Amat di San Filippo Cristina, Pasquali Marzia, Longo Nicola
Division of Medical Genetics, Department of Pediatrics, University of Utah, Salt Lake City, Utah, USA.
Hum Mutat. 2006 Jun;27(6):513-23. doi: 10.1002/humu.20314.
Primary carnitine deficiency is a recessive disorder caused by heterogeneous mutations in the SLC22A5 gene encoding the OCTN2 carnitine transporter. Here we extend mutational analysis to eight new families with this disorder. To determine the mechanism by which missense mutations impaired carnitine transport, the OCTN2 transporter was tagged with the green fluorescent protein and expressed in CHO cells. Analysis by confocal microscopy indicated that several missense mutants (M1I, R169W, T232 M, G242 V, S280F, R282Q, W283R, A301D, W351R, R399Q, T440 M, E452 K, and T468R) matured normally to the plasma membrane. By contrast, other mutations (including R19P, DeltaF22, R83L, S280F, P398L, Y447C, and A142S/R488 H) caused significant retention of the mutant OCTN2 transporter in the cytoplasm. Failed maturation to the plasma membrane is a common mechanism in disorders affecting membrane transporters/ion channels, including cystic fibrosis. To correct this defect, we tested whether drugs reducing the efficiency of protein degradation in the endoplasmic reticulum (ER) (phenylbutyrate, curcumin) or capable of binding the OCTN2 carnitine transporter (verapamil, quinidine) could improve carnitine transport. Prolonged incubation with phenylbutyrate, quinidine, and verapamil partially stimulated carnitine transport, while curcumin was ineffective. These results indicate that OCTN2 mutations can affect carnitine transport by impairing maturation of transporters to the plasma membrane. Pharmacological therapy can be effective in partially restoring activity of mutant transporters.
原发性肉碱缺乏症是一种隐性疾病，由编码OCTN2肉碱转运体的SLC22A5基因的异质性突变引起。在此，我们将突变分析扩展至八个患有该疾病的新家族。为确定错义突变损害肉碱转运的机制，将OCTN2转运体用绿色荧光蛋白标记并在CHO细胞中表达。共聚焦显微镜分析表明，几个错义突变体（M1I、R169W、T232M、G242V、S280F、R282Q、W283R、A301D、W351R、R399Q、T440M、E452K和T468R）正常成熟至质膜。相比之下，其他突变（包括R19P、DeltaF22、R83L、S280F、P398L、Y447C和A142S/R488H）导致突变的OCTN2转运体在细胞质中显著滞留。未能成熟至质膜是影响膜转运体/离子通道的疾病（包括囊性纤维化）中的常见机制。为纠正这一缺陷，我们测试了降低内质网（ER）中蛋白质降解效率的药物（苯丁酸钠、姜黄素）或能够结合OCTN2肉碱转运体的药物（维拉帕米、奎尼丁）是否能改善肉碱转运。用苯丁酸钠、奎尼丁和维拉帕米长时间孵育可部分刺激肉碱转运，而姜黄素无效。这些结果表明，OCTN2突变可通过损害转运体成熟至质膜来影响肉碱转运。药物治疗可有效部分恢复突变转运体的活性。
