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J Neurochem ; 105(5): 1683-99, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18248623


We examined the mechanisms involved in protein kinase C (PKC)-dependent down-regulation of dopamine transporter (DAT) activity and cell surface expression by treating heterologously expressing cells with the clathrin-mediated endocytosis inhibitor concanavalin A (Con A) or the cholesterol depleter/membrane raft disrupter methyl-beta-cyclodextrin (MbetaC) prior to treatment with the PKC activator phorbol 12-myristate, 13-acetate (PMA). Con A blocked PMA-induced surface reductions of DAT but only partially inhibited down-regulation, while MbetaC partially blocked down-regulation but did not inhibit loss of cell surface DAT, demonstrating that PKC-induced DAT down-regulation occurs by a combination of trafficking and non-trafficking processes. Using density-gradient centrifugation, we found that DATs are distributed approximately equally between Triton-insoluble, cholesterol-rich membrane rafts and Triton-soluble non-raft membranes. DATs in both populations are present at the cell surface and are active for dopamine and cocaine binding. PMA-induced loss of cell surface DAT occurred only from non-raft populations, demonstrating that non-raft DATs are regulated by trafficking events and indicating the likelihood that the cholesterol-dependent non-trafficking regulatory mechanism occurs in rafts. PMA did not affect the DAT raft-non-raft distribution but stimulated the phosphorylation of DAT to a substantially greater level in rafts than non-rafts. These findings reveal a previously unknown role for cholesterol in DAT function and demonstrate the presence of distinct subcellular DAT populations that possess multiple regulatory differences that may impact dopaminergic neurotransmission.

Colesterol/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Microdomínios da Membrana/metabolismo , Ésteres de Forbol/farmacologia , Animais , Colesterol/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Humanos , Células LLC-PK1 , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/genética , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Ratos , Suínos
J Biol Chem ; 282(12): 8915-25, 2007 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-17255098


The site of cocaine binding on the dopamine transporter (DAT) was investigated using the photoactivatable irreversible cocaine analog [125I]3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82). The incorporation site of this compound was mapped to transmembrane domains (TMs) 4-6 using epitope-specific immunoprecipitation of trypsin fragments and further localized using cyanogen bromide (CNBr), which hydrolyzes proteins on the C-terminal side of methionine residues. CNBr hydrolysis of [125I]RTI 82-labeled rat striatal and expressed human DATs produced fragments of approximately 5-10 kDa consistent with labeling between Met(271/272) or Met(290) in TM5 to Met(370/371) in TM7. To further define the incorporation site, substitution mutations were made that removed endogenous methionines and inserted exogenous methionines in combinations that would generate labeled CNBr fragments of distinct masses depending on the labeling site. The results obtained were consistent with the presence of TM6 but not TMs 4, 5, or 7 in the labeled fragments, with additional support for these conclusions obtained by epitope-specific immunoprecipitation and secondary digestion of CNBr fragments with endoproteinase Lys-C. The final localization of [125I]RTI 82 incorporation to rat DAT Met(290)-Lys(336) and human DAT I291M to R344M provides positive evidence for the proximity of cocaine binding to TM6. Residues in and near DAT TM6 regulate transport and transport-dependent conformational states, and TM6 forms part of the substrate permeation pathway in the homologous Aquifex aeolicus leucine transporter. Cocaine binding near TM6 may thus overlap the dopamine translocation pathway and function to inhibit TM6 structural rearrangements necessary for transport.

Azidas/farmacologia , Cocaína/análogos & derivados , Proteínas da Membrana Plasmática de Transporte de Dopamina/química , Radioisótopos do Iodo/farmacologia , Animais , Cocaína/farmacologia , Brometo de Cianogênio/farmacologia , Inibidores da Captação de Dopamina/farmacologia , Epitopos/química , Humanos , Metionina/química , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Tripsina/química
J Biol Chem ; 279(35): 36621-4, 2004 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-15234968


For nearly 50 years, succinyl-CoA synthetase in animals was thought to be specific for guanine nucleotides. Recently, we purified and characterized both an ADP-forming succinyl-CoA synthetase from pigeon breast muscle and the GDP-forming enzyme from liver (Johnson, J. D., Muhonen, W. W., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27573-27579). Using the sequences of the pigeon enzymes as queries in BLAST searches, we obtained genetic evidence that both enzymes are expressed in a wide range of animal species (Johnson, J. D., Mehus, J. G., Tews, K., Milavetz, B. I., and Lambeth, D. O. (1998) J. Biol. Chem. 273, 27580-27586). Here we extend those observations by presenting data from Western and Northern blots and enzymatic assays showing that both proteins are widely expressed in mammals with the relative amounts varying from tissue to tissue. We suggest that both succinyl-CoA synthetases catalyze the reverse reaction in the citric acid cycle in which the ADP-forming enzyme augments ATP production, whereas the GDP-forming enzyme supports GTP-dependent anabolic processes. Widely accepted shuttle mechanisms are invoked to explain how transport of P-enolpyruvate across mitochondrial membranes can transfer high energy phosphate between the cytosol and mitochondrial matrix.

Succinato-CoA Ligases/biossíntese , Succinato-CoA Ligases/química , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Animais , Northern Blotting , Western Blotting , Mama/metabolismo , Membrana Celular/metabolismo , Columbidae , Citosol/metabolismo , Primers do DNA/farmacologia , Bases de Dados como Assunto , Feminino , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Fígado/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Modelos Biológicos , Miocárdio/metabolismo , Ratos , Testículo/metabolismo , Distribuição Tecidual
Am J Physiol Endocrinol Metab ; 284(2): E366-76, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12531745


This study characterized the cardiac contractile function and IGF-I response in a transgenic diabetic mouse model. Mechanical properties were evaluated in cardiac myocytes from OVE26 diabetic and FVB wild-type mice, including peak shortening (PS), time to PS (TPS), time to 90% relengthening (TR(90)) and maximal velocity of shortening/relengthening (+/-dL/dt). Intracellular Ca(2+) was evaluated as Ca(2+)-induced Ca(2+) release [difference in fura 2 fluorescent intensity (Delta FFI)] and fluorescence decay rate (tau). Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a, phospholamban (PLB), Na(+)-Ca(2+) exchanger (NCX), GLUT4, and the serine-threonine kinase Akt were assessed by Western blot. RhoA and IGF-I/IGF-I receptor mRNA levels were determined by RT-PCR and Northern blot. OVE26 myocytes displayed decreased PS, +/-dL/dt, and Delta FFI associated with prolonged TPS, TR(90), and tau. SERCA2a, NCX, and Akt activation were reduced, whereas PLB and RhoA were enhanced in OVE26 hearts. GLUT4 was unchanged. IGF-I enhanced PS and Delta FFI in FVB but not OVE26 myocytes. IGF-I mRNA was increased, but IGF-I receptor mRNA was reduced in OVE26 hearts and livers. These results validate diabetic cardiomyopathy in OVE26 mice due to reduced SERCA2, NCX, IGF-I response, and Akt activation associated with enhanced RhoA level, suggesting a therapeutic potential for Akt and RhoA.

Calmodulina/genética , Cardiomiopatias/fisiopatologia , Angiopatias Diabéticas/fisiopatologia , Fator de Crescimento Insulin-Like I/genética , Proteínas Musculares , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Animais , Western Blotting , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/análise , ATPases Transportadoras de Cálcio/análise , Cardiomiopatias/metabolismo , Angiopatias Diabéticas/metabolismo , Expressão Gênica , Transportador de Glucose Tipo 4 , Fator de Crescimento Insulin-Like I/farmacologia , Fígado/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas de Transporte de Monossacarídeos/análise , Contração Miocárdica/fisiologia , Miócitos Cardíacos/química , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Proteínas Proto-Oncogênicas/análise , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/análise , Receptor IGF Tipo 1/genética , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Trocador de Sódio e Cálcio/análise