Neuropediatrics 2006; 37 - PS1_3_2
DOI: 10.1055/s-2006-943573

NOVEL LOCALIZATION OF OCTN1, AN ORGANIC CATION/CARNITINE TRANSPORTER, TO MAMMALIAN MITOCHONDRIA: IMPLICATIONS FOR CARNITINE-RESPONSIVE ENCEPHALOPATHY AND MYOPATHY

AM Lamhonwah 1, I Tein 1
  • 1Hospital for Sick Children, Toronto, ON, Canada

Objectives: To determine the cellular localization of the organic cation/carnitine transporter, OCTN1. Carnitine is a zwitterion essential for the ß-oxidation of fatty acids.

Methods: We made GFP- and RFP-human OCTN1 cDNA constructs and transfected them into several mammalian cell lines (e.g.HepG2, human skin fibroblasts, HEK 293, MEF-3T3). We immunostained the GFP-hOCTN1 transfected cells with different intracellular markers and then applied confocal fluorescent microscopy. We measured L-[3H]-carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2. We also measured uptake in mutant human skin fibroblasts having <1% of carnitine acylcarnitine translocase (CACT) activity (alternate mitochondrial carnitine transporter).

Results: Immunostaining of GFP-hOCTN1 transfected cells with different intracellular markers and confocal fluorescent microscopy analysis demonstrated mitochondrial expression of OCTN1. There was striking co-localization of an RFP-hOCTN1 fusion protein and a mitochondrial-GFP marker construct in transfected MEF-3T3 and no co-localization of GFP-hOCTN1 in transfected human skin fibroblasts with other intracellular markers. L- [3H]-carnitine uptake in freshly isolated mitochondria of GFP-hOCTN1 transfected HepG2 demonstrated a Km of 422µM and western blot with an anti-GFP antibody identified a GFP-hOCTN1 fusion protein (90kDa). We showed endogenous expression of native OCTN1 in HepG2 mitochondria with anti-GST-hOCTN1 antibody. Further, we definitively confirmed intact L-[3H]-carnitine uptake (Km 1324µM) solely attributable to OCTN1 in isolated mitochondria of mutant human skin fibroblasts having <1% of CACT activity.

Conclusion: This mitochondrial localization suggests an important yet different role for OCTN1 from other OCTN family members in intracellular carnitine homeostasis. The predicted clinical phenotype for a defect in the OCTN1 transporter may parallel the myopathic and episodic hypoketotic hypoglycemic encephalopathy phenotypes of the high-affinity plasmalemmal carnitine transporter, OCTN2 and CACT defects.