Abnormal Sodium Stimulation of Carnitine Transport in Primary Carnitine Deficiency

Abstract

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 sodium-dependent carnitine cotransporter OCTN2. The majority of natural mutations identified in this and other Na+/solute symporters introduce premature termination codons or impair insertion of the mutant transporter on the plasma membrane. Here we report that a missense mutation (E452K) identified in one patient with primary carnitine deficiency did not affect membrane targeting, as assessed with confocal microscopy of transporters tagged with the green fluorescent protein, but reduced carnitine transport by impairing sodium stimulation of carnitine transport. The natural mutation increased the concentration of sodium required to half-maximally stimulate carnitine transport (K Na) from the physiological value of 11.6 to 187 mm. Substitution of Glu452 with glutamine (E452Q), aspartate (E452D), or alanine (E452A) caused intermediate increases in the K Na. Carnitine transport decreased exponentially with increased K Na. The E452K mutation is the first natural mutation in a mammalian cotransporter affecting sodium-coupled solute transfer and identifies a novel domain of the OCTN2 cotransporter involved in transmembrane sodium/solute transfer. 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 sodium-dependent carnitine cotransporter OCTN2. The majority of natural mutations identified in this and other Na+/solute symporters introduce premature termination codons or impair insertion of the mutant transporter on the plasma membrane. Here we report that a missense mutation (E452K) identified in one patient with primary carnitine deficiency did not affect membrane targeting, as assessed with confocal microscopy of transporters tagged with the green fluorescent protein, but reduced carnitine transport by impairing sodium stimulation of carnitine transport. The natural mutation increased the concentration of sodium required to half-maximally stimulate carnitine transport (K Na) from the physiological value of 11.6 to 187 mm. Substitution of Glu452 with glutamine (E452Q), aspartate (E452D), or alanine (E452A) caused intermediate increases in the K Na. Carnitine transport decreased exponentially with increased K Na. The E452K mutation is the first natural mutation in a mammalian cotransporter affecting sodium-coupled solute transfer and identifies a novel domain of the OCTN2 cotransporter involved in transmembrane sodium/solute transfer. Chinese hamster ovary green fluorescent protein Primary carnitine deficiency (On-line Mendelian Inheritance in Man (OMIM) no. 212140) is a recessively inherited disorder of fatty acid oxidation due to defective carnitine transport (1.Roe C.R. Coates P.M. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill Inc., New York1995: 1501-1533Google Scholar, 2.Scaglia F. Longo N. Semin. Perinatol. 1999; 23: 152-161Crossref PubMed Scopus (83) 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 heart myopathy with insidious onset later in life. The gene for primary carnitine deficiency encodes a functional carnitine transporter named OCTN2 (3.Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (322) Google Scholar, 4.Tamai 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 (629) Google Scholar) that maps to chromosome 5q31.1–32 (3.Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (322) Google Scholar, 5.Shoji Y. Koizumi A. Kayo T. Ohata T. Takahashi T. Harada K. Takada G. Am. J. Hum. Genet. 1998; 63: 101-108Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). OCTN2 is a novel organic cation transporter and operates a sodium-dependent transport of carnitine. It belongs to a family of transporters believed important in drug absorption and detoxification, although the physiological substrate has been identified only for OCTN2. Human fibroblasts express the carnitine transporter defective in primary carnitine deficiency (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). This transporter is not inhibited by amino acids, but is competitively inhibited by acetylcarnitine, palmitoylcarnitine, butyrobetaine, and betaine. Noncompetitive inhibition is observed with verapamil and quinidine (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). The carnitine transporter is energized by the sodium electrochemical potential gradient; and in the absence of sodium, only minimal amounts of carnitine enter the cell through a nonsaturable process (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). Mutations in the OCTN2 gene have been reported in patients with primary carnitine deficiency (7.Nezu J. Tamai I. Oku A. Ohashi R. Yabuuchi H. Hashimoto N. Nikaido H. Sai Y. Koizumi A. Shoji Y. Takada G. Matsuishi T. Yoshino M. Kato H. Ohura T. Tsujimoto G. Hayakawa J. Shimane M. Tsuji A. Nat. Genet. 1999; 21: 91-94Crossref PubMed Scopus (486) Google Scholar, 8.Wang Y. Ye J. Ganapathy V. Longo N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2356-2360Crossref PubMed Scopus (177) Google Scholar, 9.Tang N.L. Ganapathy V. Wu X. Hui J. Seth P. Yuen P.M. Fok T.F. Hjelm N.M. Hum. Mol. Genet. 1999; 8: 655-660Crossref PubMed Scopus (137) Google Scholar, 10.Burwinkel B. Kreuder J. Schweitzer S. Vorgerd M. Gempel K. Gerbitz K.D. Kilimann M.W. Biochem. Biophys. Res. Commun. 1999; 261: 484-487Crossref PubMed Scopus (57) Google Scholar, 11.Vaz F.M. Scholte H.R. Ruiter J. Hussaarts-Odijk L.M. Pereira R.R. Schweitzer S. de Klerk J.B. Waterham H.R. Wanders R.J. Hum. Genet. 1999; 105: 157-161Crossref PubMed Scopus (45) Google Scholar), most of whom presented early in life with a severe metabolic decompensation. Identified mutations result in premature termination codons or in nonfunctional transporters when missense mutations have been expressed in mammalian cells (9.Tang N.L. Ganapathy V. Wu X. Hui J. Seth P. Yuen P.M. Fok T.F. Hjelm N.M. Hum. Mol. Genet. 1999; 8: 655-660Crossref PubMed Scopus (137) Google Scholar). We have recently identified a missense mutation (E452K) associated with residual carnitine transport activity in a patient who presented at 7 years of age with severe cardiomyopathy and carnitine deficiency (10.Burwinkel B. Kreuder J. Schweitzer S. Vorgerd M. Gempel K. Gerbitz K.D. Kilimann M.W. Biochem. Biophys. Res. Commun. 1999; 261: 484-487Crossref PubMed Scopus (57) Google Scholar). Preliminary experiments indicated that this mutation reduced carnitine transport without affecting the K m for carnitine recognition (12.Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar). Since the majority of mutations reported in the sodium/glucose cotransporter SGLT1 impair membrane trafficking (13.Wright E.M. Loo D.D. Panayotova-Heiermann M. Hirayama B.A. Turk E. Eskandari S. Lam J.T. Acta Physiol. Scand. Suppl. 1998; 643: 257-264PubMed Google Scholar), it was proposed that the E452K substitution could impair insertion of OCTN2 carnitine transporters to the plasma membrane, with the transporters reaching the plasma membrane having normal affinity for carnitine (12.Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar). Here we report that the E452K mutation does not affect membrane insertion of carnitine transporters, but impairs activation of carnitine transport by the cotransported sodium. This is the first natural mutation in a sodium cotransporter affecting energization of substrate transport and identifies a novel domain involved in sodium recognition or transfer. Chinese hamster ovary (CHO)1 cells were grown in Ham's F-12 medium supplemented with 6% fetal bovine serum. Carnitine transport was measured at 37 °C with the cluster-tray method as described previously (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar, 8.Wang Y. Ye J. Ganapathy V. Longo N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2356-2360Crossref PubMed Scopus (177) Google Scholar, 12.Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar). Cells were grown to confluence in 24-well plates (Costar Corp.) and depleted of intracellular amino acids by incubation for 90 min in Earle's balanced salt solution containing 5.5 mmd-glucose and supplemented with 0.1% bovine serum albumin. Carnitine (0.5 μm, 0.5 μCi/ml) was then added to the cells for 10 min. Nonsaturable carnitine transport was measured in the presence of 2 mm unlabeled carnitine. The transport reaction was stopped by rapidly washing the cells four times with ice-cold 0.1 m MgCl2. Intracellular carnitine was then corrected for intracellular water content and expressed as nmol/ml of cell water (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). Saturable carnitine transport was calculated by subtracting sodium-independent carnitine transport from total transport, and values are reported as means ± S.E. of three to six independent determinations. Carnitine transport in the absence of sodium was measured substituting methylglucamine for sodium so that the sum of methylglucamine and sodium remained constant at 150 mm (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). Initial experiments on transfected cells indicated that carnitine accumulation at 0.5 μm was linear for up to 30 min in cells expressing the normal OCTN2 transporter and for up to 4 h in cells expressing mutant transporters, with a roughly inverse correlation between transport activity and time during which transport remained linear (data not shown). Kinetic constants for carnitine transport were determined by nonlinear regression analysis according to a Michaelis-Menten equation (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). Na+-independent carnitine transport was determined in parallel trays and subtracted from total transport to obtain Na+-dependent carnitine transport. Nonlinear parameters are expressed as means ± 95% confidence intervals. The K m for sodium (K Na) was calculated from the intersection (−1/K Na) of linear regressions of 1/v versus 1/[sodium] at three different carnitine concentrations (14.Segel I.H. Enzyme Kinetics. John Wiley & Sons, Inc., New York1975: 274-345Google Scholar). The OCTN2 cDNA (3.Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (322) Google Scholar) cloned in pcDNA3 (8.Wang Y. Ye J. Ganapathy V. Longo N. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2356-2360Crossref PubMed Scopus (177) Google Scholar) was amplified by polymerase chain reaction using high-fidelity Pfu polymerase and the primers 5′-GCGAATTCCCAGACCCCAGGCCGCGCT-3′ and 5′-GCGGATCCAGAAGGCTGTGCTTTTAAGG-3′, corresponding to the 5′- and 3′-primers of the OCTN2 cDNA with attached EcoRI andBamHI linkers, respectively. The 3′-primer removed the physiological stop codon of OCTN2 (TAA). The polymerase chain reaction product was digested with EcoRI and BamHI and ligated in the corresponding restriction sites of pEGFP-N2 (CLONTECH). The resulting plasmid had the OCTN2 cDNA fused in frame with the green fluorescent protein under control of the cytomegalovirus promoter. The final vector was sequenced, confirming the absence of polymerase chain reaction artifacts and the correct fusion with the green fluorescent protein. This plasmid was transfected into CHO cells by LipofectAMINE (Life Technologies, Inc.) according to the manufacturer's instructions. Cells were selected with 0.8 mg/ml G418 (Life Technologies, Inc.) for 2 weeks, and several independent clones of transfected cells were isolated. The E452K, E452A, E452Q, and E452D mutations were introduced by site-directed mutagenesis using the QuickChange system (Stratagene) following the manufacturer's instructions. The final plasmids were sequenced to confirm the presence of the mutations and the absence of polymerase chain reaction artifacts. The plasmids obtained were then transfected into CHO cells as described above. Cellular RNA was extracted with guanidinium thiocyanate, separated by formaldehyde-agarose gel electrophoresis, blotted onto nylon, and hybridized under high-stringency conditions with the OCTN2 and actin cDNAs. cpm in each band were determined using a micro-array radioactivity detector (Instant Imager, Packard Instrument Co.). Lane-specific background was subtracted from each lane. OCTN2 cpm were normalized for actin cpm to correct for variations in RNA loading. Linearity of the assay was determined using increasing amounts of control RNA (12.Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar). The gel was then exposed to film. Subcellular distribution of normal and mutant OCTN2 carnitine transporters conjugated with the green fluorescent protein was analyzed by confocal microscopy (Zeiss LSM 410 laser scanning confocal microscope). Cells were seeded on glass slides and covered with medium and a coverslip whose borders were chemically sealed. Images of the cells were obtained at 1-μm sections. Multiple cells were scanned, and the ones shown are representative of the The experiments indicated that the E452K mutation decreased the for carnitine transport, without affecting the K m (12.Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar). This was with the that the mutation could impair of the transporter to the plasma membrane, as described for mutations in the sodium/glucose cotransporter SGLT1 (13.Wright E.M. Loo D.D. Panayotova-Heiermann M. Hirayama B.A. Turk E. Eskandari S. Lam J.T. Acta Physiol. Scand. Suppl. 1998; 643: 257-264PubMed Google Scholar). this the OCTN2 transporter was tagged with the green fluorescent protein to with the green fluorescent protein did not affect carnitine transport which remained in CHO cells expressing the OCTN2 cDNA and the normal cDNA with a of the green fluorescent protein m for sodium-dependent carnitine transport was ± μm in CHO cells transfected with a value to that in CHO cells expressing the OCTN2 cDNA (12.Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar) and in fibroblasts (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). the for sodium-dependent carnitine transport was in the measured in cells expressing the OCTN2 cDNA (12.Wang Y. Kelly M.A. Cowan T.M. Longo N. Hum. Mutat. 2000; 15: 238-245Crossref PubMed Scopus (52) Google Scholar) different clones of CHO cells expressing the E452K mutant carnitine transporter and normal K m values for carnitine ± and ± and although values for sodium-dependent carnitine transport were reduced with that of normal OCTN2 that of the of the carnitine transporter does not affect carnitine recognition and constants for carnitine transport in CHO cells expressing normal and mutant carnitine ± ± ± ± ± ± ± ± ± ± ± ± constants for carnitine transport were determined in the absence or presence of sodium Na+-independent carnitine transport was subtracted from total transport to obtain Na+-dependent carnitine transport. are shown as means ± 95% confidence intervals. in a Kinetic constants for carnitine transport were determined in the absence or presence of sodium Na+-independent carnitine transport was subtracted from total transport to obtain Na+-dependent carnitine transport. are shown as means ± 95% confidence intervals. of the cells with a confocal indicated that CHO cells transfected with the vector had a distribution of green the was at the of the cell on the plasma membrane in cells in which was conjugated with normal OCTN2 2 cells expressing the E452K mutant OCTN2 transporter had a distribution of to that of cells expressing normal OCTN2 2 that this mutation does not impair insertion of the transporter to the plasma membrane. The carnitine transporter defective in primary carnitine deficiency is energized by the sodium electrochemical potential (6.Scaglia F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). We determined the constants of normal and mutant OCTN2 for sodium to the E452K mutation energization of the carnitine transporter by the sodium electrochemical was by methylglucamine in the medium The of carnitine transport on the sodium concentration was measured at different carnitine concentrations to the K the intersection of the (14.Segel I.H. Enzyme Kinetics. John Wiley & Sons, Inc., New York1975: 274-345Google Scholar). Na+-independent transport was subtracted from transport to CHO cells expressing the normal OCTN2 cDNA had a K of 11.6 mm This value that measured in fibroblasts using the 2 of F. Wang Y. Longo N. Arch. Biochem. Biophys. 1999; 364: 99-106Crossref PubMed Scopus (35) Google Scholar). Cells expressing the E452K mutant transporter had a K of 187 which was times that of cells expressing the normal OCTN2 transporter K values were obtained in CHO cells transfected with the E452K mutant OCTN2 cDNA not conjugated to the green fluorescent protein (data not that the K is not an due to the of the mutant protein with the green fluorescent protein. that the E452K mutation impairs sodium stimulation of carnitine transport by the OCTN2 a of one substrate affect the affinity of the transporter for the other substrate (14.Segel I.H. Enzyme Kinetics. John Wiley & Sons, Inc., New York1975: 274-345Google Scholar). Since the E452K mutation increased the K we the K m for carnitine at different sodium concentrations the E452K mutation were to the at concentrations of sodium, the recognition of carnitine could have been and the K m could have been shown in for carnitine transport remained constant at sodium concentrations in independent clones of CHO cells expressing the normal OCTN2 transporter and in cells the K carnitine decreased as the sodium concentration increased in cells expressing the normal and E452K mutant carnitine most of the at concentrations of sodium and was at mm sodium This that only minimal amounts of sodium are required to recognition of carnitine by this the in the K m for carnitine was in cells expressing the normal and E452K mutant transporters, that the E452K mutation does not affect of sodium to the transporter and carnitine The of the carnitine transporter is analysis of the amino acid of OCTN2 by the method of and J. J. Mol. Biol. PubMed Scopus Google Scholar) as described by Wu (3.Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (322) Google Scholar) or with 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 (629) Google Scholar) transmembrane OCTN2 is according to other membrane transporters, the amino and the (3.Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (322) Google Scholar, P. Wu X. Leibach F.H. Ganapathy V. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). This on the correct of the membrane potential and protein sites as as the transporter protein and the (3.Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (322) Google Scholar, 4.Tamai 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 (629) Google Scholar, P. Wu X. Leibach F.H. Ganapathy V. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). to this the E452K mutation an intracellular between transmembrane 10 and of the increased K of the E452K mutant transporter and the normal in m for carnitine with increased sodium concentration we the that Glu452 was for the correct transfer of the the the of Glu452 was required for this alanine glutamine (E452Q), or aspartate was in by site-directed and the resulting plasmids were expressed in CHO that mutant transporters a m for but for sodium-dependent carnitine transport decreased as OCTN2 E452D and E452K The lack of in the K m for carnitine that Glu452 is not involved in carnitine the K decreased as E452K E452D normal Glu452 The K of normal and mutant carnitine transporters were then the for carnitine transport normalized for OCTN2 was a in for carnitine transport with the in K 2 The inverse correlation between carnitine transport and the that decreased carnitine transport by the E452K mutation is due to sodium stimulation of carnitine transfer by OCTN2. It that in this is aspartate for sodium and that the of the chain is important the of this glutamine was to aspartate in normal between carnitine transport in CHO cells expressing normal and mutant carnitine RNA was extracted from CHO cells and analyzed by analysis with to the OCTN2 cDNA and to a actin cDNA cpm in each band were on an and normalized for actin for carnitine transport was normalized for OCTN2 of normal OCTN2 as a and K Na. the of the to an equation with analysis of 2 of of sodium/solute from the of and the cotransporter SGLT1 (13.Wright E.M. Loo D.D. Panayotova-Heiermann M. Hirayama B.A. Turk E. Eskandari S. Lam J.T. Acta Physiol. Scand. Suppl. 1998; 643: 257-264PubMed Google Scholar). this protein, the to sodium recognition and the a for (13.Wright E.M. Loo D.D. Panayotova-Heiermann M. Hirayama B.A. Turk E. Eskandari S. Lam J.T. Acta Physiol. Scand. 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H. 1998; PubMed Scopus Google Scholar), the with the E452K a of Glu452 in the of sodium to the a is that Glu452 in the of the transporter during the transport transporters as and the of the and of acid and in the of transmembrane to a in the transporter between an and A. H. H. V. A. PubMed Scopus Google Scholar). Substitution of of amino acids can the transporter in an or as assessed by the of for the of the transporter A. H. H. V. A. PubMed Scopus Google Scholar). The OCTN2 carnitine transporter has a between transmembrane 2 and (3.Wu X. Prasad P.D. Leibach F.H. Ganapathy V. Biochem. Biophys. Res. Commun. 1998; 246: 589-595Crossref PubMed Scopus (322) Google Scholar, 4.Tamai I. Ohashi R. Nezu J. Yabuuchi H. Oku A. Shimane M. Sai Y. Tsuji A. J. Biol. 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