研究者詳細 - 表　弘志

論文 - 表　弘志

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Identification of a vesicular nucleotide transporter 査読

Keisuke Sawada, Noriko Echigo, Narinobu Juge, Takaaki Miyaji, Masato Otsuka, Hiroshi Omote, Akitsugu Yamamoto, Yoshinori Moriyama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 105 ( 15 ) 5683 - 5686 2008年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

ATP is a major chemical transmitter in purinergic signal transmission. Before secretion, ATIP is stored in secretory vesicles found in purinergic cells. Although the presence of active transport mechanisms for ATP has been postulated for a long time, the proteins responsible for its vesicular accumulation remains unknown. The transporter encoded by the human and mouse SLC17A9 gene, a novel member of an anion transporter family, was predominantly expressed in the brain and adrenal gland. The mouse and bovine counterparts were associated with adrenal chromaffin granules. Proteoliposomes containing purified transporter actively took up ATP, ADP, and GTP by using membrane potential as the driving force. The uptake properties of the reconstituted transporter were similar to that of the ATP uptake by synaptic vesicles and chromaffin granules. Suppression of endogenous SLC17A9 expression in PC12 cells decreased exocytosis of ATP. These findings strongly suggest that SLC17A9 protein is a vesicular nucleotide transporter and should lead to the elucidation of the molecular mechanism of ATP secretion in purinergic signal transmission.

DOI： 10.1073/pnas.0800141105

Role of glutamate residues in substrate recognition by human MATE1 polyspecific H+/organic cation exporter 査読

Takuya Matsumoto, Takuji Kanamoto, Masato Otsuka, Hiroshi Omote, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 294 ( 4 ) C1074 - C1078 2008年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

Human multidrug and toxic compound extrusion 1 ( hMATE1) is an electroneutral H+/organic cation exchanger responsible for the final excretion step of structurally unrelated toxic organic cations in kidney and liver. To elucidate the molecular basis of the substrate recognition by hMATE1, we substituted the glutamate residues Glu273, Glu278, Glu300, and Glu389, which are conserved in the transmembrane regions, for alanine or aspartate and examined the transport activities of the resulting mutant proteins using tetraethylammonium ( TEA) and cimetidine as substrates after expression in human embryonic kidney 293 ( HEK- 293) cells. All of these mutants except Glu273Ala were fully expressed and present in the plasma membrane of the HEK- 293 cells. TEA transport activity in the mutant Glu278Ala was completely absent. Both Glu300Ala and Glu389Ala and all aspartate mutants exhibited significantly decreased activity. Glu273Asp showed higher affinity for cimetidine, whereas it has reduced affinity to TEA. Glu278Asp showed decreased affinity to cimetidine. Both Glu300Asp and Glu389Asp had lowered affinity to TEA, whereas the affinity of Glu389Asp to cimetidine was fourfold higher than that of the wildtype transporter with about a fourfold decrease in V-max value. Both Glu273Asp and Glu300Asp had altered pH dependence for TEA uptake. These results suggest that all of these glutamate residues are involved in binding and/ or transport of TEA and cimetidine but that their individual roles are different.

DOI： 10.1152/ajpcell.00504.2007

A novel variant of mouse MATE-1 H+/organic cation antiporter with a long hydrophobic tall 査読

Ayumi Kobara, Miki Hiasa, Takuya Matsumoto, Masato Otsuka, Hiroshi Omote, Yoshinori Moriyama

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 469 ( 2 ) 195 - 199 2008年1月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

Mammalian multidrug and toxic compound extrusion 1 (MATE1) are polyspecific H+-coupled exporters of organic cations (OCs) and responsible for excretion of metabolic waste products and xenobiotics. Here, we report a novel variant of mouse MATE1, mMATE1b, that has a long carboxyl terminal hydrophobic tail homologous to other MATE1 transporter proteins. Mouse MATE1b mediates tetraethylammonium (TEA) uptake with properties similar to that of mMATE1 and is localized in renal brush border membranes. Thus, mMATE1b is a functional variant of mMATE1 and seems to be the true counterpart to other MATE1 transporters. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.abb.2007.10.010

[Identification, structure and function of a drug transporter at the final step of extrusion]. 査読

Hiasa M, Omote H, Moriyama Y

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 53 ( 1 ) 52 - 58 2008年1月

Multidrug and toxic compound extrusion (MATE)-type proteins as anchor transporters for the excretion of metabolic waste products and xenobiotics 査読

Y. Moriyama, M. Hiasa, T. Matsumoto, H. Omote

XENOBIOTICA 38 ( 7-8 ) 1107 - 1118 2008年

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：INFORMA HEALTHCARE

1. Multidrug and toxic compound extrusion (MATE)-type transporters, which were first identified as a bacterial drug transporter family, are present in almost all prokaryotes and eukaryotes, and are thus one of the mostly conserved transporter families in nature.
2. Recently, a mammalian MATE transporter was shown to be a long hypothesized electroneutral H(+)/organic cation exporter that is responsible for the excretion of metabolic waste products and xenobiotics at renal brush border membranes and bile canaliculi. Plant MATE-type transporters are involved in the detoxification of metals and secondary metabolites such as phenols through their vesicular storage or extrusion at the plasma membrane.
3. Thus, MATE transporters are involved in one of the basic mechanisms that maintain homeostasis through the excretion of metabolic waste products and xenobiotics in nature.

DOI： 10.1080/00498250701883753

Functional characterization of testis-specific rodent multidrug and toxic compound extrusion 2, a class III MATE-type polyspecific H+/organic cation exporter 査読

Miki Hiasa, Takuya Matsumoto, Toshinori Komatsu, Hiroshi Omote, Yoshinori Moriyama

AMERICAN JOURNAL OF PHYSIOLOGY-CELL PHYSIOLOGY 293 ( 5 ) C1437 - C1444 2007年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER PHYSIOLOGICAL SOC

Mammalian multidrug and toxic compound extrusion ( MATE) proteins are classified into three subfamilies: classes I, II, and III. We previously showed that two of these families act as polyspecific H+-coupled transporters of organic cations (OCs) at final excretion steps in liver and kidney ( Otsuka et al. Proc Natl Acad Sci USA 102: 17923-17928, 2005; Omote et al. Trends Pharmacol Sci 27: 587-593, 2006). Rodent MATE2 proteins are class III MATE transporters, the molecular nature, as well as transport properties, of which remain to be characterized. In the present study, we investigated the transport properties and localization of mouse MATE2 (mMATE2). On expression in human embryonic kidney (HEK)-293 cells, mMATE2 localized to the intracellular organelles and plasma membrane. mMATE2 mediated pH-dependent TEA transport with substrate specificity similar to, but distinct from, that of mMATE1, which prefers N-methylnicotinamide and guanidine as substrates. mMATE2 expressed in insect cells was solubilized and reconstituted with bacterial H+-ATPase into liposomes. The resultant proteoliposomes exhibited ATP-dependent uptake of TEA that was sensitive to carbonyl cyanide 3-chlorophenylhydrazone but unaffected by valinomycin in the presence of K+. Immunologic techniques using specific antibodies revealed that mMATE2 was specifically expressed in testicular Leydig cells. Thus mMATE2 appears to act as a polyspecific H+/OC exporter in Leydig cells. It is concluded that all classes of mammalian MATE proteins act as polyspecific and electroneutral transporters of organic cations.

DOI： 10.1152/ajpcell.00280.2007

Molecular mechanism of vesicular glutamate transporter 査読

Hiroshi Omote, Narinobu Juge

SEIKAGAKU 79 ( 10 ) 956 - 960 2007年10月

Vesicular glutamate transporter contains two independent transport machineries 査読

Narinobu Juge, Yumi Yoshida, Shouki Yatsushiro, Hiroshi Omote, Yoshinori Moriyama

JOURNAL OF BIOLOGICAL CHEMISTRY 281 ( 51 ) 39499 - 39506 2006年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of L-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial F0F1-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated L-glutamate uptake in a membrane potential (Delta psi)-dependent fashion. The ATP-dependent L-glutamate uptake exhibited an absolute requirement for similar to 4 mM Cl-, was sensitive to Evans blue, but was insensitive to D, L-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg(184), His(128), and Glu(191) showed a dramatic loss in L-glutamate transport activity, whereas Na+-dependent inorganic phosphate (Pi) uptake remained comparable to that of the wild type. Furthermore, Pi transport did not require Cl- and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a Delta psi-dependent L-glutamate uptake and a Na+-dependent Pi uptake.

DOI： 10.1074/jbc.M607670200

The MATE proteins as fundamental transporters of metabolic and xenobiotic organic cations 査読

Hiroshi Omote, Miki Hiasa, Takuya Matsumoto, Masato Otsuka, Yoshinori Moriyama

TRENDS IN PHARMACOLOGICAL SCIENCES 27 ( 11 ) 587 - 593 2006年11月

記述言語：英語出版者・発行元：ELSEVIER SCIENCE LONDON

Multidrug and toxic compound extrusion (MATE) proteins, comprising the most recently designated family of multidrug transporter proteins, are widely distributed in all kingdoms of living organisms, although their function is far from understood. The bacterial MATE-type transporters that have been characterized function as exporters of cationic drugs, such as norfloxacin and ethidium, through H+ or Na+ exchange. Plant MATE-type transporters are involved in the detoxification of secondary metabolites, including alkaloids. Mammalian MATE-type transporters are responsible for the final step in the excretion of metabolic waste and xenobiotic organic cations in the kidney and liver through electroneutral exchange of H+. Thus, we propose that members of the MATE family are organic cation exporters that excrete metabolic or xenobiotic organic cations from the body.

DOI： 10.1016/j.tips.2006.09.001

Secretion of L-glutamate from osteoclasts through transcytosis 査読

Riyo Morimoto, Shunsuke Uehara, Shouki Yatsushiro, Narinobu Juge, Zhaolin Hua, Shigenori Senoh, Noriko Echigo, Mitsuko Hayashi, Toshihide Mizoguchi, Tadashi Ninomiya, Nobuyuki Udagawa, Hiroshi Omote, Akitsugu Yamamoto, Robert H. Edwards, Yoshinori Moriyama

EMBO JOURNAL 25 ( 18 ) 4175 - 4186 2006年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATURE PUBLISHING GROUP

Osteoclasts are involved in the catabolism of the bone matrix and eliminate the resulting degradation products through transcytosis, but the molecular mechanism and regulation of transcytosis remain poorly understood. Upon differentiation, osteoclasts express vesicular glutamate transporter 1 (VGLUT1), which is essential for vesicular storage and subsequent exocytosis of glutamate in neurons. VGLUT1 is localized in transcytotic vesicles and accumulates L-glutamate. Osteoclasts secrete L-glutamate and the bone degradation products upon stimulation with KCl or ATP in a Ca2+-dependent manner. KCl-and ATP-dependent secretion of L-glutamate was absent in osteoclasts prepared from VGLUT1(-/-) knockout mice. Osteoclasts express mGluR8, a class III metabotropic glutamate receptor. Its stimulation by a specific agonist inhibits secretion of L-glutamate and bone degradation products, whereas its suppression by a specific antagonist stimulates bone resorption. Finally, it was found that VGLUT1(-/-) mice develop osteoporosis. Thus, in bone-resorbing osteoclasts, L-glutamate and bone degradation products are secreted through transcytosis and the released L-glutamate is involved in autoregulation of transcytosis. Glutamate signaling may play an important role in the bone homeostasis.

DOI： 10.1038/sj.emboj.7601317

破骨細胞によるトランスサイトーシスを介したグルタミン酸分泌とその生理的意義

上原俊介, 森本理代, 八代聖基, 樹下成信, 林美都子, 妹尾繁範, 溝口利英, 二宮禎, 宇田川信之, Hua Zhaolin, 表弘志, 山本章嗣, Edwards Robert H., 森山芳則

松本歯学 32 ( 2 ) 171 - 172 2006年8月

Interaction of transported drugs with the lipid bilayer and beta-glycoprotein through a solvation exchange mechanism 査読

H Omote, MK Al-Shawi

BIOPHYSICAL JOURNAL 90 ( 11 ) 4046 - 4059 2006年6月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：BIOPHYSICAL SOCIETY

Broad substrate specificity of human P-glycoprotein (ABCB1) is an essential feature of multidrug resistance. Transport substrates of P-glycoprotein are mostly hydrophobic and many of them have net positive charge. These compounds partition into the membrane. Utilizing the energy of ATP hydrolysis, P-glycoprotein is thought to take up substrates from the cytoplasmic lea. et of the plasma membrane and to transport them to the outside of the cell. We examined this model by molecular dynamics simulation of the lipid bilayer, in the presence of transport substrates together with an atomic resolution structural model of P-glycoprotein. Taken together with previous electron paramagnetic resonance studies, the results suggest that most transported drugs are concentrated near the surface zone of the inner lea. et of the plasma membrane. Here the drugs can easily diffuse laterally into the drug-binding site of P-glycoprotein through an open cleft. It was concluded that the initial high-affinity drug-binding site was located in the interfacial surface area of P-glycoprotein in contact with the membrane interface. Based on these results and our recent kinetic studies, a "solvation exchange'' drug transport mechanism of P-glycoprotein is discussed. A molecular basis for the improved colchicine transport efficiency by the much-studied colchicine-resistance G185V mutant human P-glycoprotein is also provided.

DOI： 10.1529/biophysj.105.077743

Examination of drug resistance activity of human TAP-like (ABCB9) expressed in yeast 査読

A Ohashi-Kobayashi, K Ohashi, WB Du, H Omote, R Nakamoto, M Al-shawi, M Maeda

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 343 ( 2 ) 597 - 601 2006年5月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE

A half-type ABC transporter, human TAP-like (hTAPL) tagged with histidine cluster, was expressed in budding yeast protease-deficient strain BJ5457, and the effect of expression for resistance to peptide compounds including antibiotics and proteinase inhibitor was examined. Among these Compounds, the yeast expressing hTAPL exhibits high sensitivity to valinomycin, a monovalent cation ionophore. A mutation in Walker A motif, which lost ATP-binding activity of hTAPL, eliminated the enhanced sensitivity to valinomycin. These findings suggest that the transport activity of hTAPL is important for conferring high valinomycin-sensitive phenotype to yeast. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2006.03.002

Transmembrane topology of vesicular glutamate transporter 2 査読

SK Jung, R Morimoto, M Otsuka, H Omote

BIOLOGICAL & PHARMACEUTICAL BULLETIN 29 ( 3 ) 547 - 549 2006年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：PHARMACEUTICAL SOC JAPAN

Vesicular glutamate transporter (VGLUT) plays an essential role in L-glutamate signaling in neurons and some peripheral tissues through vesicular storage of L-glutamate in secretory vesicles. To investigate the topology of VGLUT in membranes, we prepared site-directed antibodies against the amino-terminal (anti-N), 1st putative loop (anti-L), and carboxyl terminal (anti-C) regions. None of the antibodies reacted with VGLUT2 expressed in COS cells because they could not gain access to the antigen. However, both the anti-N and anti-C antibodies recognized VGLUT2 when the cells were permeabilized with digitonin, while the anti-L antibodies did not. Immunological reactivity to anti-L-antibodies appeared when the cells were permeabilized with Triton X-100. These results suggest that both the amino-terminal and carboxyl-terminal regions of VGLUT2 in membranes face the cytoplasm while the 1st loop faces the lumen.

DOI： 10.1248/bpb.29.547

A human transporter protein that mediates the final excretion step for toxic organic cations 査読

M Otsuka, T Matsumoto, R Morimoto, S Arioka, H Omote, Y Moriyama

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 102 ( 50 ) 17923 - 17928 2005年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：NATL ACAD SCIENCES

In mammals, toxic electrolytes of endogenous and exogenous origin are excreted through the urine and bile. Before excretion, these compounds cross numerous cellular membranes in a transporter-mediated manner. However, the protein transporters involved in the final excretion step are poorly understood. Here, we show that MATE1, a human and mouse orthologue of the multidrug and toxin extrusion family conferring multidrug resistance on bacteria, is primarily expressed in the kidney and liver, where it is localized to the luminal membranes of the urinary tubules and bile canaliculi. When expressed in HEK293 cells, MATE1 mediates H+-coupled electroneutral exchange of tetraethylammonium and 1-methyl-4-phenylpyridinium. its substrate specificity is similar to those of renal and hepatic H+-coupled organic cations (OCs) export. Thus, MATE1 appears to be the long searched for polyspecific OC exporter that directly transports toxic OCs into urine and bile.

DOI： 10.1073/pnas.0506483102

The remarkable transport mechanism of P-glycoprotein: A multidrug transporter 査読

MK Al-Shawi, H Omote

JOURNAL OF BIOENERGETICS AND BIOMEMBRANES 37 ( 6 ) 489 - 496 2005年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：SPRINGER/PLENUM PUBLISHERS

Human P-glycoprotein (ABCB1) is a primary multidrug transporter located in plasma membranes, that utilizes the energy of ATP hydrolysis to PUMP toxic xenobiotics out of cells. P-glycoprotein employs a Most unusual molecular mechanism to perform this drug transport function. Here we review Our work to elucidate the molecular mechanism of drug transport by P-glycoprotein. High level heterologous expression of human P-glycoprotein. in the yeast Saccharomyces cerevisiae, has facilitated biophysical Studies in Purified proteoliposome preparations. Development of novel spin-labeled transport substrates has allowed for quantitative and rigorous measurements of drug transport in real time by EPR spectroscopy. We have developed a new drug transport model of P-glycoprotein from the results Of mutagenic, quantitative thermodynamic and kinetic studies. This model satisfactorily accounts for most of the unusual kinetic, coupling, and physiological features of P-glycoprotein. Additionally, an atomic detail structural model of P-glycoprotein has been devised 10 place Our results within a proper structural context.

DOI： 10.1007/s10863-005-9497-5

Proteolipid of vacuolar H+-ATPase of Plasmodium falciparum: cDNA cloning, gene organization and complementation of a yeast null mutant 査読

Shouki Yatsushiro, Shinya Taniguchi, Toshihide Mitamura, Hiroshi Omote, Yoshinori Moriyama

Biochimica et Biophysica Acta - Biomembranes 1717 ( 2 ) 89 - 96 2005年11月

記述言語：英語掲載種別：研究論文（学術雑誌）

Vacuolar H+-ATPase (V-ATPase), an electrogenic proton pump, is highly expressed in Plasmodium falciparum, the human malaria parasite. Although V-ATPase-driven proton transport is involved in various physiological processes in the parasite, the overall features of the V-ATPase of P. falciparum, including the gene organization and biogenesis, are far less known. Here, we report cDNA cloning of proteolipid subunit c of P. falciparum, the smallest and most highly hydrophobic subunit of V-ATPase. RT-PCR analysis as well as Northern blotting indicated expression of the proteolipid gene in the parasite cells. cDNA, which encodes a complete reading frame comprising 165 amino acids, was obtained, and its deduced amino acid sequence exhibits 52 and 57% similarity to the yeast and human counterparts, respectively. Southern blot analysis suggested the presence of a single copy of the proteolipid gene, with 5 exons and 4 introns. Upon transfection of the cDNA into a yeast null mutant, the cells became able to grow at neutral pH, accompanied by vesicular accumulation of quinacrine. In contrast, a mutated proteolipid with replacement of glutamate residue 138 with glutamine did not lead to recovery of the growth ability or vesicular accumulation of quinacrine. These results indicated that the cDNA actually encodes the proteolipid of P. falciparum and that the proteolipid is functional in yeast. © 2005 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.bbamem.2005.08.011

Proteolipid of vacuolar H(+)-ATPase of Plasmodium falciparum: cDNA cloning, gene organization and complementation of a yeast null mutant. 査読

Yatsushiro S, Taniguchi S, Mitamura T, Omote H, Moriyama Y

Biochimica et biophysica acta 1717 ( 2 ) 89 - 96 2005年11月

Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus 査読

M Otsuka, M Yasuda, Y Morita, C Otsuka, T Tsuchiya, H Omote, Y Moriyama

JOURNAL OF BACTERIOLOGY 187 ( 5 ) 1552 - 1558 2005年3月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC MICROBIOLOGY

NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.

DOI： 10.1128/jb.187.1552-1558.2005

Improved energy coupling of human P-glycoprotein by the glycine 185 to valine mutation 査読

H Omote, RA Figler, MK Polar, MK Al-Shawi

BIOCHEMISTRY 43 ( 13 ) 3917 - 3928 2004年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER CHEMICAL SOC

A glycine 185 to valine mutation of human P-glycoprotein (ABCB1, MDR1) has been previously isolated from high colchicine resistance cell lines. We have employed purified and reconstituted P-glycoproteins expressed in Saccharomyces cerevisiae [Figler et al. (2000) Arch. Biochem. Biophys. 376, 34-46] and devised a set of thermodynamic analyses to reveal the mechanism of improved resistance. Purified G185V enzyme shows altered basal ATPase activity but a strong stimulation of colchicine- and etoposide-dependent activities, suggesting a tight regulation of ATPase activity by these drugs. The mutant enzyme has a higher apparent K,, for colchicine and a lower K,, for etoposide than that of wild type. Kinetic constants for other transported drugs were not significantly modified by this mutation. Systematic thermodynamic analyses indicate that the G185V enzyme has modified thermodynamic properties of colchicine- and etoposide-dependent activities. To improve the rate of colchicine or etoposide transport, the G185V enzyme has lowered the Arrhenius activation energy of the transport rate-limiting step. The high transition state energies of wild-type P-glycoprotein, when transporting etoposide or colchicine, increase the probability of nonproductive degradation of the transition state without transport. G185V P-glycoprotein transports etoposide or colchicine in an energetically more efficient way with decreased enthalpic and entropic components of the activation energy. Our new data fully reconcile the apparently conflicting results of previous studies. EPR analysis of the spin-labeled G185C enzyme in a cysteine-less background and kinetic parameters of the G185C enzyme indicate that position 185 is surrounded by other residues and is volume sensitive. These results and atomic detail structural modeling suggest that residue 185 is a pivotal point in transmitting conformational changes between the catalytic sites and the colchicine drug binding domain. Replacement of this residue with a bulky valine alters this communication and results in more efficient transport of etoposide or colchicine.

DOI： 10.1021/bi0353651

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