Laboratory of Clinical Pharmaceutics and Therapeutics, Division of Pharmasciences, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
PLoS One. 2013 Jul 31;8(7):e67690. doi: 10.1371/journal.pone.0067690. Print 2013.
Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4.
Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4.
In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization.
Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.
单羧酸转运蛋白（MCTs）转运单羧酸，如乳酸、丙酮酸和酮体。这些转运蛋白是癌症治疗的极具吸引力的靶点。阐明 MCTs 的功能和结构对于开发靶向这些蛋白质的有效药物是必要的。然而，与 MCT1 相比，关于 MCT4 的功能部分的位置以及哪些组成氨基酸控制 MCT4 的转运功能的信息较少。本工作的目的是确定 hMCT4 中 L-乳酸转运的分子机制。
通过使用 hMCT4 cRNA 注射的非洲爪蟾卵母细胞来确定 L-乳酸通过 hMCT4 的转运。在不存在和存在化学修饰剂和 4,4'-二异硫氰基二苯乙烯-2,2'-二磺酸盐（DIDS）的情况下，测量 hMCT4 介导的 L-乳酸摄取。此外，通过 hMCT4 精氨酸突变体测量 L-乳酸摄取。免疫组织化学研究揭示了 hMCT4 的定位。
在表达 hMCT4 的卵母细胞中，用苯乙二醛（PGO）处理，一种针对精氨酸残基的化合物，完全消除了 hMCT4 的转运活性，尽管存在 L-乳酸可以防止这种消除。另一方面，除 PGO 处理之外的化学修饰对 hMCT4 的转运活性没有影响。该转运体有六个保守的精氨酸残基，两个在跨膜结构域（TMDs）中，四个在细胞内环中。在 hMCT4-R278 突变体中，L-乳酸的摄取没有任何转运活性，而 hMCT4 的定位没有改变。
我们的结果表明，跨膜结构域 8 中的 Arg-278 是 hMCT4 识别底物 L-乳酸的关键残基。
