Functional and pharmacological characterization of human Na+-carnitine cotransporter hOCTN2

Carsten A. Wagner1, Ulrike Lükewille1, Simone Kaltenbach1, Ivano Moschen1, Angelika Bröer1, Teut Risler2, Stefan Bröer1, and Florian Lang1

Departments of 1 Physiology I and 2 Internal Medicine, University of Tübingen, 72076 Tübingen, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
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L-Carnitine is essential for the translocation of acyl-carnitine into the mitochondria for beta -oxidation of long-chain fatty acids. It is taken up into the cells by the recently cloned Na+-driven carnitine organic cation transporter OCTN2. Here we expressed hOCTN2 in Xenopus laevis oocytes and investigated with two-electrode voltage- clamp and flux measurements its functional and pharmacological properties as a Na+-carnitine cotransporter. L-carnitine transport was electrogenic. The L-carnitine-induced currents were voltage and Na+ dependent, with half-maximal currents at 0.3 ± 0.1 mM Na+ at -60 mV. Furthermore, L-carnitine-induced currents were pH dependent, decreasing with acidification. In contrast to other members of the organic cation transporter family, hOCTN2 functions as a Na+-coupled carnitine transporter. Carnitine transport was stereoselective, with an apparent Michaelis-Menten constant (Km) of 4.8 ± 0.3 µM for L-carnitine and 98.3 ± 38.0 µM for D-carnitine. The substrate specificity of hOCTN2 differs from rOCT-1 and hOCT-2 as hOCTN2 showed only small currents with classic OCT substrates such as choline or tetraethylammonium; by contrast hOCTN2 mediated transport of betaine. hOCTN2 was inhibited by several drugs known to induce secondary carnitine deficiency. Most potent blockers were the antibiotic emetine and the ion channel blockers quinidine and verapamil. The apparent IC50 for emetine was 4.2 ± 1.2 µM. The anticonvulsant valproic acid did not induce a significant inhibition of carnitine transport, pointing to a different mode of action. In summary, hOCTN2 mediates electrogenic Na+-dependent stereoselective high-affinity transport of L-carnitine and Na+. hOCTN2 displays transport properties distinct from other members of the OCT family and is directly inhibited by several substances known to induce systemic carnitine deficiency.

carnitine transport; human sodium-driven organic cation transporter 2; pharmacology; secondary carnitine deficiency


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

3-HYDROXY-4-N-TRIMETHYLAMINOBUTYRIC acid (L-carnitine) is essential for the translocation of acyl-carnitine esters into the mitochondria for beta -oxidation of long-chain fatty acids. Moreover, L-carnitine is important for regenerating the cytosolic coenzyme A pools (3, 21). Even though L-carnitine is synthesized by some cells, particularly skeletal and heart muscle cells are not able to synthesize L-carnitine and are therefore fully dependent on the uptake of L-carnitine across the plasma membrane (3, 21). Carnitine transport is mediated by an almost ubiquitious high-affinity Na+-dependent transport mechanism (2, 4, 7, 8, 12, 21, 22, 28, 36). Furthermore, the transport mechanism allows the intestinal absorption and renal reabsorption of L-carnitine. Inborn or acquired defects of carnitine transport lead to primary or secondary systemic carnitine deficiency (21). The primary carnitine deficiency syndrome is characterized by the impaired intestinal and muscular uptake and increased renal loss of L-carnitine (21, 23, 31). The resulting reduction of carnitine levels lead to muscular weakness, progressive skeletal and heart myopathy, the failure to thrive in children, and hypoketotic hypoglycemic encephalopathy (21). Without carnitine-replacement therapy, the defect eventually leads to the death of the patients. Besides a number of described enzymopathies, some forms of secondary carnitine deficiency may be caused by interaction of drugs with carnitine transport or metabolism. Clinically important are the antibiotics emetine and pivalic acid and the anticonvulsant valproic acid (1, 10, 16, 30). Long-term treatment with these drugs often leads, particularly in children, to the development of secondary carnitine deficiency. The mechanism of interaction between these substances and carnitine transport or metabolism has remained elusive.

Recently, the rat, murine, and human L-carnitine transporters [Na+-driven organic cation transporter 2 (OCTN2)] were cloned (25, 26, 29, 35). OCTN2 is widely expressed in human tissues such as heart and skeletal muscle, proximal and distal renal tubules, some brain areas, the placenta, and the small intestine (25, 26, 29, 34, 35). OCTN2 belongs to the large superfamily of organic cation transporters (OCT) and shares high homology to most transporters from this family (12). The highest homology is found with OCTN1, which is thought to function as an organic cation/proton exchanger (36). Mutations in hOCTN2 were found in patients with primary carnitine deficiency (18, 24, 33) and in the juvenile visceral steatosis (JVS) mouse line (17). Whereas the properties of OCTN2 as an organic cation transporter have been investigated (35), only little is known about the functional and pharmacological properties as a Na+-carnitine cotransporter. Moreover, OCTN2 has the unique feature of being the only Na+-dependent transporter within the OCT superfamily. To study the functional properties of hOCTN2, we expressed the transporter in Xenopus laevis oocytes and examined the Na+-carnitine cotransport properties with the two-electrode-voltage- clamp technique and tracer fluxes. As a result, hOCTN2 conveys electrogenic high-affinity cotransport of Na+ and L-carnitine. hOCTN2 functions as symporter, but there is no evidence for exchange with organic cations or protons. Furthermore, carnitine transport by hOCTN2 is inhibited by emetine and pivalic acid but not by valproic acid, drugs known to interfere with carnitine uptake or metabolism.


    MATERIAL AND METHODS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cloning of hOCTN2. hOCTN2 cDNA was cloned by high-fidelity RT-PCR. For this purpose, mRNA, isolated from cultured HeLa cells, was reverse transcribed with Superscript II RT. For PCR two primers were constructed that flanked the coding sequence of OCTN2 (accession no. AB015050). Oligonucleotide hOCTN2s (5' CTC TGT GGG CCT CTG AGG G 3') corresponded to bases 101-119, and oligonucleotide hOCTN2a (5' CTC CCT TAC TGG AAG CGA TG 3') corresponded to bases 1798-1817 of the hOCTN2 cDNA sequence (29). The cDNA was amplified in a 30-cycle PCR reaction (45 min, 98°C-45 min, 50°C-480 min, 72°C) by using Pfu-Polymerase (Promega, Mannheim, Germany) with 1 M formamide and 5% DMSO (final concentrations) as additives. The amplified band was extracted from the agarose gel and ligated into the Srf I site of the cloning vector PCRscript (Stratagene, Heidelberg, Germany). For oocyte expression, the cloned cDNA was excised with EcoR I and Not I, blunted with T4 DNA polymerase and cloned into the Sma I site of vector pGEM-He-Juel (16). The orientation of the cDNA was verified by digestion with restriction enzymes. For in vitro transcription, plasmid DNA was linearized with Not I and transcribed in vitro with T7-RNA polymerase in the presence of a cap analog. The protocol supplied with the polymerase was followed, except that all nucleotides and the cap analog were used at twofold concentrations (1 mM) to increase the yield of cRNA. Template plasmids were removed by digestion with RNase-free DNase. The cRNA was purified by phenol/chloroform extraction followed by precipitation with 1/2 vol of 7.5 M ammonium acetate and 2 vol of ethanol to remove unincorporated nucleotides. After determination of the amount of cRNA by measuring absorption at 260 nm, the integrity of the transcript was verified by denaturing agarose gel electrophoresis.

Preparation of X. laevis oocytes. cRNA encoding the human Na+-carnitine cotransporter hOCTN2, the rat organic cation transporter rOCT1, and the human organic cation transporter hOCT2, were synthesized in vitro as previously described (5, 6). Dissection of X. laevis ovaries and collection and handling of the oocytes have been described in detail elsewhere (5). Oocytes were injected with either 25 ng cRNA hOCTN2 or 15 ng rOCT1 or hOCT2 · 50 nl water-1 · oocytes-1; noninjected oocytes served as controls. All experiments were performed at room temperature 3-8 days after injection.

Electrophysiology. Two-electrode voltage-clamp recordings were performed at a holding potential of -60 mV if not otherwise specified. The data were filtered at 10 Hz and recorded with a MacLab digital-to-analog converter and software for data acquisition and analysis (AD Instruments, Castle Hill, Australia). The external control solution (superfusate/ND-96) contained (in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4. For experiments studying the dependence from external sodium, sodium was replaced by N-methyl-D-glucamine. When necessary, osmolarity was adjusted by adding glucose. Carnitine and all other substrates were added to the solutions at the indicated concentrations. The final solutions were titrated to the pH indicated by using HCl or KOH. The flow rate of the superfusion was 20 ml/min, and a complete exchange of the bath solution was reached within ~10 s. The currents given are the maximal values measured during a 30-s substrate superfusion. Usually, the maximal current was reached after 25-s substrate superfusion. All chemicals were obtained from Sigma and were of analytic grade.

pH-sensitive electrodes. pH-sensitive electrodes were made and calibrated as described previously (30). In brief, borosilicate electrodes were pulled, silanized, (hexamethyldisilazane, Fluka Chemicals) and baked at 200°C for 15 min. A column of H+ cocktail (hydrogen ionophore II-cocktail A, Fluka Chemicals) of ~300 µm in length was established at the tip of the electrode. The electrode was backfilled with a solution of 100 mM KCl buffered with 10 mM HEPES at pH 7.0. The electrode was calibrated by using solutions with pH 6.0, 7.0, and 8.0. Only electrodes with a linear slope >50 mV/pH unit and stable calibration before and after the experiment were used. Signals were recorded with an electrometer (WPI model FD223). On the basis of the calibration curve for the pH-sensitive electrode, the intracellular pH of oocytes was calculated as the difference between the membrane potential in millivolts measured simultaneously with a 3 M KCl microelectrode and the potential of the pH-sensitive electrode.

Flux measurements. For each determination, groups of seven cRNA-injected or noninjected oocytes were washed twice with 4 ml ND-96 buffer. They were then incubated at room temperature in a 5-ml polypropylene tube containing 100 µl of the same buffer containing 10 kBq L-[3H]carnitine or [14C]betaine (obtained from Biotrend, Cologne, Germany) plus unlabeled substrates as indicated. Transport was stopped after the appropiate interval by washing oocytes three times with 4 ml ice-cold ND-96 buffer. Single oocytes were placed in scintillation vials and lysed by addition of 200 µl 10% SDS. After lysis, 3 ml of scintillation fluid were added, and the radioactivity was determined by liquid scintillation counting. [14C]betaine and [3H]carnitine uptake were linear over at least 30 min. To investigate trans stimulation of the transporter, seven oocytes were preloaded with 10 µM [3H]carnitine for 30 min. Subsequently, oocytes were washed three times with 4 ml ice-cold incubation buffer followed by an addition of 1 ml incubation buffer, supplemented with the indicated substrates, at room temperature. At the indicated times 100-µl aliquots were removed, and the released radioactivity was determined. The released radioactivity was divided by the number of oocytes and integrated over time.

Calculations. Curves were obtained by using the Hill equation I = Imax * [Sn]/([Sn] + Kmn) where n and [S] are the Hill coefficient and the substrate concentration, respectively, Imax is the extrapolated maximal current (I) , and Km is the apparent concentration needed for half-maximal current. Curves were fitted for data from each oocyte, and the values obtained for Km and Imax were used for statistical analysis. Data are provided as means ± SE, and n represents the number of oocytes investigated. The magnitude of the induced currents varied two- to fivefold, depending on the time period after cRNA injection and on the batch of oocytes (from different animals). Therefore, throughout the paper we show experimental data obtained on the same day for each specific set of experiments. All experiments were repeated with at least two to three batches of oocytes; in all repetitions qualitatively similar data were obtained. All data were tested for significance by using the paired Students t-test and ANOVA, and only results with P < 0.05 were considered as statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

Superfusion of X. laevis oocytes expressing the human Na+-carnitine cotransporter hOCTN-2 with 1 mM L-carnitine caused an inward current of -10.3 ± 0.9 nA at -50 mV holding potential (n = 5) in the voltage-clamp modus. In the current-clamp modus, this was paralleled by a depolarization of 5.2 ± 0.7 mV (n = 5). The L-carnitine (30 µM and 1 mM, respectively)-induced current was voltage dependent, decreasing with depolarization (Fig. 1A). However, the voltage dependence was more pronounced at the lower L-carnitine concentration. L-carnitine-induced currents (1 mM) were strictly Na+ dependent and followed a hyperbolic curve, with an apparent Km for Na+ of 2.3 ± 0.6 mM at -60 mV and 2.1 ± 1.8 mM at -90 mV holding potential (n = 7). Because 10 mM Na+ almost saturated the transporter we performed further kinetics at -60 mV with more Na+ concentrations between 0 and 10 mM. This gained an apparent Km for Na+ of 0.4 ± 0.1 mM (n = 5, Fig. 1B). The Hill coefficient under all conditions was close to one, suggesting the cotransport of one Na+ per carnitine.


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Fig. 1.   A: voltage dependence of L-carnitine-induced currents transport. At higher L-carnitine concentrations the voltage dependence is less pronounced. B: Na+ dependence of L-carnitine-induced currents at -60-mV holding potential. The curve was obtained by fitting the data to the Hill equation. Imax, the extrapolated maximal current; Km, Michaelis-Menten coefficient.

L-Carnitine-induced currents exhibited saturable kinetics that followed the Hill equation with an apparent affinity of 4.8 ± 0.3 µM at -60 mV holding potential and 2.5 ± 0.4 µM at -90 mV holding potential (n = 7, Fig. 2A). The Hill coefficient was 1.3 ± 0.1 and 1.1 ± 0.2, respectively, pointing to the transport of one carnitine per transport cycle. The transport of carnitine was stereoselective. The maximum current for both stereoisomers was similar, i.e., -14.8 ± 0.4 nA for D-carnitine and -12.8 ± 0.8 nA for L-carnitine. However, the apparent affinity for D-carnitine was much lower than that for the L-stereoisomer with 98.3 ± 38.0 µM (Fig. 2B, n = 5).


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Fig. 2.   Concentration dependence and stereoselectivity of L- and D-carnitine-induced currents. A: concentration dependence curve for L-carnitine at -60 mV. B: concentration dependence curve for D-carnitine-induced currents as obtained by fitting the data to the Hill equation.

The pH dependence was determined to investigate the possibility of an organic cation or H+ antiport mechanism of hOCTN2, which has been proposed for the closely related putative organic cation transporter OCTN1 (34, 36). Carnitine-induced currents decreased with extracellular acidification by 45.1 ± 5.4% from -17.7 ± 2.3 nA at pH 8.5 to -9.5 ± 2.1 nA at pH 6.5 (Fig. 3A, n = 5). In addition, intracellular pH electrodes were used to measure whether the substrate-induced current was paralleled by a change in the intracellular pH. Neither choline, TEA, nor carnitine at 1 mM for 15 min caused a change in intracellular pH. To test further for a possible H+ antiport mechanism, the intracellular pH was acidified by using 25 mM HCO3-. During this treatment the intracellular pH acidified from pH 7.12 ± 0.03 to pH 6.73 ± 0.04 after 10 min, and the L-carnitine-induced current (1 mM) was reduced by 22.3 ± 4.5% (Fig. 3B, n = 4). Taken together it seemed unlikely that uptake of carnitine is accompanied by an efflux of protons but that the transporter is sensitive to acidification of the intra- as well as the extracellular milieu. L-Carnitine-induced currents were not affected by the presence of other organic cations on the trans side. Preincubation with 1 mM choline for 10 min or overnight did not alter L-carnitine currents (n = 4).


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Fig. 3.   pH dependence of L-carnitine-induced currents through human organic cation transporter 2 (hOCTN2). A: dependence from external pH. Decreasing external pH decreased L-carnitine currents. B: internal acidification of oocytes with 25 mM HCO3- from pH 7.1 to 6.7 also decreased L-carnitine currents.

Several substrates, either precursors of carnitine synthesis as methionine or lysine, or derivatives or chemically related substrates such as D-carnitine, palmitoyl-DL-carnitine, beta -hydroxybutyrate, gamma -aminobutyric acid, betaine, or potential blockers of L-carnitine transport such as emetine, valproic acid, and pivalic acid, and substrates of organic cation transporters such as TEA, choline, verapamil, and quinidine were tested as to whether they could induce transporter currents or inhibit L-carnitine-induced currents (Table 1, n = 5). Besides L-carnitine, only D-carnitine (see above), lysine, TEA, choline, betaine, and methionine evoked currents in hOCTN2-expressing oocytes significantly different from control oocytes. In comparison, in X. laevis oocytes expressing the organic cation transporters rOCT1 and hOCT2, carnitine did not induce any currents whereas choline and TEA produced significant inward currents (30-60 nA) as previously described (data not shown) (5, 6). Furthermore, we tested some compounds for their ability to inhibit L-carnitine-induced currents. Among these substances we tested the pharmacologically important substrates emetine, pivalic acid, and valproic acid as their use is often accompanied and limited by secondary systemic carnitine deficiency (1, 11, 14, 21, 23, 30). The most potent inhibitors of L-carnitine-induced currents (1 mM) were the antibiotic emetine (by 74.6 ± 1.8%) and the L-type calcium channel blocker verapamil (by 72.3 ± 3.3%). The inhibition by the antibiotic pivalic acid (500 µM) was small but significant with 36.3 ± 6.8% (Fig. 4A, n = 4). To test whether the inhibition of L-carnitine-induced currents reflected inhibition of carnitine transport rather than inhibition of unspecific carnitine-induced conductances, flux measurements with L-[3H]carnitine were performed. As shown in Fig. 4B, 500 µM emetine, quinidine, betaine, and cysteine strongly inhibited the uptake of 50 µM L-[3H]carnitine whereas the inhibition by pivalic acid was only moderate (n = 7). As emetine plays an important role in secondary carnitine deficiency, we determined the IC50 value for inhibition, which was found to be 4.1 ± 1.2 µM (n = 7, Fig. 4C). Because both emetine and betaine induced small currents in OCTN2-expressing oocytes and therefore were likely to be substrates of the transporter, we tested whether these and other substances were able to elicit counterexchange of preloaded [3H]carnitine. Addition of 1 mM carnitine or betaine to the medium led to a rapid release of labeled carnitine (Fig. 5, n = 7 for each condition). Emetine and cysteine, however, induced only a very small release, suggesting that both are not substrates of the transporter. Transport of betaine was further directly demonstrated by measuring the uptake of [14C]betaine. Oocytes expressing OCTN2 took up [14C]betaine (82 µM, concentration caused by carrier) at a rate of 36.7 ± 7.5 pmol/30 min (n = 7), which was significantly faster than carnitine transport (13 ± 0.9 pmol/30 min at a substrate concentration of 10 µM, n = 7). Noninjected oocytes took up 1.6 ± 0.1 pmol/30 min betaine (n = 7) and 0.9 ± 0.13 pmol/30 min L-carnitine (n = 7) under the same conditions.

                              
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Table 1.   Substrate specificity and inhibition of hOCTN2



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Fig. 4.   A: inhibition of L-carnitine (1 mM)-induced currents by various substrates (500 µM). B: inhibition of L-[3H]carnitine (50 µM) uptake by several inhibitors at 500 µM concentration. C: the IC50 value for emetine as determined by inhibition of L-[3H]carnitine (50 µM) uptake.



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Fig. 5.   Release of preloaded L-[3H]carnitine from hOCTN2-expressing oocytes by counterexchange. Addition of 1 mM L-carnitine and 1 mM betaine stimulated release of L-[3H]carnitine whereas 1 mM L-cysteine, emetine or control solution did not (n = 7 for each condition). cpm, Counts/min.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study is the first report demonstrating electrogenicity of Na+ -dependent carnitine transport through hOCTN2. It displayed all characteristics of Na+-dependent high-affinity carnitine transport as reported from various tissues (2, 4, 7, 8, 11, 12, 22, 26, 28, 37). Taken together with the fact that hOCTN2 is the only Na+-dependent transporter within the OCT superfamily (13, 14), the most remarkable feature of this transporter is the very high affinity for Na+ with an apparent Km of ~0.5 mM. It may be speculated that Na+ does not only supply the driving force for transport but may also be needed to create the positive charge for the binding of carnitine to an ancient organic cation binding site. Recent data from Seth et al. (27) indicate that organic cations and carnitine may have different binding sites in the protein that may resemble two different cation binding sites, a true and an ancient one (27). At higher carnitine concentrations the voltage dependence was less pronounced than at lower carnitine concentrations, which might be caused by a displacement of Na+ from its binding site.

The substrate specificity of OCTN2 partially overlaps with the related organic cation transporters OCT-1 and OCT-2. No carnitine-induced currents were found with rat OCT-1 and human OCT-2 but small choline and TEA currents with hOCTN2. However, the choline- and TEA-induced currents were smaller than the carnitine-induced currents, in good agreement with a previous study showing lower uptake rates for TEA than carnitine with the hOCTN2 (34, 35). This points to a specialized function of hOCTN2 as a Na+-dependent carnitine transporter. We found also transport of betaine by hOCTN2 but not of cysteine. Accordingly, betaine has been shown to act as an inhibitor of carnitine transport (24). This may be interesting as recently a non- betaine-sensitive carnitine uptake system in the blood-brain barrier has been described, suggesting carnitine transport systems other than OCTN2 (18). Cysteine, which is not transported by OCTN2, may inhibit the transporter by reacting with -SH groups.

OCTN1, the closest related OCT family member, is thought to function as an organic cation/proton exchanger (35, 36). Therefore, we investigated the transport mechanism of hOCTN2. The size of the L-carnitine currents depended on extracellular pH. However, any involvement of protons in the transport mechanism can be most likely ruled out as no evidence for H+ or organic cation co- or antiport could be found. No intracellular acidification was observed on application of choline, TEA, or carnitine, and intracellular acidification even decreased carnitine currents. The electrogenic nature of the transport mechanism in addition is not compatible with a Na+/H+ exchange coupled to carnitine transport. Accordingly, our results suggest a symport mechanism for Na+ and carnitine rather than an exchange mechanism. The equivalent decrease in L-carnitine currents caused by extracellular as well as intracellular acidification suggests an allosteric regulation of carnitine transport by protons.

Several drugs are known to induce secondary systemic carnitine deficiency. Among these drugs, the antibiotics emetine and pivalic acid and the anticonvulsant valporic acid cause the strongest effects (1, 10, 15, 21, 23, 30). Here, we tested these drugs on L-carnitine transport and found a strong inhibition by emetine and a moderate inhibition by pivalic acid. No effect was observed for valproic acid. Emetine, an oral emetic and antamoebic, has been shown to cause carnitine deficiency and myopathy in patients (15, 21). However, the underlying mechanism has not been clear as yet (15). Our results indicate that emetine is a potent inhibitor of L-carnitine uptake via the Na+-carnitine transporter, with a similar affinity as L-carnitine. Emetine, however, is not itself a substrate of the transporter. The small currents induced by emetine and the very small counterexchange activity, however, suggest that emetine could be regarded as a marginally transported substrate that therefore should competitively inhibit carnitine transport. Emetine-induced inhibition of carnitine transport would cause intracellular carnitine depletion, with subsequent disruption of mitochondrial beta -oxidation. Pivalic acid is contained as pivaloyloximethyl ester in several antibiotics routinely used for the treatment of respiratory and urinary tract infections and serves to facilitate intestinal absorption. After uptake it is subsequently liberated (1, 10). In patients, pivalic acid leads to an increase in urinary pivaloylcarnitine, acylcarnitine, and free carnitine excretion and a fall in serum and intracellular carnitine levels (1, 10). Patients with long-term treatment with pivalic acid-containing drugs are reported to display symptoms similar to systemic carnitine deficiency (1, 10). The effects of pivalic acid have been partially ascribed to the production of abnormal organic acids, affecting carnitine transport and metabolism (10). The small inhibition observed here was found only at concentrations much higher than usually achieved under therapy, indicating that the direct inhibition may not play an important role in vivo (10).

The anticonvulsant valproic acid had no effect on L-carnitine-induced currents, which is in agreement with previous studies in cell cultures showing an inhibition of L-carnitine transport only after an incubation for 24 h (30). This may indicate that valproic acid interferes with the regulation or biosynthesis of carnitine transporter itself or that metabolites of valproic acid are responsible for the inhibition of L-carnitine transport observed in cell culture. Similar results with respect to emetine and valproic acid have been found by Ohashi et al. (20) during the course of our work.

In summary, when expressed in X. laevis oocytes hOCTN2 operates as an electrogenic high-affinity Na+-carnitine cotransporter. Na+ and carnitine are cotransported without the involvement of any other ions or substrates in the transport mechanism. The transport of Na+/carnitine is inhibited by the antibiotic emetine, explaining the side effect of this drug on carnitine metabolism. hOCTN2 may therefore play also an important role in the absorption of drugs in the small intestine and kidney (14, 38).


    ACKNOWLEDGEMENTS

The technical support of B. Noll is acknowledged. We thank H. Koepsell for the critical reading of the manuscript.


    FOOTNOTES

This study was supported by Deutsche Forschungsgemeinschaft Grant La 315/4-3 (to F. Lang), a grant from the Federal Ministry of Education, Science, Research and Technology (Fö. 01KS9602), and the Interdisciplinary Center for Clinical Research (IZKF), Tübingen (to F. Lang and C. A. Wagner). C. A. Wagner is a Feodor Lynen fellow of the Humboldt-Stiftung.

Address for reprint requests and other correspondence: C. A. Wagner, Dept. of Cellular and Molecular Physiology, School of Medicine, Yale Univ., 333 Cedar St., New Haven, CT 06520 (E-mail: wagnerca{at}hotmail.com).

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. §1734 solely to indicate this fact.

Received 14 December 1999; accepted in final form 23 May 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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