RESUMEN
Recombinant nucleotide-binding domains (NBDs) from human multidrug resistance protein MRP1 were overexpressed in bacteria and purified to measure their direct interaction with high-affinity flavonoids, and to evaluate a potential correlation with inhibition of MRP1-mediated transport activity and reversion of cellular multidrug resistance. Among different classes of flavonoids, dehydrosilybin exhibited the highest affinity for both NBDs, the binding to N-terminal NBD1 being prevented by ATP. Dehydrosilybin increased vanadate-induced 8-N3-[alpha-32P]ADP trapping, indicating stimulation of ATPase activity. In contrast, dehydrosilybin strongly inhibited leukotriene C4 (LTC4) transport by membrane vesicles from MRP1-transfected cells, independently of reduced glutathione, and chemosensitized cell growth to vincristine. Hydrophobic C-isoprenylation of dehydrosilybin increased the binding affinity for NBD1, but outsite the ATP site, lowered the increase in vanadate-induced 8-N3-[alpha-32P]ADP trapping, weakened inhibition of LTC4 transport which became glutathione dependent, and induced some cross-resistance. The overall results indicate multiple binding sites for dehydrosilybin and its derivatives, on both cytosolic and transmembrane domains of MRP1.
Asunto(s)
Flavonoides/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Adenosina Difosfato/metabolismo , Animales , Antineoplásicos Fitogénicos/farmacología , Sitios de Unión , Clonación Molecular , Cricetinae , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/efectos de los fármacos , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Estructura Terciaria de Proteína , Vincristina/farmacologíaRESUMEN
Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the '9CA' mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5-10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wild-type channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by PKC and for its modulation of CFTR responses to PKA.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , AMP Cíclico/análogos & derivados , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteína Quinasa C/metabolismo , Alcaloides , Secuencia de Aminoácidos , Animales , Benzofenantridinas , Sitios de Unión , Bovinos , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Cricetinae , AMP Cíclico/farmacología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Electrofisiología , Inhibidores Enzimáticos/farmacología , Immunoblotting , Yoduros/metabolismo , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Mutación/fisiología , Técnicas de Placa-Clamp , Fenantridinas/farmacología , Fosforilación , Ratas , Tionucleótidos/farmacologíaRESUMEN
Although the CFTR protein alone is sufficient to generate a regulated chloride channel, it is unknown how many of the polypeptides form the channel. Using biochemical and functional assays, we demonstrate that the CFTR polypeptide is a monomer. CFTR sediments as a monomer in a linear, continuous sucrose gradient. Cells co-expressing different epitope-tagged CFTR provide no evidence of co-assembly in immunoprecipitation and nickel affinity binding experiments. Co-expressed wild-type and DF508 CFTR are without influence on each other in their ability to progress through the secretory pathway, suggesting they do not associate in the endoplasmic reticulum. No hybrid conducting single channels are seen in planar lipid bilayers with which membrane vesicles from cells co-expressing similar amounts of two different CFTR conduction species have been fused.
Asunto(s)
Canales de Cloruro/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Expresión Génica , Membrana Dobles de Lípidos/química , Animales , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/aislamiento & purificación , Epítopos , Humanos , Riñón/fisiología , Potenciales de la Membrana/fisiología , Microsomas/química , Microsomas/fisiología , Estructura Cuaternaria de Proteína , Proteínas Recombinantes , Especificidad de la EspecieRESUMEN
ATP-binding cassette (ABC) transporters couple the binding and hydrolysis of ATP to the translocation of solutes across biological membranes. The so-called "Walker motifs" in each of the nucleotide binding domains (NBDs) of these proteins contribute directly to the binding and the catalytic site for the MgATP substrate. Hence mutagenesis of residues in these motifs may interfere with function. This is the case with the MRP1 multidrug transporter. However, interpretation of the effect of mutation in the Walker B motif of NBD1 (D792L/D793L) was confused by the fact that it prevented biosynthetic maturation of the protein. We have determined now that this latter effect is entirely due to the D792L substitution. This variant is unable to mature conformationally as evidenced by its remaining more sensitive to trypsin digestion in vitro than the mature wild-type protein. In vivo, the core-glycosylated form of that mutant is retained in the endoplasmic reticulum and degraded by the proteasome. A different substitution of the same residue (D792A) had a less severe effect enabling accumulation of approximately equal amounts of mature and immature MRP1 proteins in the membrane vesicles but still resulted in defective nucleotide interaction and organic anion transport, indicating that nucleotide hydrolysis at NBD1 is essential to MRP1 function.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Mutación , Transportadoras de Casetes de Unión a ATP/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Cricetinae , Cisteína Endopeptidasas/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Complejos Multienzimáticos/antagonistas & inhibidores , Complejos Multienzimáticos/metabolismo , Mutagénesis Sitio-Dirigida , Mutación Missense , Nucleótidos/metabolismo , Complejo de la Endopetidasa Proteasomal , Conformación Proteica , Procesamiento Proteico-Postraduccional , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TripsinaRESUMEN
Multidrug resistance protein MRP1 is an ATP-dependent drug efflux pump that confers resistance in human cancer cells to various chemotherapeutic drugs. We have reconstituted purified MRP1 in lipid vesicles. The reconstituted protein conserves ATPase and drug transport activity. Structural analysis of MRP1 was investigated by infrared spectroscopy for the first time. This technique offers a unique opportunity to determine structural parameters characterizing a membrane protein in its lipid environment. Addition of different ligands (MgATP, MgATPgammaS, MgADP and P(i), and MgADP) did not significantly affect the MRP1 secondary structure, which is made of 46% alpha-helix, 26% beta-sheet, 12% beta-turns, and 17% random coil. Binding of MgATP increased the protein accessibility to the solvent, suggesting a modification in the tertiary organization of the protein. Hydrolysis of MgATP to MgADP and P(i) did not significantly change the global accessibility of the protein. Release of P(i), after hydrolysis, caused a decrease in the accessibility of MRP1 to the water phase which brings the protein back to its initial conformation. All together, the data demonstrate that MRP1 adopts different structures during its catalytic cycle.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Resistencia a Múltiples Medicamentos , Liposomas/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Adenosina Difosfato/farmacología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Animales , Transporte Biológico , Línea Celular , Cricetinae , Deuterio/metabolismo , Cinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Estructura Secundaria de Proteína/genética , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , TransfecciónRESUMEN
We have examined the influence of a novel missense mutation in the fourth extracytoplasmic loop (EL4) of CFTR detected in a patient with cystic fibrosis. This substitution (T908N) creates a consensus sequence (N X S/T) for addition of an N-linked oligosaccharide chain near the C-terminal end of EL4. Oligosaccharyl transferase generally does not have access to this consensus sequence if it is closer than about twelve amino acids from the membrane. However, the T908N site is used, even though it is within four residues of the predicted membrane interface and the oligosaccharide chain added binds calnexin, a resident chaperone of the ER membrane. The chloride channel activity of this variant CFTR is abnormal as evidenced by a reduced rate of (36)Cl(-) efflux and a noisy single channel open state. This may reflect some displacement of the membrane spanning sequence C-terminal of EL4 since it contains residues influencing the ion pore.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/genética , Oligosacáridos/aislamiento & purificación , Secuencia de Aminoácidos , Proteínas de Unión al Calcio/metabolismo , Calnexina , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
CFTR possesses a large cluster of strict dibasic consensus sites for phosphorylation by protein kinase A (PKA) in the R-domain and an obligatory dependence on phosphorylation is a hallmark of CFTR Cl(-) channel function. Removal of as many as 11 of these sites reduces the conformational change in the R-domain and the degree of channel activation in response to PKA. However, until recently a completely PKA-unresponsive CFTR variant has not been reported, leaving open the possibility that the residual response may be mediated by associating ancillary phosphoproteins. We traced the residual PKA-catalyzed (32)P-labelling of the variant with 11 sites mutagenized (11SA) to distinct CNBr phosphopeptides within the R-domain. Mutagenesis of 4 additional monobasic sites in these segments produced a 15SA variant in which Cl(-) channel response to PKA was abolished. Therefore, it can be concluded that ancillary phosphoproteins do not contribute to CFTR activation by PKA. Notably, however, the 15SA protein did exhibit a low level of constitutive channel activity not dependent on PKA, which might have reflected a down-regulating effect of phosphorylation of one or two of the 15 sites as suggested by others. However, this did not prove to be the case.Since immature CFTR has been claimed to be active in the endoplasmic reticulum (ER), we also examined whether it can be phosphorylated in cells and what influence if any this might have on its susceptibility to degradation. Teleologically, activation by phosphorylation of CFTR Cl(-) channels in the ER might be undesirable to the cell. Using various phosphorylation site mutants and kinase and phosphatase inhibitors in pulse-chase experiments, we have found that although nascent CFTR can be phosphorylated at the ER, this is without effect on its ability to mature and avoid proteolysis. Furthermore, we found that microsomes from cells expressing CFTR processing mutants such as DeltaF508 do not generate Cl(-) active channels when fused with planar bilayers unless maturation is promoted, e.g. by growth of cells at reduced temperature or other means. We conclude that the ER-retained mutant nascent chains which are incapable of maturation may be phosphorylated but do not form active channels. Stimulation by PKA of the insertion of CFTR containing vesicles into the plasma membrane as part of the mechanism of stimulation of chloride secretion has been reported, as has an influence of CFTR on the balance between endocytosis and exocytosis but these findings have not been universally confirmed.
Asunto(s)
Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/química , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Retículo Endoplásmico/metabolismo , Activación Enzimática , Humanos , Activación del Canal Iónico , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Conformación ProteicaRESUMEN
Many cystic fibrosis transmembrane conductance regulator (CFTR) mutants are recognized as aberrant by the quality control apparatus at the endoplasmic reticulum (ER) and are targeted for degradation. The mechanism whereby nascent chains are distinguished as either competent or incompetent for ER export has not been elucidated. Here we show that export-incompetent chains display multiple arginine-framed tripeptide sequences like the one recently identified in ATP-sensitive K+ channels. Replacement of arginine residues at positions R29, R516, R555, and R766 with lysine residues to inactivate four of these motifs simultaneously causes delta F508 CFTR, present in approximately 90% of CF patients, to escape ER quality control and function at the cell surface. Interference with recognition of these signals may be helpful in the management of CF.
Asunto(s)
Arginina/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Células Cultivadas , Canales de Cloruro/genética , Cloruros/metabolismo , Retículo Endoplásmico/metabolismo , Técnica del Anticuerpo Fluorescente , Humanos , Lisina/genética , Mutagénesis Sitio-Dirigida , Mutación , Transducción de Señal/genéticaRESUMEN
Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Complejos Multienzimáticos/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/biosíntesis , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Antibacterianos/farmacología , Benzoquinonas , Células CHO , Línea Celular , Cricetinae , Proteínas HSP70 de Choque Térmico/metabolismo , Lactamas Macrocíclicas , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Complejo de la Endopetidasa Proteasomal , Quinonas/farmacologíaRESUMEN
Membrane vesicles prepared from cells expressing the multidrug resistance-associated protein (MRP) transport glutathione S-conjugates of hydrophobic substrates in an ATP dependent manner. Purified MRP possesses ATPase activity which can be further stimulated by anticancer drugs or leukotriene C4. However, the detailed relationship between ATP hydrolysis and drug transport has not been established. How the ATPase activity of MRP is regulated in the cell is also not known. In this report, we have examined the effects of different nucleotides on the ATPase activity of purified MRP. We have found that pyrimidine nucleoside triphosphates have little effect on enzymatic activity. In contrast, purine nucleotides dATP, dGTP, and adenosine 5'-(beta,gamma-imido)triphosphate function as competitive inhibitors. Somewhat unexpectedly, low concentrations of all the nucleoside diphosphates (NDPs) tested, except UDP, stimulate the ATPase activity severalfold. ADP or GDP at higher concentrations was inhibitory, reflecting NDP binding to the substrate site. On the other hand, the enhancement of hydrolysis at low NDP concentrations must reflect interactions with a separate site. Therefore, we postulate the presence of at least two types of nucleotide binding sites on the MRP, a catalytic site(s) to which ATP preferentially binds and is hydrolyzed and a regulatory site to which NDPs preferentially bind and stimulate hydrolysis. Interestingly, the stimulatory effects of drugs transported by MRP and NDPs are not additive, i.e. drugs are not able to further stimulate the NDP-activated enzyme. Hence, the two activation pathways intersect at some point. Since both nucleotide binding domains of MRP are likely to be required for drug stimulation of ATPase activity, the two sites that we postulate may also involve both domains.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Difosfato/farmacología , Adenosina Trifosfatasas/metabolismo , Desoxirribonucleótidos/farmacología , Resistencia a Múltiples Medicamentos , Ribonucleótidos/farmacología , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Animales , Línea Celular , Cricetinae , Activación Enzimática , GTP Fosfohidrolasas/metabolismo , Humanos , Cinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TransfecciónAsunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Animales , Baculoviridae , Células CHO , Células COS , Línea Celular , Supervivencia Celular/efectos de los fármacos , Canales de Cloruro/biosíntesis , Canales de Cloruro/fisiología , Clonación Molecular/métodos , Cricetinae , Femenino , Humanos , Activación del Canal Iónico , Potenciales de la Membrana/fisiología , Metotrexato/toxicidad , Oocitos/fisiología , Plásmidos , Proteínas Recombinantes/biosíntesis , Mapeo Restrictivo , Saccharomyces cerevisiae , Spodoptera , Transfección/métodos , Xenopus laevisRESUMEN
1. The relationship between phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel and its gating by nucleotides was examined using the patch clamp technique by comparing strongly phosphorylated wild-type (WT) channels with weakly phosphorylated mutant channels lacking four (4SA) or all ten (10SA) dibasic consensus sequences for phosphorylation by protein kinase A (PKA). 2. The open probability (Po) of strongly phosphorylated WT channels in excised patches was about twice that of 4SA and 10SA channels, after correcting for the number of functional channels per patch by addition of adenylylimidodiphosphate (AMP-PNP). The mean burst durations of WT and mutant channels were similar, and therefore the elevated Po of WT was due to its higher bursting rate. 3. The ATP dependence of the 10SA mutant was shifted to higher nucleotide concentrations compared with WT channels. The relationship between Po and [ATP] was noticeably sigmoid for 10SA channels (Hill coefficient, 1.8), consistent with positive co-operativity between two sites. Increasing ATP concentration to 10 mM caused the Po of both WT and 10SA channels to decline. 4. Wild-type and mutant CFTR channels became locked in open bursts when exposed to mixtures of ATP and the non-hydrolysable analogue AMP-PNP. The rate at which the low phosphorylation mutants became locked open was about half that of WT channels, consistent with Po being the principal determinant of locking rate in WT and mutant channels. 5. We conclude that phosphorylation at 'weak' PKA sites is sufficient to sustain the interactions between the ATP binding domains that mediate locking by AMP-PNP. Phosphorylation of the strong dibasic PKA sites controls the bursting rate and Po of WT channels by increasing the apparent affinity of CFTR for ATP.
Asunto(s)
Canales de Cloruro/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Nucleótidos/farmacología , Adenosina Trifosfato/farmacología , Animales , Células CHO , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/genética , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/química , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Cinética , Mutación , FosforilaciónRESUMEN
Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (P/P) sequence NO > Cl > HCO > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (P/P < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of approximately 5.3 A. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.
Asunto(s)
Aniones/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulación de la Expresión Génica , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Mutagénesis , Técnicas de Placa-Clamp , PermeabilidadRESUMEN
Human multidrug resistance protein (MRP) was expressed at high levels in stably transfected baby hamster kidney (BHK-21) cells. These cells exhibited a pattern of cross-resistance to several different drugs typical of an MRP-mediated phenotype despite the addition of 10 histidine residues at the C terminus to facilitate purification. Consistent with this functional evidence of the presence of MRP at the surface of these transfectants, strong signals were detected by immunoblotting and immunofluorescence using a specific monoclonal antibody to MRP. There was intense uniform staining of the cell surface as well as weaker staining of intracellular membranes. MRP-containing membranes were solubilized in 1% N-dodecyl-beta-D-maltoside in the presence of 0.4% sheep brain phospholipids. Two sequential affinity purification steps on Ni-NTA agarose and wheat germ agglutinin agarose provided substantial enrichment, and contaminating bands were not detected. ATPase activity of the purified protein was assayed in the presence of the phospholipids, which had been maintained throughout all purification steps. ATP was hydrolyzed in proportion to the amount of purified protein assayed, and typical Michaelis-Menten behavior was exhibited, yielding estimations of Km of approximately 3.0 mM and Vmax of 0.46 micromol mg-1 min-1. This activity was moderately stimulated by the drugs that others have shown to be transported by MRP-containing membrane vesicles. This stimulation was enhanced by reduced glutathione as is its drug transport, and oxidized glutathione, itself a substrate for transport, caused a strong stimulation. These data describe the first purification of MRP and provide the first direct evidence that the molecule possesses drug-stimulated ATPase activity.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/metabolismo , Resistencia a Múltiples Medicamentos , Proteínas de Neoplasias/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Animales , Clonación Molecular , Cricetinae , ADN Complementario/química , Células HeLa , Humanos , Cinética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , TransfecciónRESUMEN
Patch-clamp, iodide efflux, and biochemical techniques were used to evaluate the ability of phenylimidazothiazoles to open normal and mutated cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels and to investigate the mechanism of activation. As reported previously for bromotetramisole, levamisole activated wild-type CFTR channels stably expressed in Chinese hamster ovary cells in the absence of other secretagogues and without elevating intracellular cAMP or calcium. The protein kinase A (PKA) inhibitor N - (2-(p-bromocinnamylamino)ethyl)-5-isoquinolinesul-fonamid e abolished activation by forskolin but only partially inhibited stimulation by levamisole, suggesting the involvement of other kinases. CFTR channels bearing mutations at multiple phosphorylation sites, in the membrane domains, and in the first nucleotide binding domain (including the disease-causing mutations G551D and DeltaF508) all responded to phenylimidazothiazoles. Moreover, levamisole and bromotetramisole increased the activity of wild-type and mutant channels already exposed to PKA + MgATP, consistent with the inhibition of a constitutive, membrane-associated phosphatase activity. We conclude that phenylimidazothiazole drugs can open normal and mutated CFTR channels by stabilization of phosphoforms of CFTR that are produced by basal activity of PKA and alternative protein kinases. If similar stimulation is observed in humans in vivo, phenylimidazothiazoles may be useful in the development of pharmacological therapies for cystic fibrosis.
Asunto(s)
Calcio/metabolismo , Colforsina/farmacología , AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/fisiología , Levamisol/farmacología , Sulfonamidas , Tetramisol/análogos & derivados , Adenosina Trifosfato/farmacología , Animales , Sitios de Unión , Células CHO , Calcimicina/farmacología , Cricetinae , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Imidazoles/farmacología , Isoquinolinas/farmacología , Cinética , Potenciales de la Membrana/efectos de los fármacos , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Mutación Puntual , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Tetramisol/farmacología , Tiazoles/farmacología , TransfecciónRESUMEN
The cystic fibrosis transmembrane conductance regulator (CFTR) in an ATP-dependent channel which mediates cAMP-stimulated chloride secretion by epithelia, particularly those of the pancreas, airways, and intestine. CFTR homologues have been found in all higher vertebrates examined to date and also in some lower vertebrates, although only the human, shark, and Xenopus genes have been heterologously expressed and shown to generate protein kinase A-activated Cl channels. Once phosphorylated, CFTR channels require hydrolyzable nucleotides to be active, but they can be locked in an open burst state when exposed to mixtures of ATP and its hydrolysis-resistant analogue AMP-PNP. This locking requires low-level phosphorylation at unidentified sites that are not among the ten "strong" (dibasic) PKA consensus sequences on CFTR. Mutagenesis of the dibasic PKA sites, which reduces in vitro phosphorylation by > 98%, reduces open probability (Po) by about 50% whilst having no effect on burst duration. Thus, incremental phosphorylation of these sites under normal conditions does not increase Po by slowing down ATP hydrolysis and stabilizing the open burst state, although locking does strictly require low-level phosphorylation at one or more cryptic sites. In addition to serving as a Cl channel, there is compelling evidence that CFTR inhibits the amiloride-sensitive, epithelial sodium channel (ENaC). The mechanism of coupling is not known but most likely involves physical interactions between the channels, perhaps mediated by an intermediate protein that impinges on other transport proteins. CFTR does not function as a conductive channel for ATP; however, extracellular ATP does regulate epithelial channels through activation of P2U purinergic receptors and, after being hydrolyzed extracellularly, through activation of adenosine receptors which elevate cAMP.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Regulador de Conductancia de Transmembrana de Fibrosis Quística/química , Humanos , Activación del Canal Iónico/fisiología , Datos de Secuencia Molecular , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Proteínas Quinasas/metabolismo , TiburonesRESUMEN
Hormonal regulation of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is largely mediated via cAMP-dependent protein kinase (PKA). CFTR contains 10 dibasic consensus sites for potential PKA phosphorylation ((R/K) (R/K)X(S*/T*)). Previous studies (Chang, X.-B., Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon, N., Hanrahan, J. W., and Riordan, J.R (1993) J. Biol. Chem. 268, 11304-11311) showed that approximately 25% of the CFTR wild-type response to PKA activation remained upon inhibition of most detectable phosphorylation by in vitro mutagenesis of all 10 dibasic consensus sites (10SA CFTR). To identify potential additional sites responsible for the residual activity, large amounts of this mutant CFTR were phosphorylated with PKA using high specific activity [gamma-32P]ATP. Cyanogen bromide cleavage indicated that a large portion of the observed PKA phosphorylation occurred within a 5.8-kDa fragment of the R domain between residues 722-773. Removal of serines at potential PKA sites in this fragment showed that Ser-753 accounted for all of the gamma-32P labeling of the 5.8-kDa peptide. Replacement of Ser-753 with alanine reduced the level of residual CFTR activity by a further 40%, indicating that phosphorylation at this previously unidentified site contributes to the activation of 10SA CFTR.
Asunto(s)
Canales de Cloruro/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de la Membrana/metabolismo , Fosfoserina/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Secuencia de Consenso , Cricetinae , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Humanos , Técnicas In Vitro , Activación del Canal Iónico , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosforilación , Relación Estructura-ActividadRESUMEN
Mutation of potential cAMP dependent protein kinase sites in the R domain of the cystic fibrosis transmembrane conductance regulator has significant effects on protein function. Mutation of the potential phosphorylation sites from serine to alanine, to abolish the site, reduced sensitivity to activation, or to glutamic acid, to mimic phosphorylation, caused some constitutive activity. To explore the structural effects of these mutations, recombinant R domain peptides were studied: the wild type, a mutant with nine serine residues changed to alanine, and a mutant with eight serine residues changed to glutamic acid. As assessed by C.D. spectroscopy, the mutants have substantially different secondary structure than the wild type, in agreement with the predictive algorithm of Gascuel and Golmard. The results show that mutagenesis of residues alters the polypeptide structurally as well as preventing it from serving as a phosphorylation substrate. Hence, the functional consequences of the mutations may not be entirely due to effects on phosphorylation.
Asunto(s)
Canales de Cloruro/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Dicroismo Circular , Regulador de Conductancia de Transmembrana de Fibrosis Quística , Proteínas de la Membrana/biosíntesis , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fosforilación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Serina , Especificidad por Sustrato , TreoninaRESUMEN
Metabolic labeling experiments followed by immunoprecipitation were performed to investigate the kinetics, location and inhibitor sensitivity of degradation of both wild-type (wt) and mutant (delta F508) cystic fibrosis conductance transmembrane regulator (CFTR). At the earliest stages of the biosynthetic process, both wt and delta F508 CFTR were found to be susceptible to degradation by endogenous proteases. Virtually all delta F508 CFTR and 45-80% of wt CFTR were rapidly degraded with a similar half-life (t1/2 approximately 0.5 h). The remaining wt CFTR attained a protease-resistant configuration regardless of whether traffic between the endoplasmic reticulum (ER) and Golgi was operational. Metabolic energy is required for the conformational transition, but not to maintain the stability of the protease-resistant wt CFTR. Intracellular degradation of delta F508 CFTR and of incompletely folded wt CFTR occurs in a non-lysosomal, pre-Golgi compartment, as indicated by the sensitivity of proteolysis to different inhibitors and temperature. Accordingly, products of the degradation of delta F508 CFTR could be detected by immunoblotting in isolated ER, but not in the Golgi. Together, these results suggest a dynamic equilibrium between two forms of wt CFTR in the ER: an incompletely folded, protease-sensitive form which is partially converted by an ATP-dependent process to a more mature form that is protease-resistant and capable of leaving the ER. The inability delta F508 CFTR to undergo such a transition renders it susceptible to complete and rapid degradation in a pre-Golgi compartment.