MEMBRANE REMODELING DURING VIRAL INFECTION, PARASITE INVASION, AND APOPTOSIS

我们继续研究致病过程的膜生物学。采用快速冷冻、冷冻置换、薄层电子显微镜、全细胞导纳和荧光同步记录等方法，研究了流感血凝素糖基化磷脂酰肌醇连接胞外区(GPI-HA)诱导的膜融合中间产物。在触发后，先前分离的膜在电子显微镜下形成许多沙漏状的膜接触点(腰围约10-130 nm)。立体对在互补突起的峰处显示紧密的膜接触，这些突起来自每个膜。HA组接触较少，融合孔较宽。生理测量显示酸化后细胞间脂类染料快速混合，形成融合孔或缺乏融合孔(真正的半融合)。对于最早的孔，HA和GPI-HA的电导分布和闪烁孔的频率相似。对于GPI-HA，在开孔前、开孔期间或开孔后检测到脂质混合，而对于HA，只有在开孔后才能看到脂质混合。我们的发现与这样一种途径是一致的，即HA胞外结构域的构象变化将膜相互拉向形成接触部位，然后在这些接触部位中的一小部分开始半融合和孔形成。最后，需要HA的跨膜区来完成膜融合，以进行大分子含量的混合。疟疾寄生虫在人类红细胞(RBC)中的生长伴随着对许多溶质的摄取增加，包括阴离子、糖、嘌呤、氨基酸和有机阳离子。尽管这种摄取的药理性质和选择性表明涉及氯离子通道，但确切的机制尚未确定。此外，这种摄取在受感染的红细胞中的位置尚不清楚，因为示踪剂研究因可能通过液相吞饮或膜性导管摄取而变得复杂。在这个项目中，我们用全细胞电压钳方法研究了感染红细胞的通透性，这是以前从未在感染细胞上进行过的。用这种方法，未感染的红细胞具有低于100ps的欧姆全细胞电导，这与它们低的示踪剂渗透性是一致的。相比之下，滋养体感染的红细胞表现出电压依赖的非饱和电流，电流大150倍，主要由阴离子携带，并被通道阻滞剂突然消除。膜片钳测量和光谱分析证实，受感染的红细胞表面存在一个小的(&lt；10ps)离子通道，每个细胞大约有1000个拷贝，是造成这些电流的原因。由于其药理特性和底物选择性与示踪剂研究相匹配，这一通道解释了受感染红细胞对小溶质摄取的增加。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，它可能在寄生虫获得营养物质的一系列扩散途径中起主要作用。这一渠道可能会被阻断，因此它是药物开发的一个有吸引力的目标。

We continued our work on the membrane biology of pathogenic processes. Membrane fusion intermediates induced by the glycosylphosphatidylinositol-linked ectodomain of influenza hemagglutinin (GPI-HA) were investigated by rapidly freeze, freeze-substitution, thin section electron microscopy, and with simultaneous recordings of whole-cell admittance and fluorescence. Upon triggering, the previously separated membranes developed, when viewed by electron microscopy, numerous hourglass shaped points of membrane contact sites (~10-130 nm waist). Stereo pairs showed close membrane contact at peaks of complementary protrusions, arising from each membrane. With HA, there were fewer contacts, with wide fusion pores. Physiological measurements showed fast lipid dye mixing between cells after acidification, and either fusion pore formation or the lack thereof (true hemifusion). For the earliest pores a similar conductance distribution and frequency of flickering pores were detected for both HA and GPI-HA. For GPI-HA, lipid mixing was detected prior to, during, or after pore opening, whereas for HA lipid mixing is seen only after pore opening. Our findings are consistent with a pathway wherein conformational changes in the ectodomain of HA pulls membranes towards each other to form a contact site, then hemifusion and pore formation initiate in a small percentage of these contact sites. Finally, the transmembrane domain of HA is needed to complete membrane fusion for macromolecular content mixing. Growth of the malaria parasite in human red blood cells (RBCs) is accompanied by an increased uptake of many solutes including anions, sugars, purines, amino acids and organic cations. Although the pharmacological properties and selectivity of this uptake suggest that a chloride channel is involved, the precise mechanism has not been identified. Moreover, the location of this uptake in the infected RBC is unknown because tracer studies are complicated by possible uptake through fluid-phase pinocytosis or membranous ducts. In this project, we have studied the permeability of infected RBCs using the whole-cell voltage-clamp method, never before performed on infected cells. With this method, uninfected RBCs had ohmic whole-cell conductances of less than 100 pS, consistent with their low tracer permeabilities. In contrast, trophozoite-infected RBCs exhibited voltage-dependent, non-saturating currents that were 150-fold larger, predominantly carried by anions and abruptly abolished by channel blockers. Patch-clamp measurements and spectral analysis confirmed that a small (< 10 pS) ion channel on the infected RBC surface, present at about 1,000 copies per cell, is responsible for these currents. Because its pharmacological properties and substrate selectivities match those seen with tracer studies, this channel accounts for the increased uptake of small solutes in infected RBCs. The surface location of this new channel and its permeability to organic solutes needed for parasite growth indicate that it may have a primary role in a sequential diffusive pathway for parasite nutrient acquisition. This channel may be blocked, and so it is an attractive target for drug development.