Abstract
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 microM, 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 gamma-butyrobetaine with high affinity and by glycinebetaine with lower affinity, whereas choline, beta-hydroxybutyric acid, gamma-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.
Highlights
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]
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)
TEA is a good substrate of OCTN1 [17], no significant increase of [14C]TEA uptake was observed in human OCTN2-transfected cells
Summary
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). 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. The criterion of significance was taken to be p Ͻ 0.05
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