Amat di San Filippo Cristina, Longo Nicola
Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, Utah 84132, USA.
J Biol Chem. 2004 Feb 20;279(8):7247-53. doi: 10.1074/jbc.M309171200. Epub 2003 Dec 9.
Primary carnitine deficiency is a disorder of fatty acid oxidation caused by mutations in the Na+-dependent carnitine/organic cation transporter OCTN2. Studies with tyrosyl group-modifying reagents support the involvement of tyrosine residues in Na+ binding by sodium-coupled transporters. Here we report two new patients with carnitine deficiency caused by mutations affecting tyrosyl residues (Y447C and Y449D) close to a residue (Glu-452) previously shown to affect sodium stimulation of carnitine transport. Kinetic analysis indicated that the Y449D substitution, when expressed in Chinese hamster ovary cells, increased the concentration of sodium required to half-maximally stimulate carnitine transport from 14.8 +/- 1.8 to 34.9 +/- 5.8 mM (p<0.05), whereas Y447C completely abolished carnitine transport. Substitution of these tyrosine residues with phenylalanine restored normal carnitine transport in Y449F but resulted in markedly impaired carnitine transport by Y447F. This was associated with an increase in the concentration of sodium required to half-maximally stimulate carnitine transport to 57.8 +/- 7.4 mM (p<0.01 versus normal OCTN2). The Y447F and Y449D mutant transporters retained their ability to transport the organic cation tetraethylammonium indicating that their effect on carnitine transport was specific and likely associated with the impaired sodium stimulation of carnitine transport. By contrast, the Y447C natural mutation abolished the transport of organic cations in addition to carnitine. Confocal microscopy of OCTN2 transporters tagged with green fluorescent protein indicated that the Y447C mutant transporters failed to reach the plasma membrane, whereas Y447F, Y449D, and Y449F had normal membrane localization. These natural mutations identify tyrosine residues possibly involved in coupling the sodium electrochemical gradient to transmembrane solute transfer in the sodium-dependent co-transporter OCTN2.
原发性肉碱缺乏症是一种脂肪酸氧化紊乱疾病，由钠依赖性肉碱/有机阳离子转运体OCTN2的突变引起。用酪氨酸基团修饰试剂进行的研究支持酪氨酸残基参与钠偶联转运体与钠的结合。在此，我们报告了两名新的肉碱缺乏症患者，其病因是影响酪氨酸残基（Y447C和Y449D）的突变，这些残基靠近先前已证明会影响钠刺激肉碱转运的一个残基（Glu-452）。动力学分析表明，当在中国仓鼠卵巢细胞中表达时，Y449D替代增加了半最大刺激肉碱转运所需的钠浓度，从14.8±1.8 mM增加到34.9±5.8 mM（p<0.05），而Y447C则完全消除了肉碱转运。用苯丙氨酸替代这些酪氨酸残基可使Y449F恢复正常的肉碱转运，但导致Y447F的肉碱转运明显受损。这与半最大刺激肉碱转运所需的钠浓度增加到57.8±7.4 mM有关（与正常OCTN2相比，p<0.01）。Y447F和Y449D突变转运体保留了转运有机阳离子四乙铵的能力，表明它们对肉碱转运的影响是特异性的，并且可能与钠刺激肉碱转运受损有关。相比之下，Y447C自然突变除了消除肉碱转运外，还消除了有机阳离子的转运。用绿色荧光蛋白标记的OCTN2转运体的共聚焦显微镜检查表明，Y447C突变转运体未能到达质膜，而Y447F、Y449D和Y449F具有正常的膜定位。这些自然突变确定了酪氨酸残基可能参与钠依赖性共转运体OCTN2中钠电化学梯度与跨膜溶质转运的偶联。
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