Membrane Transport Proteins of the Malaria Parasite

疟疾寄生虫在人红细胞（RBC）内的生长伴随着许多溶质（包括阴离子、糖、嘌呤、氨基酸和有机阳离子）的摄取增加。 虽然这种增加的摄取已经知道了20多年，但其确切的机制和受感染RBC内的摄取部位一直存在强烈争议。在过去的一年中，我们用全细胞和单通道膜片钳技术解决了这些重要问题。 使用这些方法，未感染的RBC具有< 100 pS的欧姆全细胞电导，这与它们的低示踪剂渗透性一致。 相比之下，滋养体感染的红细胞表现出电压依赖性，非饱和电流是150倍大，主要由阴离子携带，并突然取消通道阻断剂。 膜片钳测量和光谱分析证实，感染的RBC表面上的小（<10 pS）离子通道（约1000拷贝/细胞）负责这些电流。 这个通道（称为疟原虫表面阴离子通道，PSAC）完全解释了感染红细胞中小营养素的吸收增加。 这一新通道的表面位置及其对寄生虫生长所需的有机溶质的渗透性表明，在寄生虫营养获取的连续扩散途径中起主要作用。 我们最近开发了一种生物化学方法，用于分离和纯化受感染细胞表面的寄生虫蛋白;这些蛋白质将用最先进的质谱方法进行测序，并应导致克隆PSAC？我们还致力于通过高通量筛选250，000个候选通道阻断剂来鉴定PSAC的高亲和力阻断剂。该筛选将使用我们实验室开发的通道活性的分光光度测定法。

Growth of the malaria parasite within human red blood cells (RBC) is accompanied by increased uptake of many solutes including anions, sugars, purines, amino acids, and organic cations. While this increased uptake has been known for more than 20 years, its precise mechanism and the site of uptake within the infected RBC have been strongly debated. In the past year, we resolved these important problems with the whole-cell and single channel patch-clamp methods. With these methods, uninfected RBCs had ohmic whole-cell conductances of < 100 pS, consistent with their low tracer permeabilities. In contrast, trophozoite-infected RBCs exhibited voltage-dependent, non-saturating currents which are 150-fold larger, predominantly carried by anions, and abruptly abolished by channel blockers. Patch-clamp measurements and spectral analysis confirm that a small (<10 pS) ion channel on the infected RBC surface, present at ~1000 copies/cell, is responsible for these currents. This channel (called the plasmodial surface anion channel, PSAC) fully accounts for the increased uptake of small nutrients in infected RBCs. The surface location of this new channel and its permeability to organic solutes needed for parasite growth suggest a primary role in a sequential diffusive pathway for parasite nutrient acquisition.At present, our laboratory is focused on cloning the gene responsible for PSAC. We recently developed a biochemical approach for isolating and purifying parasite proteins on the surface of infected cells; these proteins will be sequenced with state-of-the-art mass spectroscopy methods and should lead to cloning PSAC?s gene.We are also working on identifying high-affinity blockers of PSAC with a high-throughput screen of 250,000 candidate channel blockers. This screen will use a spectrophotometric assay for channel activity developed in our laboratory.