Introduction

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Recent studies have identified several members of a relatively new multispecific organic ion transporter superfamily (Koepsell, 1998; Zhang et al., 1998). This family consists of transporters for organic cations as well as organic anions. The members of this family exhibit broad substrate specificity and are likely to play an important role in the handling of a wide variety of structurally diverse xenobiotics and endobiotics in tissues such as the kidney, liver, intestine, and placenta (Pritchard and Miller, 1993). The transporters identified thus far as the members of this family are the organic cation transporters (OCTs)1 (Grundemann et al., 1994), OCT2 (Okuda et al., 1996), OCT3 (Kekuda et al., 1998), novel organic cation transporter (OCTN) 1 (Tamai et al., 1997), and OCTN2 (Wu et al., 1998c) and the organic anion transporters (OATs) 1 (Sekine et al., 1997; Sweet et al., 1997) and OAT2 (Sekine et al., 1998a).

Among the OCTs, OCT1 and OCT2 are expressed predominantly in the kidney and/or liver, depending on the animal species (Grundemann et al., 1994; Okuda et al., 1996; Gorboulev et al., 1997; Zhang et al., 1997). OCT3, on the other hand, is expressed most abundantly in the placenta and, to a lesser extent, in several other tissues (Kekuda et al., 1998). We have shown recently (Wu et al., 1998b) that OCT3 is identical with the extraneuronal monoamine transporter (uptake₂), which is known to be present in several tissues and to transport the monoamines norepinephrine and dopamine (Trendelenburg, 1988). All of these OCTs are potential-sensitive, and their function is independent of Na⁺, Cl, and H⁺.

OCTN1 and OCTN2 are structurally related much more closely to each other than to OCT1, OCT2, or OCT3. These two transporters are widely expressed in mammalian tissues. There is very little known regarding the functional characteristics of OCTN1, except that it can transport organic cations (Tamai et al., 1997). Our initial report on the cloning of OCTN2 indicated that this transporter can also transport organic cations (Wu et al., 1998c). However, a recent study by Tamai et al. (1998) has shown that OCTN2 is a Na⁺-coupled carnitine transporter. Thus, OCTN2 is the first member of this family of transporters that can function as a Na⁺-coupled transporter. The function of OCTN2 as a carnitine transporter is further supported by the recent findings that the OCTN2 gene and the gene for the genetic disorder primary systemic carnitine deficiency map to the same chromosomal location 5q31.1-32 (Shoji et al., 1998; Wu et al., 1998c) and that mutations in the OCTN2 gene are responsible for the disorder (Tang et al., 1999; Wang et al., 1999). The ability of this Na⁺-coupled transporter to interact with a variety of organic cations is very surprising and may have significant physiological and pharmacological implications in humans. This is particularly relevant because of the occurrence of loss-of-function mutations in this gene in humans and the association of these mutations with serious, and often fatal, clinical consequences such as cardiac myopathy and skeletal myopathy.

The present investigation was therefore undertaken to analyze in detail the functional characteristics of OCTN2 with regard to its ability to transport carnitine and organic cations. The results of this investigation provide evidence that OCTN2 is a Na⁺-coupled carnitine transporter and a Na⁺-independent OCT. We have also investigated the tissue-distribution pattern of OCTN2 mRNA by in situ hybridization in the rat. OCTN2 mRNA is expressed not only in the heart, kidney, and placenta, the tissues in which the function of OCTN2 as a carnitine transporter is easily recognizable, but also in the brain, a tissue in which a specific role for carnitine in cellular metabolism has not been implicated. The widespread expression of OCTN2 message in the brain is intriguing and raises the possibility that OCTN2 may have additional functions. The findings of the present investigation that OCTN2 is not only a carnitine transporter but also is a transporter for a wide variety of organic cations may be relevant to identify additional, hitherto unrecognized, functions of OCTN2.

	Experimental Procedures

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Materials. [Ethyl-1-¹⁴C]tetraethylammonium (TEA) bromide (specific radioactivity, 55 mCi/mmol) was obtained from American Radiolabeled Chemicals, Inc. (St. Louis, MO). L-[³H]Carnitine (specific radioactivity, 65 Ci/mmol), acetyl-L-[³H]carnitine (specific radioactivity, 65 Ci/mmol), and propionyl-L-[³H]carnitine (specific radioactivity, 65 Ci/mmol) were obtained from Moravek Biochemicals, Inc. (Brea, CA). Unlabeled organic cations, carnitine, and carnitine esters were obtained from Research Biochemicals International (Natick, MA) or Sigma. Cell culture media and Lipofectin were obtained from Life Technologies, Inc. (Gaithersburg, MD). Restriction enzymes were obtained from Promega (Madison, WI). Magna nylon transfer membranes were purchased from Micron Separations, Inc. (Westboro, MA). Human retinal pigment epithelial (HRPE) cells were originally provided by Dr. M. A. Del Monte (University of Michigan, Ann Arbor, MI) and have been in use in our laboratory for several years (Huang et al., 1997). The Ready-To-Go oligolabeling kit used in the preparation of cDNA probes for library screening was purchased from Amersham Pharmacia Biotech (Piscataway, NJ).

Screening of cDNA Libraries. A 0.65-kilobase pair (kbp) fragment of human OCTN (hOCTN) 1 cDNA (Tamai et al., 1997) obtained by digestion with PstI was used to screen a rat placental cDNA library (Prasad et al., 1998). This screening resulted in the isolation of a full-length rat OCTN (rOCTN) 2 cDNA clone. To obtain a full-length mouse OCTN (mOCTN) 2 cDNA clone, a 1.7-kbp fragment of rat OCTN2 cDNA obtained by digestion with ApaI/HincII was used as the probe to screen a mouse kidney cDNA library (Seth et al., 1997). The probes were labeled with [-³²P]dCTP using the Ready-To-Go oligolabeling kit. The cDNA libraries were screened at low-stringency conditions as described (Seth et al., 1997; Prasad et al., 1998). Hybridization was carried out for 20 h at 60°C in a solution containing 5× saline-sodium phosphate-EDTA (SSPE) (1× SSPE = 0.15 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA), 5× Denhardt's solution, 0.5% SDS, and 100 µg/ml denatured salmon sperm DNA. Posthybridization washing involved extensive washes with 3× SSPE/0.5% SDS at room temperature. Positive clones were identified, and the colonies were purified by secondary screening.

DNA Sequencing. Both sense and antisense strands of the cDNAs were sequenced by primer walking. Sequencing by the dideoxynucleotide chain-termination method was performed by Taq DyeDeoxy terminator cycle sequencing with an automated Perkin-Elmer Applied Biosystems 377 Prism DNA sequencer. The sequence was analyzed using the GCG sequence analysis software package GCG version 7.B (Genetics Computer Group, Inc., Madison, WI).

Functional Expression of OCTN2 cDNAs in HRPE Cells. The cloned human, rat, and mouse OCTN2 cDNAs were oriented in the pSPORT plasmid in such a way that their expression was under the control of the T7 promoter. The cDNAs were heterologously expressed in HRPE cells by vaccinia virus expression system as described (Kekuda et al., 1998; Wu et al., 1998b). Transport measurements were made at room temperature using [¹⁴C]TEA or [³H]carnitine. The transport buffer was composed of 25 mM Tris/HEPES (pH 8.5) or 25 mM HEPES/Tris (pH 7.5) supplemented with 140 mM NaCl (or 140 mM N-methyl-D-glucamine chloride or 280 mM mannitol), 5.4 mM KCl, 1.8 mM CaCl₂, 0.8 mM MgSO₄, and 5 mM glucose. After incubation for 30 min, transport was terminated by aspiration of the buffer followed by two washes with 2 ml of ice-cold transport buffer. The cells were then solubilized with 0.5 ml of 1% SDS in 0.2 N NaOH and transferred to vials for quantitation of the radioactivity associated with the cells. HRPE cells transfected with vector alone under similar conditions served as control. In experiments dealing with saturation kinetics, data were analyzed by nonlinear regression and confirmed by linear regression.

Site-Directed Mutagenesis. The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to generate the hOCTN2 mutants according to the manufacturer's protocol. The procedure uses Pfu DNA polymerase, which replicates both strands of DNA with high fidelity and without displacing the mutant primers. Two synthetic oligonucleotide primers (sense and antisense) specific for the hOCTN2 cDNA and containing the desired mutation in the middle were used. After PCR with the wild-type hOCTN2 cDNA as the template, the product was digested with DpnI to digest the parental DNA template, leaving behind the nicked double-stranded DNA containing the mutation introduced in the primers. The resultant product was then transformed into Escherichia coli for repair of the nicks and amplification. The entire coding region of the mutant cDNAs was sequenced to confirm the presence of the introduced mutations and the absence of any unwanted mutations arising from PCR. Two mutant hOCTN2 cDNAs were made, M352R and P478L. The sense and antisense primers for the generation of the M352R mutant were 5'-accatcatgtccataatgctgtggAGGaccatatcagtgg-3' and 5'-ccactgatatggtCCTccacagcattatggacatgatgg-3' (mutated codon is given in uppercase letters). The sense and antisense primers for the generation of the P478L mutant were 5'-gcatcctgtctCTCtacttcgtttaccttg-3' and 5'-caaggtaaacgaagtaGAGagacaggatgc-3' (mutated codon is given in uppercase letters).

Addition of c-myc Epitope Tag at the N Terminus of Wild-Type hOCTN2. The Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA) was used to add the c-myc epitope tag at the N terminus of the wild-type hOCTN2. The primers used were 5'-CGCGTTGGTGCGGAATTCTCGGTAGTGGGA-3' (selection primer) and 5'-TCTGAGGGCGGCATGGAACAGAAACTGATCTCCGAAGAAGACCTGCGGGACTACGAC-3' (epitope tag mutagenic primer). The underlined portion of the mutagenic primer indicates the codons for the c-myc epitope sequence (EQKLISEEDL), which is added to the N terminus of the wild-type hOCTN2 between the first (M) and second (R) amino acid residues. The primers were phosphorylated by incubation with T4 polynucleotide kinase. The selection primer converted a unique restriction site for EcoRV in the pSPORT-hOCTN2 cDNA to a restriction site for EcoRI. The plasmid was denatured, and the selection primer and the mutagenic primer were annealed to the denatured plasmid DNA. The second strand was synthesized with T4 DNA polymerase and circularized with T4 DNA ligase. The product, containing the wild-type and the mutant strands, was digested with EcoRV, which linearized only the wild-type strand. The mixture was then transformed into MutS E. coli competent cells. Plasmid DNA was isolated from the transformed cells and digested once again with EcoRV to linearize the wild-type DNA. This digest was transformed into E. coli DH5 competent cells. Plasmid DNA was isolated from individual transformants and the mutant clones were identified by restriction analysis. The addition of the epitope tag to the N terminus was confirmed by sequencing.

Addition of c-myc Epitope Tag to Mutants. Because the M352R and P478L mutations are in the C-terminal half of the hOCTN2 protein, we were able to add the c-myc epitope tag to the mutants by exchanging a 1.2-kbp 5' fragment in the mutant cDNAs with the corresponding 5' fragment from the epitope-tagged wild-type cDNA. This was possible because of the presence of a unique restriction site for BstBI in the 5'-half of the wild-type and mutant cDNAs 5' to the mutation sites. The fragment containing the tag was released by digestion of the epitope-tagged wild-type clone with EcoRI/BstBI and ligated to the EcoRI/BstBI-digested mutant clones.

Immunolocalization of c-myc Epitope-Tagged hOCTN2. HRPE cells were cultured in chamber slides, and tagged and nontagged hOCTN2 cDNAs (wild-type and mutants) were expressed in these cells heterologously using the vaccinia virus expression technique. Twelve hours after transfection, medium was removed and cells were washed with PBS. Cells were then fixed with 2.5% paraformaldehyde for 20 min. The fixative was removed, and the cells were permeabilized with 0.1% Triton X-100 for 5 min. Cells were subsequently washed thrice with PBS for 5 min each and then exposed to 4% goat serum for 10 min at room temperature. After this, cells were incubated with anti-c-myc monoclonal antibody (Invitrogen, San Diego, CA) for 3 h at room temperature and overnight at 4°C. Cells were subsequently washed with PBS three times for 5 min each and then incubated overnight at 4°C with fluorescein isothiocyanate-coupled antimouse IgG. Cells were again washed, covered with Vectashield and coverslipped, and analyzed under a fluorescent microscope.

In Situ Hybridization. Four-week-old adult male rat kidneys, hearts, and whole brains were collected and immediately frozen in liquid nitrogen. Similarly, rat term placentas were also collected and frozen. Unfixed 12-µm serial sections were prepared on a cryostat, mounted on 2% 3-aminopropyltriethoxysilane-coated slides, air dried for 10 min, and stored at 70°C until required. Nonradioactive in situ hybridization using digoxigenin UTP-labeled probes was performed on tissue sections following the protocol of Schaeren-Wiemers and Gerfin-Moser (1993) with the following modifications. Cryostat sections were fixed in 4% paraformaldehyde in 0.1 M PBS (pH 7.3) at 4°C for 20 min, washed, and permeabilized with proteinase K (10 µg/ml in PBS containing 0.1% Tween 20) for 10 min at room temperature. Sections were then refixed in 4% paraformaldehyde in PBS, prehybridized for 1 h at 65°C in 50% formamide, and incubated with probes for 16 h at 65°C. For the preparation of the rOCTN2-specific riboprobe, a 0.66-kbp fragment of the rOCTN2 cDNA, obtained by the digestion of pSPORT-rOCTN2 cDNA by PstI, was subcloned into pBluescript vector. The orientation of the cDNA insert in the pBluescript vector was determined by sequencing. Antisense and sense riboprobes were synthesized with T7 RNA polymerase or T3 RNA polymerase after linearization of the plasmid with appropriate restriction enzymes. The riboprobes were labeled using a digoxigenin-labeling kit (Boehringer Mannheim, Indianapolis, IN).

Statistics. Each uptake experiment was done in triplicate and repeated two or three times. Uptake values given are means ± S.E. of these replicates.

	Results

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Transport of TEA and Carnitine by Human, Rat, and Mouse OCTN2. hOCTN2 cDNA was isolated in our laboratory from a placental trophoblast cell line cDNA library (Wu et al., 1998c). We have now cloned the rat and mouse OCTN2 from rat placenta and mouse kidney, respectively (Genbank accession numbers for nucleotide sequence: AF110416 for rOCTN2 cDNA and AF110417 for mOCTN2 cDNA). The human, rat, and mouse OCTN2 proteins are highly homologous (83% identity in amino acid sequence). All three cDNAs were expressed heterologously in HRPE cells, and their function with respect to the transport of the organic cation TEA and of the zwitterion carnitine was studied under identical conditions (Fig. 1). Uptake of TEA was severalfold higher in cells expressing human, rat, and mouse OCTN2 (4.3-, 16.8-, and13.5-fold, respectively) than in control cells transfected with vector alone. Similarly, the uptake of carnitine was also increased severalfold in cells expressing these three OCTN2s. The increase was 27.4-, 9.0-, and 16.1-fold for hOCTN2, rOCTN2, and mOCTN2, respectively. Even though all three OCTN2s had the ability to transport TEA as well as carnitine, the relative activity with respect to the two substances varied markedly among them. hOCTN2 showed a much higher capacity to transport carnitine than TEA, whereas rOCTN2 showed a much higher capacity to transport TEA than carnitine. The relative ability of mOCTN2 to transport TEA and carnitine was intermediate to that of hOCTN2 and rOCTN2.

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Transport of TEA and carnitine by human, rat, and mouse OCTN2s. The cDNAs were functionally expressed in HRPE cells using the vaccinia virus expression technique. Cells transfected with pSPORT vector alone served as control. Uptake of [14C]TEA (20 µM) was measured at pH 8.5 in a medium in which NaCl was replaced isoosmotically by mannitol. Uptake of [3H]carnitine (25 nM) was measured at pH 7.5 in a medium containing NaCl.

Characteristics of Transport of TEA and Carnitine by rOCTN2. Fig. 2 describes the Na⁺ dependence of rOCTN2-mediated uptake of TEA and carnitine in HRPE cells. The uptake of TEA mediated by rOCTN2 was not dependent on the presence of Na⁺. The uptake was comparable in the presence of NaCl or when NaCl was replaced isoosmotically by mannitol. The uptake was indeed significantly higher in the presence of N-methyl-D-glucamine chloride than in the presence of NaCl. Clearly, the rOCTN2-mediated TEA uptake was independent of Na⁺. In contrast, carnitine uptake via rOCTN2 was obligatorily dependent on the presence of Na⁺. In uptake buffers containing mannitol or N-methyl-D-glucamine chloride instead of NaCl, carnitine uptake was markedly reduced in rOCTN2-expressing cells.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Influence of Na+ on rOCTN2-mediated transport of TEA and carnitine. HRPE cells were transfected with either pSPORT vector alone or rOCTN2 cDNA. Functional expression was carried out using the vaccinia virus technique. Uptake of [14C]TEA (20 µM) and [3H]carnitine (25 nM) was measured at pH 8.5 and 7.5, respectively. Uptake medium contained 140 mM NaCl, 140 mM N-methyl-D-glucamine chloride (NMDG-Cl), or 280 mM mannitol. Uptake in vector-transfected cells was subtracted from corresponding uptake in cDNA-transfected cells to calculate rOCTN2-mediated uptake.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Substrate specificity of the organic cation transport process mediated by rOCTN2. rOCTN2 cDNA was functionally expressed in HRPE cells. Cells transfected with pSPORT vector alone served as control. Uptake of [14C]TEA (20 µM) was measured at pH 7.5 in a medium in which NaCl was replaced by N-methyl-D-glucamine chloride isoosmotically. Final concentration of the inhibitors was 2.5 mM. The pH of the uptake medium was adjusted to 7.5 in the presence of each inhibitor. Uptake measured in vector-transfected cells was subtracted from uptake in cDNA-transfected cells to calculate rOCTN2-mediated uptake.

View larger version (20K): [in this window] [in a new window]   Fig. 4.   Influence of Na+ on the inhibition of rOCTN2-mediated [14C]TEA uptake by carnitine and organic cations. Uptake of [14C]TEA (20 µM) was measured in HRPE cells expressing rOCTN2. Uptake measured in cells transfected with vector alone was subtracted from uptake in rOCTN2-expressing cells to calculate rOCTN2-mediated uptake. Uptake measurements were made at pH 7.5 in a medium containing either 140 mM N-methyl-D-glucamine chloride (Na+) or 140 mM NaCl (+Na+). Final concentration of the inhibitors was 500 µM. Uptake measured in the absence of inhibitors was taken as control.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Influence of Na+ on the potency of unlabeled TEA and carnitine to inhibit rOCTN2-mediated [14C]TEA uptake. Uptake of [14C]TEA (25 µM) was measured in HRPE cells expressing rOCTN2. Uptake measured in vector-transfected cells was subtracted from uptake in rOCTN2-expressing cells to calculate rOCTN2-mediated uptake. Uptake measurements were made at pH 7.5 in a medium containing either 140 mM N-methyl-D-glucamine chloride () or 140 mM NaCl (). Concentration of unlabeled TEA and carnitine was varied over the range of 0.1 µM to 1 mM and 1 µM to 10 mM, respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Saturation kinetics of rOCTN2-mediated transport of TEA (A) and carnitine (B). Uptake was measured in rOCTN2-expressing HRPE cells. Uptake in vector-transfected cells was subtracted to calculate rOCTN2-mediated uptake. Uptake of TEA was measured at pH 7.5 in a medium containing 140 mM N-methyl-D-glucamine chloride in place of 140 mM NaCl. Uptake of carnitine was measured at pH 7.5 in a medium containing 140 mM NaCl. Inset, Eadie-Hofstee plot. V, uptake in pmol/106 cells/30 min; S, substrate concentration in µM.

Interaction of hOCTN2 with Organic Cations and Carnitine. We also investigated the ability to hOCTN2 to interact with organic cations. hOCTN2 has already been shown to have the ability to transport carnitine in a Na⁺-dependent manner (Tamai et al., 1998). In the present study, we investigated the ability of organic cations to inhibit hOCTN2-mediated carnitine transport (Table 2). Several organic cations were found to inhibit the transport to a marked extent. Desipramine and verapamil were the most effective inhibitors. TEA, cimetidine, and MPTP showed moderate inhibition. Choline had a small inhibitory effect. In contrast, guanidine showed no detectable inhibition. These results demonstrate that hOCTN2 is also a transporter for carnitine as well as for organic cations.

Transport of Acetyl-L-Carnitine and Propionyl-L-Carnitine by hOCTN2 and rOCTN2. The short-chain acyl esters of L-carnitine are currently used as therapeutic agents in the treatment of a wide range of disorders (Spagnoli et al., 1991; Brevetti et al., 1992; Lowitt et al., 1995; Hagen et al., 1998; Wiseman and Brogden, 1998). Therefore, it is of interest to determine whether the cloned OCTN2s are capable of transporting the acyl esters acetyl-L-carnitine and propionyl-L-carnitine. Figure 7 shows that hOCTN2 as well as rOCTN2 are able to transport these acyl esters of carnitine. The transport of acetyl-L-carnitine in hOCTN2-expressing cells was 10-fold greater than in vector-transfected cells. Similarly, expression of rOCTN2 also led to a 10-fold increase in acetyl-L-carnitine transport. In both cases, the OCTN2-mediated transport of acetyl-L-carnitine was completely inhibited by 5 mM L-carnitine or TEA (Fig. 7, A and B). Similar results were obtained with propionyl-L-carnitine (Fig. 7, C and D). hOCTN2 and rOCTN2 were able to transport propionyl-L-carnitine, and this transport was abolished by 5 mM L-carnitine or TEA.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Transport of acetyl-L-carnitine and propionyl-L-carnitine by OCTN2 and their inhibition by carnitine and TEA. HRPE cells were transfected with either hOCTN2 cDNA (A and C) or rOCTN2 cDNA (B and D). Cells transfected with pSPORT vector alone were used to measure endogenous transport activity. Uptake of acetyl-L-[3H]carnitine (50 nM) and propionyl-L-[3H]carnitine (50 nM) was measured at pH 7.5 in a medium containing 140 mM NaCl. Uptake in OCTN2-expressing cells was measured in the absence (control) or presence of 5 mM carnitine or TEA.

Electrogenicity of OCTN2-Mediated Transport Process. Because carnitine is a zwitterion at physiological pH, the Na⁺-coupled transport of carnitine mediated by rOCTN2 and hOCTN2 is expected to be electrogenic. We investigated the electrogenic nature of the transport process by assessing the influence of K⁺-induced depolarization of the cells on the Na⁺-coupled transport of carnitine mediated by rOCTN2 and hOCTN2. We have used this approach successfully in our earlier studies to demonstrate the electrogenicity of the transport process mediated by the Na⁺-coupled dicarboxylate transporter (Kekuda et al., 1999). A similar method was used recently by Scaglia et al. (1999) to demonstrate the electrogenicity of the constitutively expressed carnitine transport system in human fibroblasts. In rOCTN2-expressing cells, the transport of carnitine (40 nM) in cells exposed to a physiological concentration of K⁺ (5.6 mM) was 0.20 ± 0.01 pmol/10⁶ cells/2 min, whereas this transport was reduced 30% to 0.14 ± 0.01 pmol/10⁶ cells/2 min in cells exposed to 70 mM K⁺. Under similar conditions, the inhibition of hOCTN2-mediated carnitine transport was 24% (0.75 ± 0.02 pmol/10⁶ cells/2 min in cells exposed to 5.6 mM K⁺ versus 0.57 ± 0.01 pmol/10⁶ cells/2 min in cells exposed to 70 mM K⁺). Thus, the Na⁺-coupled carnitine transport mediated by rOCTN2 and hOCTN2 is electrogenic. The inhibition of transport by depolarization suggests that the transport process is associated with the transport of positive charge into the cells.

Carnitine Transport Function of Human OCTN2 Mutants. We identified recently a mutation in the octn2 gene in a patient with primary systemic carnitine deficiency (Tang et al., 1999). This mutation changes the amino acid at position 478 from P to L in the OCTN2 protein. We have already shown that the carnitine transport activity of the P478L mutant is less than 5% of the activity of the wild-type OCTN2. However, whether the observed loss of transport activity was due to loss of function or due to alterations in the stability of the protein was not determined. Another mutation, L352R, was recently identified in the mOCTN2 in jvs mouse that is genetically defective in the Na⁺-coupled high-affinity carnitine transport system (Lu et al., 1998). The transport function of this mutant was, however, not investigated. To analyze the possible effects of these two mutations, P478L and L352R, on the transport function and protein expression, we introduced these mutations in hOCTN2 by site-directed mutagenesis and measured the carnitine transport function of the resultant mutants. The amino acid at position 352 in hOCTN2 is M instead of L. Therefore, the corresponding mutation in hOCTN2 is M352R. Because anti-hOCTN2 antibodies are not currently available for the assessment of protein expression, we added the c-myc epitope tag to the N terminus of the wild-type and mutant hOCTN2s. Figure 8 describes the transport function of these wild-type and mutant hOCTN2s. In the absence of the c-myc tag, the expression of the wild-type OCTN2 in HRPE cells increased carnitine transport 23-fold compared to that of vector-transfected cells. The OCTN2-specific transport was 3.77 ± 0.19 pmol/10⁶ cells/30 min at 20 nM carnitine. Compared to the activity of the wild-type OCTN2, the activity of the two mutants was reduced >95%. The OCTN2-specific transport was 0.13 ± 0.06 pmol/10⁶ cells/30 min in the case of the P478L mutant and 0.02 ± 0.01 pmol/10⁶ cells/30 min in the case of the M352R mutant. The addition of the c-myc tag to the wild-type OCTN2 reduced the transport activity significantly, but the transport activity could still be measured. The expression of the c-myc-tagged wild-type OCTN2 in HRPE cells increased carnitine transport 7-fold compared to that of vector-transfected cells. The value for carnitine transport that was specific to the tagged wild-type OCTN2 was 1.02 ± 0.03 pmol/10⁶ cells/30 min at 20 nM carnitine. The tagged mutants exhibited <5% of this control activity (0.03 ± 0.01 and 0.02 ± 0.01 pmol/10⁶ cells/30 min for the P478L and M352R mutants, respectively).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Carnitine transport activity of wild-type and mutant hOCTN2s without (A) or with (B) the c-myc epitope tag. HRPE cells were transfected with pSPORT vector, tagged/untagged wild-type (WT) hOCTN2 cDNAs or tagged/untagged mutant hOCTN2 cDNAs. The mutants used were P478L (P  L) and M352R (M  R). Uptake of [3H]carnitine (20 nM) was measured at pH 7.5 in a medium containing 140 mM NaCl.

View larger version (43K): [in this window] [in a new window]   Fig. 9.   Analysis of protein expression of the wild-type and mutant hOCTN2s. HRPE cells were used to express heterologously the c-myc-tagged wild-type hOCTN2 (B), c-myc-tagged M352R mutant hOCTN2 (C), or c-myc-tagged P478L mutant hOCTN2 (D). Cells expressing the untagged wild-type hOCTN2 were used as the negative control (A). Expression of the c-myc epitope-tagged proteins was detected using a monoclonal antibody specific for the epitope and fluorescein isothiocyanate-labeled secondary antibody.

Regional Distribution of OCTN2-Specific mRNA Transcripts in Rat Tissues. We determined the expression of OCTN2 mRNA in various tissues in the rat by in situ hybridization (Fig. 10). The tissues analyzed were the kidney, heart, placenta, and brain. In the kidney, OCTN2 is expressed predominantly in the cortex, with very little expression present in the medulla. In the cortical region, the expression of OCTN2 is evident in the absorptive cells of both proximal and distal tubules. The glomeruli are also positive for OCTN2 expression. In the heart, OCTN2 is expressed throughout the myocardium and the lamina fibrosa of the heart valves. The blood vessels in the heart are also positive for OCTN2 expression. In the placenta, expression is evident throughout the labyrinthine zone and also in the branched villous structures surrounding the lacuna filled with maternal blood. There is also some expression in the visceral yolk sac and chorionic plate. In the brain, OCTN2 expression is abundant throughout the cerebral cortex, in the cornu ammonis pyramidal neurons and with the highest levels of expression in the granular cell layer and the molecular cell layer. Expression is evident also within the Purkinje cell bodies.

View larger version (73K): [in this window] [in a new window]   Fig. 10.   Detection of OCTN2 mRNA transcripts in rat tissues by in situ hybridization. Dark blue or black structures represent positive staining for OCTN2 mRNA. A and B (low power and high power, respectively), hybridization signals in the kidney. Expression is positive in the cortex (Cx) with very little positive signal in the medulla (M). In the cortex,hybridization signals are present in the proximal and distal tubules as well as in the glomeruli (G). C and D (low power and high power, respectively), hybridization signals in the heart. Positive signals are seen in the myocardium (My) and in the lamina fibrosa of the heart valves (AoV). Expression is evident throughout the myocardium as well as in intermuscular arterioles (arrowhead). F-H, hybridization signals in the placenta. E, H&E staining of the placenta. Positive signals are detectable in the labyrinthine zone (Lb) but are absent in the venous sinusoids (Vs). There is also some expression in the chorionic plate (Ch). Positive signals are abundant in the villous structures surrounding the lacuna filled with maternal blood (arrows in G and * in H). I and J (low power and high power, respectively), hybridization signals in the brain. Positive signals are evident in cortex (C), cornu ammonis pyramidal neurons (CA), granular (G), and molecular (ML) layers of cerebellum and the Purkinje cell bodies of cerebellum (arrows).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In this article, we present unequivocal evidence to support OCTN2 as an OCT as well as a carnitine transporter. OCTN2 mediates the transport of organic cations without involving Na⁺ in the transport process, whereas it mediates the transport of carnitine only in the presence of Na⁺. Thus, OCTN2 is a Na⁺-independent OCT and a Na⁺-dependent carnitine transporter. To our knowledge, this is a unique finding among the various Na⁺-coupled organic solute transporters thus far cloned. No other transporter has been shown to transport some substrates in a Na⁺-dependent manner and other substrates in a Na⁺-independent manner. Even within the family of the OCTs to which OCTN2 belongs, no other member of the family exhibits this interesting and intriguing characteristic. We have demonstrated this unique property using OCTN2 cDNAs cloned from three different species. However, there may be significant differences between rOCTN2 and hOCTN2 in their affinities for various organic cations as evidenced from the relative effectiveness of organic cations to inhibit OCTN2-mediated carnitine transport. Our data differ from that reported by Tamai et al. (1998) and Sekine et al. (1998b) in that these studies failed to detect the organic cation transport function of human and rat OCTN2s. The reasons for this discrepancy are not readily apparent at present.

The significance of the carnitine transport function of OCTN2 is readily recognizable, especially in the light of the recent findings, that loss-of-function mutations in hOCTN2 are the cause of the genetic disease primary systemic carnitine deficiency (Tang et al., 1999; Wang et al., 1999). OCTN2 is expressed in several tissues including the heart, skeletal muscle, kidney, intestine, and placenta, but not in the liver. Defects in the function of OCTN2 result in a marked urinary loss of carnitine (because of the inability of the kidneys to reabsorb carnitine), a drastic decrease in plasma levels of carnitine (because of the failure of the endogenous biosynthetic process to compensate for the urinary loss), and a severe decrease in intracellular levels of carnitine in tissues such as the heart and skeletal muscle (because of decreased blood carnitine levels as well as the impairment in the concentrative uptake of carnitine from the blood into the tissues). Consequently, the clinical symptoms of primary systemic carnitine deficiency include progressive cardiomyopathy and skeletal myopathy. The present study has clearly shown the expression of OCTN2 mRNA in the myocardium and in the cortical region of the kidney, where Na⁺-coupled high-affinity carnitine transport has been described using cultured cells or isolated plasma membrane vesicles (Angelini et al., 1992; Kerner and Hoppel, 1998).

The significance of the organic-cation transport function of OCTN2 is not known. Many of the organic cations recognized as substrates by OCTN2 are pharmacologically active and are currently used as therapeutic agents. It is likely that OCTN2 plays a significant role in the disposition and pharmacokinetics of these drugs in the body. The expression of OCTN2 in the kidney may be particularly relevant to this potential function. In the kidney, the Na⁺-coupled high-affinity carnitine transporter is present in the brush border membrane (Rebouche and Mack, 1984; Stieger et al., 1995; Roque et al., 1996). It is this transport system that is defective in primary systemic carnitine deficiency. Even though the localization of OCTN2 to the brush border membrane in the kidney has not yet been demonstrated, it is almost certain that OCTN2 is responsible for carnitine reabsorption across the brush border membrane of renal tubular cells based on the Na⁺-coupled high-affinity carnitine transport function of OCTN2 and the association of loss-of-function mutations in OCTN2 with primary systemic carnitine deficiency. This localization of OCTN2 would suggest that this transporter has the potential to mediate the reabsorption of organic cations in the kidney. Such a function is significant because OCT1 and OCT2, which are OCTs and are expressed in the kidney, are located in the basolateral membrane of the renal tubular cells and are likely to be involved in the renal elimination of organic cations rather than in the reabsorption (Meyer-Wentrup et al., 1998; Urakami et al., 1998). OCT3, another OCT, is also expressed in the kidney (Wu et al., 1998a), but the location of this transporter in the renal tubular cells is not known. Therefore, in addition to the readily recognizable physiological function of OCTN2 in the transport of carnitine, this transporter may have hitherto-unrecognized functions of significant pharmacological and therapeutic relevance. Because functional defects in OCTN2 are the cause of primary systemic carnitine deficiency, questions arise as to the possible consequences of the loss of the organic cation transport function in these patients.

Many of the mutations in OCTN2 so far identified in primary systemic carnitine deficiency result in premature stop codons in the mRNA and consequently produce truncated proteins (Lamhonwah and Tein, 1998; Tang et al., 1999; Wang et al., 1999; Nezu et al., 1999). Until now, there have been only two mutations reported that cause amino acid substitution in OCTN2; they are the P478L mutation in hOCTN2 (Tang et al., 1999) and L352R mutation in mOCTN2 (Lu et al., 1998). Even though we have shown previously that the P478L mutant does not transport carnitine, it was not known whether the absence of transport activity was due to mutation-induced loss of transport function or due to mutation-induced alteration in the stability of the protein. Similarly, even though the L352R mutation was identified in the jvs mouse, which is an animal model for primary systemic carnitine deficiency (Koizumi et al., 1988; Horiuchi et al., 1994), the transport function of this mutant has not been studied. In present study, we introduced these two mutations in hOCTN2 by site-directed mutagenesis and have clearly shown that the mutants have only <5% the carnitine transport activity of the wild-type transporter. Furthermore, we have demonstrated that the loss of transport activity of these two mutants is not due to a decrease in the protein expression. Interestingly, certain mutations in OCTN2 produce differential effects on the carnitine transport function and the organic-cation transport function. The two mutations in hOCTN2 described in the present study, P478L and M352R, cause complete loss of the carnitine transport function. However, when the organic-cation transport function was studied, only the M352R mutant was found to be associated with a complete loss of transport function, whereas the P478L mutant had in fact higher organic cation transport activity than the wild-type transporter (Seth et al., 1999). Detailed structure-function studies have shown that the binding site for carnitine and the binding site for organic cations in OCTN2 exhibit significant overlap but are not identical (Seth et al., 1999). The differential influence of certain mutations in OCTN2 on the carnitine transport function versus the organic cation transport function is of significant clinical relevance to patients with primary carnitine deficiency because there may be considerable differences in the ability of the mutant OCTN2s to transport organic cations in different patients depending on the individual mutation.

Another finding of potential significance reported in the present paper is the ability of OCTN2 to transport the acetyl and propionyl esters of carnitine. These carnitine esters have been shown to possess therapeutic potential in the treatment of a wide variety of neurological disorders (Spagnoli et al., 1991; Brevetti et al., 1992; Lowitt et al., 1995; Hagen et al., 1998; Wiseman and Brogden, 1998). The ability of OCTN2 to transport acetylcarnitine and propionylcarnitine and the widespread expression of this transporter in the central nervous system are therefore relevant to the therapeutic potential of these carnitine esters.

The present study also shows that OCTN2 mRNA is expressed in the labyrinthine zone of the rat placenta. This region of the rodent placenta is responsible for the exchange of nutrients and metabolic waste products between mother and fetus. The expression of OCTN2 mRNA is clearly evident in the cells lining the lacuna filled with maternal blood. This location agrees with the potential function of OCTN2 in the placental transfer of carnitine from the maternal blood into the developing fetus. Available evidence indicates that developing fetuses and neonates are not capable of synthesizing adequate amounts of carnitine (Schiff et al., 1979; Penn et al., 1980; Hahn, 1981; Shenai and Borum, 1984). However, carnitine levels in neonatal and umbilical cord blood are much higher than those in maternal blood (Hahn et al., 1977; Novak et al., 1979). The inability of the developing fetus to synthesize carnitine and the higher concentration of carnitine in the fetal circulation than in the maternal circulation suggest that the placenta is capable of active transfer of carnitine from mother to fetus. Our present study shows that OCTN2 expressed in the placenta is likely to be responsible for this active transfer of carnitine across the placenta. Our earlier studies have shown that a Na⁺-coupled high-affinity carnitine transporter is present in the human placental brush border membrane (Roque et al., 1996) and in human placental choriocarcinoma cells (Prasad et al., 1996).

In summary, we have demonstrated that OCTN2 is not only a carnitine transporter but also an OCT. Although it is easy to understand the physiological relevance of the carnitine transport function of OCTN2, the potential implications of the organic cation transport function of OCTN2 to the pharmacological and therapeutic relevance of this transporter remain to be assessed.