Research School of Biology, The Australian National University, Canberra, ACT 0200, Australia.
Proc Natl Acad Sci U S A. 2014 Apr 29;111(17):E1759-67. doi: 10.1073/pnas.1322965111. Epub 2014 Apr 11.
Mutations in the chloroquine resistance transporter (PfCRT) are the primary determinant of chloroquine (CQ) resistance in the malaria parasite Plasmodium falciparum. A number of distinct PfCRT haplotypes, containing between 4 and 10 mutations, have given rise to CQ resistance in different parts of the world. Here we present a detailed molecular analysis of the number of mutations (and the order of addition) required to confer CQ transport activity upon the PfCRT as well as a kinetic characterization of diverse forms of PfCRT. We measured the ability of more than 100 variants of PfCRT to transport CQ when expressed at the surface of Xenopus laevis oocytes. Multiple mutational pathways led to saturable CQ transport via PfCRT, but these could be separated into two main lineages. Moreover, the attainment of full activity followed a rigid process in which mutations had to be added in a specific order to avoid reductions in CQ transport activity. A minimum of two mutations sufficed for (low) CQ transport activity, and as few as four conferred full activity. The finding that diverse PfCRT variants are all limited in their capacity to transport CQ suggests that resistance could be overcome by reoptimizing the CQ dosage.
疟原虫 PfCRT 中的突变是导致氯喹（CQ）耐药的主要决定因素。在世界不同地区，许多不同的 PfCRT 单倍型（包含 4 到 10 个突变）导致了 CQ 耐药。在这里，我们对 PfCRT 获得 CQ 转运活性所需的突变数量（以及添加顺序）进行了详细的分子分析，同时还对 PfCRT 的多种形式进行了动力学特征分析。我们在非洲爪蟾卵母细胞表面表达了超过 100 种 PfCRT 变体，并测量了它们转运 CQ 的能力。多种突变途径可通过 PfCRT 实现 CQ 的饱和转运，但这些途径可分为两个主要谱系。此外，完全活性的获得遵循一个严格的过程，其中突变必须按特定顺序添加，以避免 CQ 转运活性的降低。至少需要两个突变才能产生（低）CQ 转运活性，而四个突变即可完全激活。这一发现表明，各种 PfCRT 变体在转运 CQ 的能力上都受到限制，这表明可以通过重新优化 CQ 剂量来克服耐药性。
