Functional Domains in the Carnitine Transporter OCTN2, Defective in Primary Carnitine Deficiency
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m307911200
ISSN1083-351X
AutoresCristina Amat Di San Filippo, Yuhuan Wang, Nicola Longo,
Tópico(s)Amino Acid Enzymes and Metabolism
ResumoPrimary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the Na+-dependent organic cation transporter, OCTN2. To define the domains involved in carnitine recognition, we evaluated chimeric transporters created by swapping homologous domains between OCTN1, which does not transport carnitine, and OCTN2. Substitution of the C terminus of OCTN2 (amino acid residues 342–557) with the corresponding residues of OCTN1 completely abolished carnitine transport. The progressive substitution of the N terminus of OCTN2 with OCTN1 resulted in a decrease in carnitine transport associated with a progressive increase in the Km toward carnitine from 3.9 ± 0.5 to 141 ± 19 μm. The largest drop in carnitine transport (and increase in Km toward carnitine) was observed with the substitution of residues 341–454 of OCTN2. An additional chimeric transporter (CHIM-9) in which only residues 341–454 of OCTN2 were substituted by OCTN1 had markedly reduced carnitine transport, with an elevated Km toward carnitine (63 ± 5 μm). Site-directed mutagenesis and introduction of residues nonconserved between OCTN1 and OCTN2 in the OCTN2 cDNA indicated that the R341A, L409W, L424Y, and T429I substitutions significantly decreased carnitine transport. Single substitutions did not increase the Km toward carnitine. By contrast, the combination of three of these substitutions (R341W + L409W + T429I) greatly decreased carnitine transport and increased the Km toward carnitine (20.2 ± 4.5 μm). The Arg-341, Leu-409, and Thr-429 residues are all located in predicted transmembrane domains. Involvement of these residues in carnitine transport was further supported by the partial restoration of carnitine transport by the introduction of these OCTN2 residues in the OCTN1 portion of CHIM-9. These studies indicate that multiple domains of the OCTN2 transporter are required for carnitine transport and identify transmembrane residues important for carnitine recognition. Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the Na+-dependent organic cation transporter, OCTN2. To define the domains involved in carnitine recognition, we evaluated chimeric transporters created by swapping homologous domains between OCTN1, which does not transport carnitine, and OCTN2. Substitution of the C terminus of OCTN2 (amino acid residues 342–557) with the corresponding residues of OCTN1 completely abolished carnitine transport. The progressive substitution of the N terminus of OCTN2 with OCTN1 resulted in a decrease in carnitine transport associated with a progressive increase in the Km toward carnitine from 3.9 ± 0.5 to 141 ± 19 μm. The largest drop in carnitine transport (and increase in Km toward carnitine) was observed with the substitution of residues 341–454 of OCTN2. An additional chimeric transporter (CHIM-9) in which only residues 341–454 of OCTN2 were substituted by OCTN1 had markedly reduced carnitine transport, with an elevated Km toward carnitine (63 ± 5 μm). Site-directed mutagenesis and introduction of residues nonconserved between OCTN1 and OCTN2 in the OCTN2 cDNA indicated that the R341A, L409W, L424Y, and T429I substitutions significantly decreased carnitine transport. Single substitutions did not increase the Km toward carnitine. By contrast, the combination of three of these substitutions (R341W + L409W + T429I) greatly decreased carnitine transport and increased the Km toward carnitine (20.2 ± 4.5 μm). The Arg-341, Leu-409, and Thr-429 residues are all located in predicted transmembrane domains. Involvement of these residues in carnitine transport was further supported by the partial restoration of carnitine transport by the introduction of these OCTN2 residues in the OCTN1 portion of CHIM-9. These studies indicate that multiple domains of the OCTN2 transporter are required for carnitine transport and identify transmembrane residues important for carnitine recognition. Primary carnitine deficiency (On-line Mendelian Inheritance in Man 212140) is a recessively inherited disorder of fatty acid oxidation due to defective carnitine transport (1Scaglia F. Longo N. Semin. Perinatol. 1999; 23: 152-161Crossref PubMed Scopus (81) Google Scholar). Carnitine is essential for the transfer of long-chain fatty acids from the cytosol to mitochondria for subsequent β oxidation and the lack of carnitine impairs the ability to use fat as fuel during periods of fasting or stress. This can result in hypoketotic hypoglycemia, Reye's syndrome, and sudden infant death in younger children or in skeletal or cardiac myopathy with insidious onset later in life (1Scaglia F. Longo N. Semin. Perinatol. 1999; 23: 152-161Crossref PubMed Scopus (81) Google Scholar). The gene for primary carnitine deficiency, SLC22A5, encodes the carnitine transporter OCTN2 1The abbreviations used are: OCTNnovel organic cation transporterhOCTNhuman OCTNCHOChinese hamster ovary. (2Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (320) Google Scholar, 3Tamai I. Ohashi R. Nezu J. Yabuuchi H. Oku A. Shimane M. Sai Y. Tsuji A. J. Biol. Chem. 1998; 273: 20378-20382Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar), and several mutations have been identified in affected patients (reviewed in Refs. 4Wang Y. Taroni F. Garavaglia B. Longo N. Hum. Mutat. 2000; 16: 401-407Crossref PubMed Scopus (59) Google Scholar and 5Wang Y. Korman S.H. Ye J. Gargus J.J. Gutman A. Taroni F. Garavaglia B. Longo N. Genet. Med. 2001; 3: 387-392Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). OCTN2 is a novel organic cation transporter and operates a sodium-dependent transport of carnitine and a sodium-independent organic cation transport (3Tamai I. Ohashi R. Nezu J. Yabuuchi H. Oku A. Shimane M. Sai Y. Tsuji A. J. Biol. Chem. 1998; 273: 20378-20382Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 6Ohashi R. Tamai I. Nezu J.J. Nikaido H. Hashimoto N. Oku A. Sai Y. Shimane M. Tsuji A. Mol. Pharmacol. 2001; 59: 358-366Crossref PubMed Scopus (169) Google Scholar, 7Wu X. Huang W. Prasad P.D. Seth P. Rajan D.P. Leibach F.H. Chen J. Conway S.J. Ganapathy V J. Pharmacol. Exp. Ther. 1999; 290: 1482-1492PubMed Google Scholar). This transporter was originally cloned for its homology to hOCTN1 (8Tamai I. Yabuuchi H. Nezu J. Sai Y. Oku A. Shimane M. Tsuji A. FEBS Lett. 1997; 419: 107-111Crossref PubMed Scopus (398) Google Scholar), the sequence of which is 88% homologous and 77% identical to that of hOCTN2 (2Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (320) Google Scholar, 3Tamai I. Ohashi R. Nezu J. Yabuuchi H. Oku A. Shimane M. Sai Y. Tsuji A. J. Biol. Chem. 1998; 273: 20378-20382Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 8Tamai I. Yabuuchi H. Nezu J. Sai Y. Oku A. Shimane M. Tsuji A. FEBS Lett. 1997; 419: 107-111Crossref PubMed Scopus (398) Google Scholar). Unlike OCTN2, hOCTN1 does not transport carnitine. Sodium-dependent carnitine transport mediated by OCTN2 is electrogenic; 1 sodium ion enters the cell with 1 molecule of carnitine (9Wagner C.A. Lukewille U. Kaltenbach S. Moschen I. Broer A. Risler T. Broer S. Lang F. Am. J. Physiol. 2000; 279: F584-F591PubMed Google Scholar, 10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). novel organic cation transporter human OCTN Chinese hamster ovary. Genes homologous to hOCTN1 and hOCTN2 have been identified in the rat and the mouse. The corresponding transporters have recognition and functional properties similar to those reported for hOCTN1 and hOCTN2 (7Wu X. Huang W. Prasad P.D. Seth P. Rajan D.P. Leibach F.H. Chen J. Conway S.J. Ganapathy V J. Pharmacol. Exp. Ther. 1999; 290: 1482-1492PubMed Google Scholar, 11Yabuuchi H. Tamai I. Nezu J. Sakamoto K. Oku A. Shimane M. Sai Y. Tsuji A. J. Pharmacol. Exp. Ther. 1999; 289: 768-773PubMed Google Scholar, 12Wu X. George R.L. Huang W. Wang H. Conway S.J. Leibach F.H. Ganapathy V. Biochim. Biophys. Acta. 2000; 1466: 315-327Crossref PubMed Scopus (186) Google Scholar, 13Tamai I. Ohashi R. Nezu J.I. Sai Y. Kobayashi D. Oku A. Shimane M. Tsuji A. J. Biol. Chem. 2000; 275: 40064-40072Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). A novel carnitine transporter, OCTN3, has been identified in the mouse (13Tamai I. Ohashi R. Nezu J.I. Sai Y. Kobayashi D. Oku A. Shimane M. Tsuji A. J. Biol. Chem. 2000; 275: 40064-40072Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). This transporter has high homology to OCTN1 and OCTN2, mediates a low-affinity sodium-independent carnitine transport, and is expressed only in the mouse testis (13Tamai I. Ohashi R. Nezu J.I. Sai Y. Kobayashi D. Oku A. Shimane M. Tsuji A. J. Biol. Chem. 2000; 275: 40064-40072Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). The corresponding human and rat genes have not yet been identified. A third carnitine transporter, CT2, was isolated from human testis (14Enomoto A. Wempe M.F. Tsuchida H. Shin H.J. Cha S.H. Anzai N. Goto A. Sakamoto A. Niwa T. Kanai Y. Anders M.W. Endou H. J. Biol. Chem. 2002; 277: 36262-36271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). This latter transporter has high affinity toward carnitine (Km 20 μm) but is only partially sodium-dependent (14Enomoto A. Wempe M.F. Tsuchida H. Shin H.J. Cha S.H. Anzai N. Goto A. Sakamoto A. Niwa T. Kanai Y. Anders M.W. Endou H. J. Biol. Chem. 2002; 277: 36262-36271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). The structure of this transporter is only partially conserved with OCTN1 and OCTN2 (14Enomoto A. Wempe M.F. Tsuchida H. Shin H.J. Cha S.H. Anzai N. Goto A. Sakamoto A. Niwa T. Kanai Y. Anders M.W. Endou H. J. Biol. Chem. 2002; 277: 36262-36271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Its exclusive presence in the testis limits its physiological role in other tissues (14Enomoto A. Wempe M.F. Tsuchida H. Shin H.J. Cha S.H. Anzai N. Goto A. Sakamoto A. Niwa T. Kanai Y. Anders M.W. Endou H. J. Biol. Chem. 2002; 277: 36262-36271Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Finally, the amino acid transporter ATB0,+, which is unrelated to the organic cation family of membrane transporters, can transport carnitine with low affinity (Km 0.8 mm) (15Nakanishi T. Hatanaka T. Huang W. Prasad P.D. Leibach F.H. Ganapathy M.E. Ganapathy V. J. Physiol. 2001; 532: 297-304Crossref PubMed Scopus (167) Google Scholar). This transporter is expressed primarily in the lungs, mammary gland, and the intestine. This latter characteristic might explain residual intestinal carnitine transport in patients with primary carnitine deficiency (15Nakanishi T. Hatanaka T. Huang W. Prasad P.D. Leibach F.H. Ganapathy M.E. Ganapathy V. J. Physiol. 2001; 532: 297-304Crossref PubMed Scopus (167) Google Scholar). Although the pharmacological and recognition properties of OCTN2 have been investigated in many systems, little is known about the domains and residues of this transporter involved in carnitine and sodium recognition. The study of natural mutations identified in patients with primary carnitine deficiency has identified domains essential for carnitine (as opposed to organic cation) transport located in transmembrane domain 11 (P478L and S467C mutations) (16Seth P. Wu X. Huang W. Leibach F.H. Ganapathy V. J. Biol. Chem. 1999; 274: 33388-33392Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Ohashi R. Tamai I. Inano A. Katsura M. Sai Y. Nezu J. Tsuji A. J. Pharmacol. Exp. Ther. 2002; 302: 1286-1294Crossref PubMed Scopus (62) Google Scholar). This domain was proposed to contain the anionic binding site for carnitine (17Ohashi R. Tamai I. Inano A. Katsura M. Sai Y. Nezu J. Tsuji A. J. Pharmacol. Exp. Ther. 2002; 302: 1286-1294Crossref PubMed Scopus (62) Google Scholar). Another natural mutation, E452K, located in the putative intracellular loop connecting transmembrane domains 10 and 11, was found to be important for transmembrane sodium/solute transfer (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Mutations in this area increased the apparent Km toward extracellular sodium for carnitine transport (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). So far, no natural mutations have been found to affect the Km toward carnitine, and no systematic studies have been performed to determine the domain(s) of OCTN2 responsible for carnitine and sodium recognition and transfer. The identification of the substrate-binding site is of general significance because it might apply to the other OCTN transporters and to organic cation transporters (OCT) in general. In this paper, we have constructed chimeric transporter molecules by swapping corresponding portions of hOCTN1 and hOCTN2. Our results identify residues located in transmembrane domains 7,8, and 10 that affect the Km toward carnitine and are likely important for carnitine recognition or transfer. Cell Culture and Carnitine Transport—Chinese hamster ovary (CHO) cells were grown in Ham's F12 medium supplemented with 6% fetal bovine serum. Carnitine transport was measured at 37 °C with the cluster tray method as described previously (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 18Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). Cells were grown to confluence in 24-well plates (Costar) and depleted of intracellular amino acids by incubation for 90 min in Earle's balanced salt solution containing 5.5 mm d-glucose and supplemented with 0.1% bovine serum albumin. Carnitine (0.5 μm, 0.5 mCi/ml) was then added to the cells for 20 min. The transport reaction was stopped by rapidly washing the cells four times with ice-cold 0.1 m MgCl2. Intracellular carnitine was normalized for intracellular water content, and transport velocity was expressed as nmol/ml cell water/h (18Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). Saturable carnitine transport was calculated by subtracting either sodium-independent carnitine transport or carnitine transport in the presence of saturating (2 mm) cold carnitine from total transport (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 18Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). The two approaches gave similar results (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Values are reported as means ± S.E. of 3–6 independent determinations. Preliminary experiments indicated that carnitine transport is linear up to 30 min in cells expressing the normal OCTN2 transporter (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). For sodium-independent transport, methylglucamine chloride was substituted for sodium chloride in the extracellular medium (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This solution was mixed with a similar one containing 150 mm sodium chloride to evaluate sodium stimulation of carnitine transport. Kinetic constants for carnitine transport were determined by nonlinear regression analysis according to a Michaelis-Menten equation (18Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). Nonsaturable carnitine transport, measured in the presence of 2 mm carnitine, was subtracted from total transport to obtain saturable carnitine transport (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Nonlinear parameters are expressed as means ± S.D. Comparisons for significance were performed using 95% (p < 0.05) or 99% (p < 0.01) confidence intervals. In previous studies (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), we used multiple carnitine concentrations to define the Km of OCTN2 toward sodium (KNa). However, we noted that the value obtained from the intercept of multiple regressions was identical to the value obtained at the lowest concentration of carnitine (0.5 μm, Ref. 10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). This is because, in a random bireactant system such as the carnitine/sodium cotransporter, the apparent Km toward the co-substrate approaches the true Km (KNa) as the concentration of the substrate (carnitine) decreases to near zero (19Segel I.H. Enzyme Kinetics. John Wiley & Son, New York1975: 274-345Google Scholar). Because our previous studies indicated experimentally that a concentration of carnitine of 0.5 μm already gave values of apparent KNa indistinguishable from those calculated from the intersection of multiple curves, in this study we used an even lower concentration of carnitine (0.1 μm) to obtain an apparent KNa that closely approaches true KNa. As shown in the results below, the values obtained with this system are similar or identical to those published previously for these cells (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Construction of Chimeric OCTN1-OCTN2 Expression Vectors—The human OCTN2 and OCTN1 cDNAs, kindly provided by Dr. Vadivel Ganapathy, Medical College of Georgia, Augusta, were amplified by PCR using Pfu high fidelity polymerase and primers to add an EcoRI site to the 5′ and a BamHI site to the 3′ ends, as described previously for OCTN2 (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The 3′ primer removed the physiologic STOP codon of these cDNAs. The PCR product was digested with EcoRI and BamHI and ligated in the corresponding restriction sites of pEGFP-N2 (Clontech). The resulting plasmid had the OCTN cDNAs fused in-frame with the green fluorescent protein under control of the cytomegalovirus promoter. The final vector was sequenced to exclude PCR artifacts. These plasmids were transfected into CHO cells by LipofectAMINE according to the manufacturer's instructions (Invitrogen). Cells were selected with 0.8 mg/ml G418 (Invitrogen) for 2 weeks, and resistant cells were isolated. The presence of green fluorescence was used to confirm successful transfection. For the construction of chimeric plasmids, unique restriction sites within each cDNA were identified (Fig. 1). The vector containing the unique restriction site was digested with that restriction enzyme and another restriction enzyme to delete part of the cDNA. For example, in the case of CHIM-1, the OCTN1-GFP plasmid was digested with AccB71 and BamHI. The larger fragment was retained after separation of the fragments on an agarose gel. The other cDNA, OCTN2 in the case of CHIM-1, was amplified by high fidelity polymerase using a 5′ primer, creating the unique restriction site, AccB71 in the case of CHIM-1, present in the main vector and the other restriction site, BamHI in the case of CHIM-1, at the 3′ end to allow amplification of the missing portion of the transporter. The PCR product was digested with the two enzymes (AccB71 and BamHI in the case of CHIM-1) and inserted in the compatible sites of the vector containing the remaining portion of cDNA. The resulting vector was sequenced to exclude PCR artifacts and to verify the correct in-frame fusion of the two cDNAs. The resulting plasmids were stably transfected into CHO cells using LipofectAMINE and G418 selection. Site-directed Mutagenesis—The indicated mutations were introduced by site-directed mutagenesis using the QuikChange system (Stratagene) following the manufacturer's instructions. The final plasmids were sequenced to confirm the presence of the mutation and the absence of PCR artifacts. The plasmids obtained were then transfected into CHO cells as above. Carnitine Transport by CHO Cells Transfected with Chimeric OCTN Transporters—OCTN1 and OCTN2 are highly conserved (Fig. 1). The regions less conserved are in the predicted intracellular loop between transmembrane domains 6 and 7 and the intracellular C-terminal domain (these regions are boxed in Fig. 1). Different chimeric transporters were constructed by fusing the N terminus of OCTN1 with the C terminus of OCTN2 (CHIM-1, CHIM-2, CHIM-3, CHIM-4). Mirror image of these transporters were also obtained by fusing the N terminus of OCTN2 with the C terminus of OCTN1 (CHIM-5, CHIM-6, CHIM-7, CHIM-8). The transport of carnitine (0.5 μm) increased about 50-fold in CHO cells transfected with the normal OCTN2 cDNA (Fig. 2). OCTN1 failed to cause any significant increase in carnitine transport. Carnitine transport increased >25-fold in cells expressing the chimeric transporter CHIM-1, 20-fold in cells expressing CHIM-2, 17-fold with CHIM-3, and less than 3-fold with CHIM-4. The dramatic decrease in carnitine transport in CHIM-4 as compared with CHIM-3 indicated that amino acid residues 341–453 of OCTN2 contained a domain essential for OCTN2 function. Carnitine transport increased only 4-fold in cells expressing CHIM-5, which contained the 453 N-terminal amino acids of OCTN2. No significant increase in carnitine transport was observed with CHIM-6, CHIM-7, and CHIM-8. The disappearance of carnitine transport with CHIM-6, CHIM-7, and CHIM-8 indicated that another essential domain for carnitine transport was contained in the C-terminal domain of the OCTN2 transporter, between amino acid residues 341 and 557. However, the complete disappearance of carnitine transport when residues 341–453 of OCTN2 were removed further supported an essential role of this region in carnitine transport. Kinetics of Carnitine Transport in CHO Cells Expressing Normal and Chimeric OCTN2 Transporters—To understand the mechanism underlying the decreased transport of carnitine in cells expressing CHIM-1, CHIM-2, CHIM-3, and CHIM-4, kinetic constants for carnitine (0.5–100 μm) transport were compared with those of cells expressing wild-type OCTN2 cDNA (Fig. 3). CHO cells overexpressing the OCTN2 cDNA transported carnitine with a Km of 2.9 ± 0.7 μm, in the range reported in previous studies (3Tamai I. Ohashi R. Nezu J. Yabuuchi H. Oku A. Shimane M. Sai Y. Tsuji A. J. Biol. Chem. 1998; 273: 20378-20382Abstract Full Text Full Text PDF PubMed Scopus (626) Google Scholar, 10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 20Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar). As the N terminus of OCTN2 was substituted by OCTN1, the Km toward carnitine increased to 5.2 ± 0.8 μm (no significant change versus OCTN2) in CHIM-1, 9.8 ± 0.8 μm in CHIM-2 (p < 0.01 as compared with wild-type OCTN2 using 99% confidence intervals), 12.5 ± 0.8 μm in CHIM-3 (p < 0.01), and 141 ± 19 μm in CHIM-4 (p < 0.01). By contrast, there were only minimal changes in Vmax among most transfectants. The Vmax was 111 ± 6 nmol/ml cell water/h with OCTN2, 105 ± 4 nmol/ml cell water/h with CHIM-1 (not significantly different from OCTN2), 108 ± 2 nmol/ml cell water/h with CHIM-2 (not significantly different from OCTN2), 133 ± 4 nmol/ml cell water/h with CHIM-3 (p < 0.05 versus OCTN2), and 215 ± 20 nmol/ml cell water/h with CHIM-4 (p < 0.01 versus OCTN2). Analysis of cells expressing CHIM-5 indicated a near normal Km toward carnitine (10.6 ± 3.9 μm, not significantly different from OCTN2) with a markedly reduced Vmax (18.1 ± 2 nmol/ml cell water/h, p < 0.01 versus OCTN2). The near normal Km of CHIM-5 indicates that the majority of residues needed for carnitine recognition are contained in the first 453 amino acids of OCTN2. This further supported the results obtained with CHIM-1, CHIM-2, CHIM-3, and CHIM-4 indicating that that the substitution of the first 453 amino acids of OCTN2 with the corresponding portion of OCTN1 caused a progressive reduction in the affinity for carnitine. The sudden increase in Km between CHIM-3 and CHIM-4 indicated that a major domain required for carnitine recognition was contained within residues 341 and 453 of OCTN2. Sodium Stimulation of Carnitine Transport in CHO Cells Expressing Normal and Chimeric OCTN2 Transporters—The binding of a co-substrate can affect the affinity of a transporter toward the substrate (19Segel I.H. Enzyme Kinetics. John Wiley & Son, New York1975: 274-345Google Scholar). We have previously seen that a marked decrease in extracellular sodium concentration increases the Km of OCTN2 toward carnitine (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Kinetic analysis of carnitine transport at different extracellular sodium concentrations indicated that sodium has at least two functions in carnitine transport (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). At low concentrations, sodium lowers the Km of OCTN2 toward carnitine (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), suggesting binding to a site close to the carnitine binding site. Higher sodium concentrations provide the electrochemical gradient to transfer the carnitine-sodium complex inside the cell (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). To exclude that the changes in the Km toward carnitine measured in CHIM-1, CHIM-2, CHIM-3, and CHIM-4 were caused by abnormal interaction with the co-transported sodium, the kinetics of sodium-stimulated carnitine transport were obtained (Fig. 4). Half-maximal stimulation of carnitine transport was obtained at a sodium concentration of 13 ± 2.7 mm in CHO cells expressing the normal OCTN2 cDNA, a value similar to that reported previously in CHO cells (11.6 mm, Ref. 10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar) and in human fibroblasts that express the same transporter (11.4 ± 2.1 mm, Ref. 18Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). KNa (the concentration of sodium at which half-maximal stimulation of carnitine transport was observed) increased to 51.7 ± 8.6 mm with CHIM-1 (p < 0.01 versus OCTN2 using 99% confidence intervals) and remained elevated in CHIM-2 (41.9 ± 3.8 mm, p < 0.01 versus OCTN2) and CHIM-3 (38.1 ± 7.9 mm, p < 0.05 versus OCTN2). KNa further increased to 90 ± 26 mm in CHIM-4 (p < 0.05 versus OCTN2). These results indicate that substitution of the N terminus of OCTN2 with OCTN1 in CHIM-1 (amino acids 1–193) leads to an increase in KNa. Therefore the mild increase in Km toward carnitine seen in CHIM-1 could be secondary to changes in the affinity toward the co-transported sodium rather than reflect primary changes of the carnitine binding site. Additional substitution of the N terminus of OCTN2 with OCTN1 in CHIM-2 and CHIM-3 did not appear to further impair the interaction with sodium, as KNa did not further increase. However, the progressive substitution of OCTN2 with OCTN1 in CHIM-2 and CHIM-3 did result in a significant progressive increase in the Km toward carnitine (Fig. 3), indicating the presence of domains interacting directly with the substrate between amino acids 194 and 340. Further substitution of OCTN2 with OCTN1 in CHIM-4 results in markedly decreased carnitine transport for both an increase in KNa (90 ± 26 mm) and a dramatic increase in the Km toward carnitine (141 ± 19 μm). Therefore, although multiple domains of the transporter contribute to carnitine recognition, the domain comprised between amino acids 341 and 453 of OCTN2 is the one with the most severe effect on carnitine (and possibly sodium) recognition. CHIM-5 had a near normal Km toward carnitine (Fig. 3). However, the Vmax for carnitine transport was markedly reduced (Fig. 3). This behavior was similar to that previously described for the E452K mutant carnitine transporter, which had a normal Km toward carnitine but markedly increased KNa (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782-20786Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 20Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar). The E452K mutation did not affect the Km of the transporter toward carnitine and remained normally sensitive to the effects of low concentrations of sodium on the Km toward carnitine, indicating that it is not located close to a carnitine binding site (10Wang Y. Meadows T.A. Longo N. J. Biol. Chem. 2000; 275: 20782
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