Functional Domains in the Carnitine Transporter OCTN2, Defective in Primary Carnitine Deficiency

Cristina Amat Di San Filippo,Yuhuan Wang,Nicola Longo

doi:10.1074/jbc.m307911200

Cristina Amat Di San Filippo, Yuhuan Wang + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m307911200

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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.

Highlights

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy
The quantitative decrease in carnitine transport obtained with R341L, L409W, and T429I was similar to that observed with CHIM-9 in which the whole region containing amino acids 341– 453 of OCTN1 was substituted in
Restoration of Carnitine Transport by the Introduction of A339R, W407L, and I427T Substitutions in OCTN1 in the Chimeric Transporter CHIM-9 —Because the substitution of only 3 residues in OCTN2 with the corresponding residues of OCTN1 had such a profound effect on carnitine transport, we evaluated whether the introduction of OCTN2 residues in the portion of OCTN1 contained in CHIM-9 was capable of restoring carnitine transport

Summary

Introduction

Primary carnitine deficiency is an autosomal recessive disorder of fatty acid oxidation characterized by hypoketotic hypoglycemia and skeletal and cardiac myopathy. These studies indicate that multiple domains of the OCTN2 transporter are required for carnitine transport and identify transmembrane residues important for carnitine recognition.

Results

Conclusion