From the Faculty of Pharmaceutical Sciences, Kanazawa
University, 13-1 Takara-machi, Kanazawa 920-0934, Japan and the
§ Chugai Research Institute for Molecular Medicine Inc.,
Ibaraki 300-4101, Japan
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ABSTRACT |
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Primary carnitine deficiency, because of a defect
of the tissue plasma membrane carnitine transporters, causes critical
symptoms. However, the transporter has not been molecularly identified. In this study, we screened a human kidney cDNA library and
assembled a cDNA-encoding OCTN2 as a homologue of the organic
cation transporter OCTN1, and then we examined the function of
OCTN2 as a carnitine transporter. OCTN2-cDNA encodes a polypeptide
of 557 amino acids with 75.8% similarity to OCTN1. Northern blot
analysis showed that OCTN2 is strongly expressed in kidney, skeletal
muscle, heart, and placenta in adult humans. When OCTN2 was expressed
in HEK293 cells, uptake of
L-[3H]carnitine was strongly enhanced
in a sodium-dependent manner with Km
value of 4.34 µM, whereas typical substrates for
previously known organic cation transporters, tetraethylammonium and
guanidine, were not good substitutes. OCTN2-mediated
L-[3H]carnitine transport was inhibited by
the D-isomer, acetyl-D,L-carnitine, and -butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline,
-hydroxybutyric acid,
-aminobutyric acid, lysine, and taurine were not inhibitory. Because
the observed tissue distribution of OCTN2 is consistent with the
reported distribution of carnitine transport activity and the
functional characteristics of OCTN2 coincide with those reported for
plasma membrane carnitine transport, we conclude that OCTN2 is a
physiologically important, high affinity sodium-carnitine cotransporter
in humans.
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INTRODUCTION |
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Carnitine (3-hydroxy-4-N-trimethylaminobutyric acid) is
a small, water soluble molecule that has important physiological roles, including involvement in the -oxidation of fatty acids by
facilitating the transport of long chain fatty acids across the
mitochondrial inner membrane as their acylcarnitine esters and
modulation of intracellular CoA homeostasis (1, 2). Carnitine
deficiency causes severe pathological symptoms such as cardiomyopathy
and muscle weakness (3-6). Primary carnitine deficiency is thought to
be because of a defect of active transport of carnitine across plasma
membranes, whereas secondary carnitine deficiency seems to be
associated with an enzymatic defect, resulting in impaired oxidation of
acyl-CoA intermediates in the mitochondria (1-3). Reduced carnitine
concentration in tissue and plasma may be caused by insufficient
carnitine uptake activity from plasma and impaired reabsorption in the
kidney, respectively (3, 5-8). Symptoms related to defective carnitine
transport have been studied in a carnitine-deficient mutant animal
model, juvenile visceral steatosis mouse, which shows several symptoms
of primary and/or secondary carnitine deficiency and lacks in high
affinity transport activity in several tissues (7, 9-12).
Although many membrane-physiological studies of carnitine transport mechanisms have been reported (3, 5-7,12-16), it is essential for a clearer understanding of the primary carnitine deficiency to identify the relevant transporter and to functionally characterize the carnitine transport in detail. We have recently cloned and characterized a novel organic cation transporter OCTN1 from human fetal liver (17). OCTN1 was expressed strongly in adult tissues such as kidney, trachea, and bone marrow and weakly in other tissues. When expressed in HEK293 cells, OCTN1 caused significant transport of tetraethylammonium (TEA),1 a typical organic cation, in a pH-dependent manner. These characteristics strongly suggested that OCTN1 acts as a proton/organic cation antiporter at the renal epithelial apical membrane (17, 18). In the present study, we identified a new transporter molecule, OCTN2, with high homology to OCTN1. We cloned full-length cDNA for this putative member of the organic cation transporter family, expressed it in HEK293 cells, and showed that OCTN2 has the characteristics of a high affinity sodium/carnitine cotransporter.
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EXPERIMENTAL PROCEDURES |
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Materials--
L-[Methyl-3H]carnitine
hydrochloride (85 Ci/mmol) and [14C]guanidine (56 mCi/mmol), [1-14C]-tetraethylammonium bromide (2.4 mCi/mmol), and [-32P]dCTP were purchased from Amersham
Pharmacia Biotech (Rockinghamshire, UK), Moravek Biochemicals Inc.
(Brea, CA), and New England Nuclear (Boston, MA), respectively.
pcDNA3 was obtained from Invitrogen (San Diego, CA). Multiple
tissue Northern blots were purchased from CLONTECH.
All other enzymes and reagents were obtained from Takara (Otsu, Japan),
Toyobo (Osaka, Japan), Wako Pure Chemical Industries (Osaka, Japan),
and Sigma Chemical Co. (St. Louis, MO). HEK293 cells were obtained from
Japanese Cancer Research Resources Bank (Tokyo, Japan).
Cloning of OCTN2 cDNA and Northern Blot Analysis--
A data
base search for matches to the cDNA sequence of the OCTN1 gene
revealed several genomic cosmid clones (GenBankTM accession
numbers L43407, L43408, L46907, L81773, and L43409), derived from human
chromosome 5q, that contain sequences highly homologous to OCTN1.
Because these genomic sequences do not cover the entire open reading
frame for this new gene, which we designated OCTN2 on the basis of its
high similarity to OCTN1, we initiated cDNA cloning. From the
genomic sequence, two primers (631RT S4 5'-GTGCTGTTGGGCTCCTTCATTTCA-3'
and 631RT A1 5'-AGCTGCATGAAGAGAAGGACACTG-3') were prepared and used in
reverse transcription-polymerase chain reaction of human kidney-derived cDNA. This afforded a 900-base pair 32 cDNA fragment of OCTN2. Screening of a human kidney cDNA library with this fragment as the
probe yielded overlapping, longer clones that provided additional sequences. A primer (631R S6 5'-AGCATCCTGTCTCCCTACTTCGTT-3') designed from the new sequence was used to amplify the 3' portion of OCTN2 by 3'
rapid amplification of cDNA ends using human kidney
Marathon-ReadyTM cDNA (CLONTECH).
Finally, the full coding sequence of OCTN2 was obtained by assembling
these sequences. To assess OCTN2 expression in human tissues, an OCTN2
cDNA fragment, amplified with the 631RT S4 and 631RT A1 primers,
was labeled with [-32P]dCTP and subjected to Northern
blotting with poly(A)+ RNA from a wide range of normal
human tissues and cancer cell lines (CLONTECH).
Hybridization was carried out in ExpressHyb hybridization solution
(CLONTECH) at 68 °C for 1 h. Membranes were
then washed in 2× SSC containing 0.1% SDS at room temperature for 60 min and finally in 0.1× SSC, 0.1% SDS at 50 °C for 20 min twice.
Transport Study in HEK293 Cells-- The full-length OCTN2 cDNA was subcloned into the BamHI sites of the expression vector pcDNA3, and the construct, pcDNA3/OCTN2 was used to transfect HEK293 cells by means of the calcium phosphate precipitation method as described previously (17). The cells were cultivated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum (Life Technologies, Inc., Tokyo, Japan), 100 units/ml penicillin, and 100 µg/ml streptomycin in tissue culture dishes in a humidified incubator at 37 °C under 5% CO2 for 24 h and then transfected with pcDNA3 plasmid carrying full-length OCTN2 cDNA or with the pcDNA3 plasmid vector alone. At 48 h after transfection, the cells were harvested by scraping with a rubber policemen and suspended in the medium for transport study, which consisted of 125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM HEPES (pH 7.4). The cell suspension and transport medium containing a radiolabeled test compound were preincubated separately for 20 min and then mixed to initiate uptake. At appropriate times, 200-µl aliquots of the mixture were withdrawn, and the cells were separated from the transport medium by centrifugation in a microtube containing a silicon oil and liquid paraffin mixture with a density of 1.03. The resultant cell pellets were solubilized in 3 N KOH and then neutralized with HCl, and the associated radioactivity was quantitated in a liquid scintillation counter (Aloka, Tokyo, Japan). Cellular protein content was determined according to the method of Bradford (19) using a Bio-Rad protein assay kit. When sodium ions were replaced with other cations, the obtained cells were suspended in sodium-free medium. The composition of sodium-free medium was the same as that of the above transport medium except that the sodium chloride was replaced isotonically with potassium chloride, choline chloride, N-methylglucamine chloride, or lithium chloride.
Usually initial uptake rates were obtained by measuring the uptake at 3 min. To estimate kinetic parameters for saturable transport, the uptake rate (v) was fitted to the following equation by means of nonlinear least squares regression analysis using WinNonlin (Scientific Consulting Inc., Cary, NC). v = Vmax × s/(Km + s), where v and s are the uptake rate and concentration of carnitine, respectively, and Km and Vmax are the half-saturation concentration (Michaelis constant) and maximum transport rate, respectively. All data were expressed as the means ± S.E., and statistical analysis was performed by use of Student's t test. The criterion of significance was taken to be p < 0.05. ![]() |
RESULTS |
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Amino Acid Sequence and Tissue Distribution of Human OCTN2-- The full-length OCTN2 cDNA appeared to encode a polypeptide of 557 amino acids and have 75.8% similarity with human OCTN1 (17) (Fig. 1A). Human OCTN2 is predicted to have twelve putative membrane-spanning domains by hydropathy analysis according to TopPred 2 (20) as well as three N-glycosylation sites and six protein kinase C phosphorylation sites. The presence of twelve membrane spanning domains agrees with that of previously known membrane transporters (21). Like human OCTN1, OCTN2 has a unique sugar transport protein signature (17). Comparison of the amino acid sequence with those of other organic ion transporters revealed that human OCTN2 has similarity with rat OCT1 (32.5%) (22), rat OCT2 (33.6%) (23), human OCT1 (33.1%) (24), human OCT2 (33.1%) (25), and rat OAT1 (28.4%) (26). No significant similarity was observed with human oatp (27) or monoamine neurotransmitter transporters such as serotonin transporter (28) or monoamine transporter (29). These data indicate that OCTN2 may be a member of the organic cation transporter family.
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Functional Analysis of OCTN2 Expressed in HEK293 Cells-- Because human OCTN1 transported the organic cation TEA in a pH-dependent manner when expressed in HEK293 cells in our previous study (17), we expressed human OCTN2 in the same cells and measured the uptake of cationic compounds for comparison with that of OCTN1. Although TEA is a good substrate of OCTN1 (17), no significant increase of [14C]TEA uptake was observed in human OCTN2-transfected cells (298 ± 60 pmol/mg protein/3 min in OCTN2-transfected HEK293 cells and 263 ± 12 in nontransfected cells at the TEA concentration of 60 µM and at 3 min). Because guanidine was suggested to be transported by a different transporter than that for TEA across the apical membrane of renal tubular epithelial cells (30), we examined the uptake of [14C]guanidine in human OCTN1- or OCTN2-expressing cells. However, neither OCTN1 (106 ± 5.6 pmol/mg protein/3 min at 10 µM guanidine) nor OCTN2 (95.1 ± 11.8 pmol/mg protein/3 min at 10 µM guanidine) exhibited increased guanidine transport activity compared with nontransfected cells (98.4 ± 5.9 pmol/mg protein/3 min at 10 µM guanidine). Zwitterionic carnitine is reabsorbed in the kidney via an active transport mechanism, although the molecular identity of the transporter remains to be established. Accordingly, we examined the transport of carnitine in the present study. Although OCTN1 showed slight but significant uptake of L-[3H]carnitine (2.5-fold increased uptake compared with nontransfected cells), a very large uptake of L-[3H]carnitine was seen with the human OCTN2-expressing HEK293 cells, as described below.
Fig. 3A shows the time course of the uptake of L-[3H]carnitine by HEK293 cells transfected with OCTN2 or with the expression vector pcDNA3 alone, in the presence or absence of sodium ions. Uptake of L-[3H]carnitine was significantly increased by OCTN2 transfection both in the presence and absence of sodium ions in the transport medium. The uptake of L-[3H]carnitine was particularly high in OCTN2-transfected cells in the presence of sodium ions, and it appears that OCTN2 is a sodium ion-dependent carnitine transporter. Because the cells transfected with expression vector alone showed a slight but significant increase of L-[3H]carnitine uptake in the presence of sodium ions compared with that in the absence of sodium ions, HEK293 cells themselves seem to have a weak activity of sodium ion-dependent carnitine transport. This is not surprising considering that HEK293 cells were originally derived from human embryonic kidney. Because sodium ion-dependent and OCTN2-mediated uptake of carnitine increased linearly up to 5 min, initial uptake rate of carnitine was determined at 3 min to characterize the transporter in all subsequent studies.
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DISCUSSION |
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Carnitine is normally maintained at a steady level in the blood, suggesting its physiological importance (1, 2). Although carnitine is biosynthesized in liver and brain (31), a significant amount of carnitine is also obtained from the diet via carrier-mediated transport across the intestinal epithelial cell membranes (32, 33) and is retained in the body through reabsorption in the kidney via active transport across the renal tubular epithelial cell membrane (1, 7, 14, 15). Furthermore, many studies have demonstrated that the tissues that extensively accumulate carnitine, such as skeletal muscle, heart, liver, and epididymis, take up and/or release carnitine via specialized transport mechanisms to maintain the steady-state tissue concentration (1). Although many of these membrane physiological studies suggested the participation of multiple sodium ion-dependent transporters with high and low affinities in carnitine movement across tissue plasma membranes (1, 13, 15), no such transporter has yet been identified at the molecular level. Accordingly, to achieve a better understanding of the biological and physiological roles of carnitine, as well as carnitine-related pathological states, it is essential to identify the carnitine transporter(s). We have previously cloned the human organic cation transporter OCTN1, which may participate at least partially in proton/organic cation antiport at the renal apical membranes, and characterized it by measuring the uptake of the typical organic cation TEA by OCTN1-transfected HEK293 cells (17). In the present study, we succeeded in obtaining cDNA of a second member of the human OCTN family, OCTN2, which has a high similarity to OCTN1, and found that OCTN2 has many of the characteristics of a high affinity, sodium ion-dependent carnitine transporter.
The idea that human OCTN2 is a sodium ion-dependent carnitine transporter is supported by the specific tissue distribution and the result of functional expression in HEK293 cells. Most adult tissues that highly express OCTN2, including skeletal muscle, kidney, placenta, and heart, have been reported to take up carnitine via a sodium ion-dependent, carrier-mediated transport mechanism (1, 15, 16, 34-36). As regards fetal tissues, we previously found that mouse embryo fibroblasts take up carnitine in a sodium ion-dependent manner with a half-saturation concentration of 5.5 µM, although the distribution of carnitine transport activity in fetal tissues was not established (13). Tissues that have apparently low affinity carnitine transporters, such as liver, brain, and intestine, with apparent half-saturation concentrations between 0.2 mM and 10 mM (1, 33, 37, 38), showed low expression of OCTN2. The distribution of OCTN2 is different from that of OCTN1, which has 75.8% sequence similarity with OCTN2 and coincides well with the functional distribution of sodium ion-dependent, high affinity carnitine transport activity as discussed above. Furthermore, OCTN2 hardly transported TEA, a good substrate of OCTN1, or guanidine, a substrate of the second renal organic cation transporter, which is distinct from that for TEA (30), OCTN2 seems likely to have some physiological role other than the renal excretion of organic cations.
When OCTN2 was expressed in HEK293 cells, a high uptake of L-[3H]carnitine was observed in the presence of sodium ions (Fig. 3, A and B). Because the cellular volume of HEK293 cells obtained from the accumulation of 3H2O is 6.3 µl/mg protein (17), L-[3H]carnitine apparently accumulated to the extent of about 320-fold at the steady-state in the cells by utilizing an inside-directed sodium ion gradient as the driving force. Lithium ions partially retained uphill transport (Fig. 3B) in a manner comparable with the carnitine transport obtained using rat renal brush border membrane vesicles (15) and human placental choriocarcinoma cells (16), which suggests that lithium ions are partially accepted as the cation for cotransport with carnitine. When sodium was replaced with choline, carnitine uptake was specifically eliminated (Fig. 3B). Although 500 µM choline was not inhibitory (Fig. 4B), choline may have a low affinity to compete with carnitine binding to OCTN2.
The half-saturation concentration of L-carnitine uptake by
OCTN2 was estimated to be 4.34 µM, which is very similar
to the values observed for high affinity carnitine transport in
membrane physiological studies in kidney, skeletal muscle, heart,
placenta, and cultured fibroblasts (1, 2, 5, 6, 15, 16, 34-36),
tissues that exhibited high expression of OCTN2 in Northern blot
analysis (Fig. 2). Carnitine transport in these tissues was reported to
be significantly inhibited by the D-isomer of carnitine, acetylcarnitine, and -butyrobetaine in a stereospecific manner (6,
15, 16, 35, 36). Furthermore, glycinebetaine and choline were low
affinity inhibitors and
-aminobutyric acid was not inhibitory
(14-16, 36). These previously reported substrate specificity
characteristics exactly coincide with the properties of OCTN2. We
conclude that OCTN2 is a high affinity, sodium
ion-dependent carnitine transporter expressed in several
tissues, including kidney, skeletal muscle, heart, and placenta.
The amino acid sequence of human OCTN2 is very similar to that of human OCTN1, although OCTN1 exhibited only a low carnitine transport activity and had the functional characteristics of a proton/organic cation antiporter (17). Furthermore, TEA, a good substrate for OCTN1, was not transported well by OCTN2. Such significant differences of substrate specificity and driving force for transport despite the similarity in amino acid sequence may suggest that the binding or recognition specificity for substrates and cotransported ions on the transporter proteins is determined in a very limited region and that OCTN proteins have both common structures as membrane transporters and distinct small regions for the recognition of the substrate and cotransported ions. It will be interesting to identify the essential amino acid sequence of the functional binding sites by constructing chimeric proteins of OCTN1 and OCTN2. The strong expression of OCTN2 in human-derived tumor cells is similar to that of OCTN1 (14). This may reflect up-regulation of expression of the gene in malignancy to meet a higher requirement for L-carnitine.
In conclusion, human OCTN2 was cloned as a new member of the family of organic cation transporters. Studies of its tissue distribution and its functional expression in HEK293 cells indicated that OCTN2 is a physiologically important, high affinity carnitine transporter that shows significant sodium ion dependence. The identification of this carnitine transporter should contribute to a better understanding of the physiological and biochemical functions of carnitine, as well as to the development of measures to treat primary carnitine deficiency. In addition, isolation of the counterpart of OCTN2 in mice and comparison with juvenile visceral steatosis mice, which show abnormal carnitine metabolism and related diseases, may help to identify the principal causes of carnitine deficiency syndromes (7, 9-12).
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FOOTNOTES |
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* This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan and by CREST Core Research for Evolutional Science and Technology of the Japan Science and Technology Corporation (JST).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB015050.
¶ To whom correspondence should be addressed. Fax: 81-76-234-4477; E-mail: tsuji{at}kenroku.ipc.kanazawa-u.ac.jp.
The abbreviation used is: TEA, tetraethylammonium.
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REFERENCES |
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