , Sequência de Aminoácidos , Ligação Competitiva , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/genética , Calmodulina/química , Proteínas de Transporte de Cátions , Membrana Celular/química , Membrana Celular/genética , Membrana Celular/metabolismo , Inibidores Enzimáticos/metabolismo , Corantes Fluorescentes/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática , Ligação Proteica , Estrutura Terciária de Proteína/genética , Deleção de Sequência , Espectrometria de Fluorescência , Triazinas/metabolismo
13.
J Assoc Res Otolaryngol ; 5(2): 99-110, 2004 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-15357414

RESUMO

In vertebrates, transduction of sound into an electrochemical signal is carried out by hair cells that rely on calcium to perform specialized functions. The apical surfaces of hair cells are surrounded by endolymphatic fluid containing calcium at concentrations that must be maintained by active transport. The mechanism of this transport is unknown, but an ATP-dependent pump is believed to participate. Mutation of the Atp2b2 gene that encodes plasma membrane calcium ATPase type 2 (PMCA2) produces the deaf, ataxic mouse: deafwaddler2J (dfw2J). We hypothesized that PMCA2 might transport calcium into the endolymph and that dfw2J mice would have low endolymph calcium concentrations, possibly contributing to their deafness and ataxia. First, using immunocytochemistry, we demonstrated that PMCA2 is present in control mice inner and outer hair cell stereocilia where it could pump calcium into the endolymph and that PMCA2 is absent in dfw2J stereocilia. Second, using an aspirating microelectrode and calcium-sensitive fluorescent dye, we found that dfw2J mice endolymph calcium concentrations are significantly lower than those of control mice. These findings suggest that PMCA2, located in hair cell stereocilia, contributes significantly to endolymph calcium maintenance.


Assuntos
ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Cálcio/metabolismo , Surdez/metabolismo , Endolinfa/metabolismo , Animais , Proteínas de Transporte de Cátions , Cóclea/fisiologia , Surdez/genética , Surdez/fisiopatologia , Potenciais Evocados Auditivos , Feminino , Células Ciliadas Auditivas Internas/metabolismo , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/patologia , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Mutantes Neurológicos , ATPases Transportadoras de Cálcio da Membrana Plasmática
14.
Neuron ; 43(2): 169-75, 2004 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-15260953

RESUMO

Rapid-onset dystonia-parkinsonism (RDP, DYT12) is a distinctive autosomal-dominant movement disorder with variable expressivity and reduced penetrance characterized by abrupt onset of dystonia, usually accompanied by signs of parkinsonism. The sudden onset of symptoms over hours to a few weeks, often associated with physical or emotional stress, suggests a trigger initiating a nervous system insult resulting in permanent neurologic disability. We report the finding of six missense mutations in the gene for the Na+/K+ -ATPase alpha3 subunit (ATP1A3) in seven unrelated families with RDP. Functional studies and structural analysis of the protein suggest that these mutations impair enzyme activity or stability. This finding implicates the Na+/K+ pump, a crucial protein responsible for the electrochemical gradient across the cell membrane, in dystonia and parkinsonism.


Assuntos
Distonia/genética , Mutação de Sentido Incorreto , Transtornos Parkinsonianos/genética , ATPase Trocadora de Sódio-Potássio/genética , Sequência de Aminoácidos , Linhagem Celular , Distonia/complicações , Distonia/metabolismo , Humanos , Conformação Molecular , Transtornos Parkinsonianos/complicações , Transtornos Parkinsonianos/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Relação Estrutura-Atividade
15.
J Biol Chem ; 278(37): 35798-804, 2003 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-12829699

RESUMO

The access of three proteases to their sites of cleavage was used as a measure of regulatory interactions in the plasma membrane Ca2+ pump isoform 4b (PMCA4b). When the proteases could not cut at their sites in the C-terminal regulatory region, the interaction was judged to be tight. This was the case in the absence of Ca2+, when chymotrypsin and caspase cut PMCA only very slowly. Ca2+ accelerated the fragmentation, but the digestion remained incomplete. In the presence of Ca2+ plus calmodulin, the digestion became nearly complete in all cases, indicating a more flexible conformation of the carboxyl terminus in the fully activated state. The acceleration of proteolysis by Ca2+ or Ca2+ plus calmodulin occurred equally at the caspase site upstream of the calmodulin-binding domain and the chymotrypsin and calpain sites downstream of that domain. Replacing Trp1093 (a key residue within the calmodulin-binding domain) with alanine had a much more specific effect, because it exposed only proteolytic sites within the calmodulin-binding domain that had previously been shielded in the native protein. At these sites, both calpain and chymotrypsin cut the Trp1093 --> Ala mutant in the absence of calmodulin. These data indicate that, in the auto-inhibited conformation, the calmodulin-binding/auto-inhibitory sequence and the regions both upstream and downstream are in close contact with the catalytic core. Trp1093 plays an essential role not only in stabilizing the Ca2+-calmodulin/calmodulin-binding domain complex but also in the formation or stability of the inhibitory conformation of that domain when it interacts with the catalytic core of PMCA4b.


Assuntos
ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Membrana Celular/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Caspase 3 , Caspases/metabolismo , Catálise , Domínio Catalítico , Retículo Endoplasmático/enzimologia , Cinética , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Retículo Sarcoplasmático/enzimologia , Transfecção
16.
J Biol Chem ; 277(39): 36146-51, 2002 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-12145294

RESUMO

The role of the plasma membrane Ca(2+) pump (PMCA) is to remove excess Ca(2+) from the cytosol to maintain low intracellular Ca(2+) levels. Asp(1080) lies within an acidic sequence between the C-terminal inhibitory region and the catalytic core of PMCAs and is part of the caspase-3 recognition site of isoform 4b. Caspase-3 cuts immediately after this residue and activates the pump by removing the inhibitory region (Pászty, K., Verma, A. K., Padányi, R., Filoteo, A. G., Penniston, J. T., and Enyedi, A. (2002) J. Biol. Chem. 277, 6822-6829). Asp(1080) had not been believed to have any other role, but here we show that it also plays a critical role in the autoinhibition and calmodulin activation of PMCA4b. Site-specific mutation of Asp(1080) to Asn, Ala, or Lys in PMCA4b resulted in a substantial increase in the basal activity in the absence of calmodulin. All Asp(1080) mutants exhibited an increased affinity for calmodulin because of an increase in the rate of activation by calmodulin. This rate was higher when the inhibition was weaker, showing that a strong inhibitory interaction slows the activation rate. In contrast, mutating the nearby Asp(1077) had no effect on basal activity or calmodulin activation. We propose that the conserved Asp(1080), even though it is neither in the regulatory domain nor in the catalytic core, plays an essential role in inhibition by stabilizing the inhibited state of the enzyme.


Assuntos
Ácido Aspártico/química , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Calmodulina/metabolismo , Adenosina Trifosfatases/metabolismo , Alanina/química , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Asparagina/química , Sítios de Ligação , Células COS , Cálcio/metabolismo , Cálcio/farmacologia , Catálise , Proteínas de Transporte de Cátions , Membrana Celular/metabolismo , Relação Dose-Resposta a Droga , Cinética , Lisina/química , Microssomos/metabolismo , Dados de Sequência Molecular , Mutação , ATPases Transportadoras de Cálcio da Membrana Plasmática , Ligação Proteica , Estrutura Terciária de Proteína , Fatores de Tempo , Transfecção
17.
J Biol Chem ; 277(20): 17728-32, 2002 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-11886854

RESUMO

Tryptophan 1093 resides in the 28-residue calmodulin-binding/autoinhibitory domain of the plasma membrane Ca(2+) pump (PMCA). Previous studies with the isolated calmodulin-binding/autoinhibitory peptide from PMCA have shown that mutations of the tryptophan residue decrease the affinity of the peptide for calmodulin and its affinity as an inhibitor of proteolytically activated pump. In this study, the PMCA mutation in which tryptophan 1093 is converted to alanine (W1093A) was constructed in the full-length PMCA isoform 4b. The mutant pump was expressed in COS cells, and its steady state and pre-steady state kinetic properties were examined. The W1093A pump exhibited an increased basal activity in the absence of calmodulin, so the activation was approximately 2-fold (it is 10-fold in the wild type). The W1093A mutation also lowered the steady state affinity for calmodulin from K(0.5) of 9 nm for wild type to 144 nm (assayed at 700 nm free Ca(2+)). Pre-steady state measurements of the rate of activation by Ca(2+)-calmodulin revealed that the W1093A mutant responded 2.5-fold faster to calmodulin. In contrast to these relatively modest effects, the half-time of inactivation of the mutant was reduced by more than 2 orders of magnitude from 41 min to 7 s. We conclude that tryptophan 1093 does not play a substantial role in Ca(2+)-calmodulin recognition; rather it functions primarily to slow the inactivation of the calmodulin-activated pump.


Assuntos
Canais de Cálcio/metabolismo , Calmodulina/metabolismo , Triptofano/fisiologia , Sequência de Aminoácidos , Animais , Células COS , Cálcio/metabolismo , Canais de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Proteínas de Transporte de Cátions , Ativação Enzimática , Cinética , Dados de Sequência Molecular , ATPases Transportadoras de Cálcio da Membrana Plasmática , Relação Estrutura-Atividade
18.
J Biol Chem ; 277(9): 6822-9, 2002 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11751908

RESUMO

The plasma membrane Ca(2+) pump (PMCA) is an essential element in the complex of mechanisms that maintain low intracellular Ca(2+) concentration in the living cell. This pump is tightly regulated by calmodulin through binding to a high affinity calmodulin-binding domain at the C terminus that also serves as an autoinhibitor of the enzyme. Inspection of the C terminus of hPMCA4b, the most widely distributed form of PMCA, revealed a caspase-3 consensus sequence ((1077)DEID(1080)) just a few residues upstream of the calmodulin-binding domain. We demonstrate here that, in the early phase of apoptosis, hPMCA4b is cleaved at aspartic acid Asp(1080) in hPMCA4b-transfected COS-7 cells or in HeLa cells that naturally express this protein. This cleavage of hPMCA4b produces a single 120-kDa fragment that is fully active in the absence of calmodulin, because the whole inhibitory region downstream of the (1077)DEID(1080) sequence is removed. Our experiments show that caspase-3 or a caspase-3-like protease is responsible for the formation of the constitutively active 120-kDa PMCA4b fragment: 1) Pretreatment of the cells with the caspase-3 inhibitor Z-DEVD-FMK (benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone) was able to block the production of the 120-kDa fragment. 2) In vitro treatment of hPMCA4b with recombinant caspase-3 also generated a 120-kDa cleavage product, consistent with that seen in cells undergoing apoptosis. 3) Mutants in which the caspase-3 consensus sequence was altered ((1077)AEID(1080), (1077)DEIA(1080), and (1077)AEIA(1080) mutants) were resistant to proteolysis. Based on these data, we conclude that hPMCA4b is a newly identified, natural caspase-3 substrate. We suggest that a constitutively active form of this protein, responding much faster to an increase in Ca(2+) concentration than the autoinhibited form, may have an important role in regulating intracellular Ca(2+) concentration in the apoptotic cell.


Assuntos
Apoptose , ATPases Transportadoras de Cálcio/química , Caspases/metabolismo , Membrana Celular/metabolismo , Sequência de Aminoácidos , Animais , Anexina A5/farmacologia , Células COS , Cálcio/química , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Calmodulina/metabolismo , Caspase 3 , Proteínas de Transporte de Cátions , Corantes/farmacologia , Citosol/química , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Células HeLa , Humanos , Dados de Sequência Molecular , Mutação , ATPases Transportadoras de Cálcio da Membrana Plasmática , Mutação Puntual , Ligação Proteica , Isoformas de Proteínas , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Sitios de Sequências Rotuladas , Fatores de Tempo , Transfecção
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