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Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency.
J Biol Chem. 2003 Nov 28; 278(48):47776-84.JB

Abstract

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the Na+-dependent organic cation transporter, OCTN2. To define the domains involved in carnitine recognition, we evaluated chimeric transporters created by swapping homologous domains between OCTN1, which does not transport carnitine, and OCTN2. Substitution of the C terminus of OCTN2 (amino acid residues 342-557) with the corresponding residues of OCTN1 completely abolished carnitine transport. The progressive substitution of the N terminus of OCTN2 with OCTN1 resulted in a decrease in carnitine transport associated with a progressive increase in the Km toward carnitine from 3.9 +/- 0.5 to 141 +/- 19 microM. The largest drop in carnitine transport (and increase in Km toward carnitine) was observed with the substitution of residues 341-454 of OCTN2. An additional chimeric transporter (CHIM-9) in which only residues 341-454 of OCTN2 were substituted by OCTN1 had markedly reduced carnitine transport, with an elevated Km toward carnitine (63 +/- 5 microM). Site-directed mutagenesis and introduction of residues nonconserved between OCTN1 and OCTN2 in the OCTN2 cDNA indicated that the R341A, L409W, L424Y, and T429I substitutions significantly decreased carnitine transport. Single substitutions did not increase the Km toward carnitine. By contrast, the combination of three of these substitutions (R341W + L409W + T429I) greatly decreased carnitine transport and increased the Km toward carnitine (20.2 +/- 4.5 microm). The Arg-341, Leu-409, and Thr-429 residues are all located in predicted transmembrane domains. Involvement of these residues in carnitine transport was further supported by the partial restoration of carnitine transport by the introduction of these OCTN2 residues in the OCTN1 portion of CHIM-9. These studies indicate that multiple domains of the OCTN2 transporter are required for carnitine transport and identify transmembrane residues important for carnitine recognition.

Authors+Show Affiliations

Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, Utah 84132, USA.No affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

14506273

Citation

Amat di San Filippo, Cristina, et al. "Functional Domains in the Carnitine Transporter OCTN2, Defective in Primary Carnitine Deficiency." The Journal of Biological Chemistry, vol. 278, no. 48, 2003, pp. 47776-84.
Amat di San Filippo C, Wang Y, Longo N. Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency. J Biol Chem. 2003;278(48):47776-84.
Amat di San Filippo, C., Wang, Y., & Longo, N. (2003). Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency. The Journal of Biological Chemistry, 278(48), 47776-84.
Amat di San Filippo C, Wang Y, Longo N. Functional Domains in the Carnitine Transporter OCTN2, Defective in Primary Carnitine Deficiency. J Biol Chem. 2003 Nov 28;278(48):47776-84. PubMed PMID: 14506273.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency. AU - Amat di San Filippo,Cristina, AU - Wang,Yuhuan, AU - Longo,Nicola, Y1 - 2003/09/23/ PY - 2003/9/25/pubmed PY - 2004/1/13/medline PY - 2003/9/25/entrez SP - 47776 EP - 84 JF - The Journal of biological chemistry JO - J Biol Chem VL - 278 IS - 48 N2 - Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. It is caused by mutations in the Na+-dependent organic cation transporter, OCTN2. To define the domains involved in carnitine recognition, we evaluated chimeric transporters created by swapping homologous domains between OCTN1, which does not transport carnitine, and OCTN2. Substitution of the C terminus of OCTN2 (amino acid residues 342-557) with the corresponding residues of OCTN1 completely abolished carnitine transport. The progressive substitution of the N terminus of OCTN2 with OCTN1 resulted in a decrease in carnitine transport associated with a progressive increase in the Km toward carnitine from 3.9 +/- 0.5 to 141 +/- 19 microM. The largest drop in carnitine transport (and increase in Km toward carnitine) was observed with the substitution of residues 341-454 of OCTN2. An additional chimeric transporter (CHIM-9) in which only residues 341-454 of OCTN2 were substituted by OCTN1 had markedly reduced carnitine transport, with an elevated Km toward carnitine (63 +/- 5 microM). Site-directed mutagenesis and introduction of residues nonconserved between OCTN1 and OCTN2 in the OCTN2 cDNA indicated that the R341A, L409W, L424Y, and T429I substitutions significantly decreased carnitine transport. Single substitutions did not increase the Km toward carnitine. By contrast, the combination of three of these substitutions (R341W + L409W + T429I) greatly decreased carnitine transport and increased the Km toward carnitine (20.2 +/- 4.5 microm). The Arg-341, Leu-409, and Thr-429 residues are all located in predicted transmembrane domains. Involvement of these residues in carnitine transport was further supported by the partial restoration of carnitine transport by the introduction of these OCTN2 residues in the OCTN1 portion of CHIM-9. These studies indicate that multiple domains of the OCTN2 transporter are required for carnitine transport and identify transmembrane residues important for carnitine recognition. SN - 0021-9258 UR - https://www.unboundmedicine.com/medline/citation/14506273/Functional_domains_in_the_carnitine_transporter_OCTN2_defective_in_primary_carnitine_deficiency_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0021-9258(20)75805-9 DB - PRIME DP - Unbound Medicine ER -