Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency
Functional domains in the carnitine transporter OCTN2, defective in primary carnitine deficiency
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DOI：
10.1074/jbc.m307911200
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发表时间：
2003-11-28
影响因子：
4.8
通讯作者：
Longo, N
中科院分区：
文献类型：
--
作者：
di San Filippo, CA;Wang, YH;Longo, N
原发性肉碱缺乏症是一种常染色体隐性遗传的脂肪酸氧化疾病，其特征是低酮性低血糖以及骨骼肌和心肌病。它是由 Na+ 依赖性有机阳离子转运蛋白 OCTN2 突变引起的。为了定义参与肉碱识别的结构域，我们评估了通过在不转运肉碱的 OCTN1 和 OCTN2 之间交换同源结构域而创建的嵌合转运蛋白。用 OCTN1 的相应残基取代 OCTN2 的 C 末端（氨基酸残基 342-557）完全消除了肉毒碱转运。 OCTN2 的 N 末端逐渐被 OCTN1 取代，导致肉毒碱转运减少，与肉毒碱 Km 从 3.9 +/- 0.5 逐渐增加到 141 +/- 19 muM 相关。通过替换 OCTN2 的残基 341-454，观察到肉碱转运的最大下降（以及朝向肉碱的 Km 增加）。另一种嵌合转运蛋白（CHIM-9），其中仅 OCTN2 的残基 341-454 被 OCTN1 取代，其肉毒碱转运显着减少，对肉毒碱的 Km 升高（63 +/- 5 muM）。定点诱变以及在 OCTN2 cDNA 中引入 OCTN1 和 OCTN2 之间的非保守残基表明 R341A、L409W、L424Y 和 T429I 取代显着降低了肉碱转运。单一取代不会增加对肉碱的 Km。相比之下，这些取代中的三个（R341W + L409W + T429I）的组合大大减少了肉碱转运并增加了对肉碱的Km（20.2 +/- 4.5 muM）。 Arg-341、Leu-409 和 Thr-429 残基均位于预测的跨膜结构域中。通过在 CHIM-9 的 OCTN1 部分中引入这些 OCTN2 残基，部分恢复肉碱运输，进一步支持了这些残基参与肉碱运输。这些研究表明，肉毒碱转运需要 OCTN2 转运蛋白的多个结构域，并鉴定出对于肉毒碱识别很重要的跨膜残基。
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 muM. 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 muM). 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 muM). 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.