Ultrastructure, pharmacologic inhibition, and transport selectivity of aquaporin channel-forming integral protein in proteoliposomes.
Ultrastructure, pharmacologic inhibition, and transport selectivity of aquaporin channel-forming integral protein in proteoliposomes.
复制标题
DOI：
10.1021/bi00172a042
复制
发表时间：
1994-02
期刊：
影响因子：
2.9
通讯作者：
M. Zeidel;S. Nielsen;S. Nielsen;S. Nielsen;Barbara L. Smith;Barbara L. Smith;Barbara L. Smith;S. Ambudkar;S. Ambudkar;S. Ambudkar;A. B. Maunsbach;A. B. Maunsbach;A. B. Maunsbach;P. Agre
中科院分区：
文献类型：
--
作者：
M. Zeidel;S. Nielsen;S. Nielsen;S. Nielsen;Barbara L. Smith;Barbara L. Smith;Barbara L. Smith;S. Ambudkar;S. Ambudkar;S. Ambudkar;A. B. Maunsbach;A. B. Maunsbach;A. B. Maunsbach;P. Agre
先前研究表明，将高纯度的水通道蛋白芯片(通道形成整合蛋白)重组到蛋白脂质体中，可以使膜具有高渗透性[Zeidel等人]。(1992)生物化学31,7436-7440]。在这里，我们报告了蛋白质脂质体中人红细胞芯片的详细的超微结构、药理和转运研究。冷冻断口和透射电子显微镜显示，切屑均匀地分布在膜中，以自然和反向的方向并入膜中。对三种不同浓度的CHIP重组膜的形态计量分析表明，膜内颗粒对应于四聚物或可能的高阶齐聚物，膜内粒子数与芯片密度成正比增加。蛋白水解法去除CHIP的4-kDa C末端胞浆结构域不会改变红细胞膜的PF或齐聚作用。当芯片被重组成不同脂肪组成的膜时，对水的电导也是相似的。芯片介导的PF对特定的巯基试剂的敏感性与已知的红细胞PF的敏感性相同，包括对对氯汞苯磺酸盐的延迟反应。CHIP不增加蛋白脂膜对H+/OH-或NH3的通透性。这些研究表明，CHIP蛋白脂质体显示了天然红细胞中水通道的所有已知特征，从而为跨膜水运动的生物物理分析提供了一个明确的系统。
Reconstitution of highly purified aquaporin CHIP (channel-forming integral protein) into proteoliposomes was previously shown to confer high osmotic water permeability (Pf) to the membranes [Zeidel et al. (1992) Biochemistry 31, 7436-7440]. Here we report detailed ultrastructural, pharmacologic, and transport studies of human red cell CHIP in proteoliposomes. Freeze-fracture and transmission electron microscopy revealed a uniform distribution of CHIP which was incorporated into the membranes in both native and inverse orientations. Morphometric analysis of membranes reconstituted at three different concentrations of CHIP revealed that the intramembrane particles correspond to tetramers or possible higher order oligomers, and the Pf increased in direct proportion to the CHIP density. Proteolytic removal of the 4-kDa C-terminal cytoplasmic domain of CHIP did not alter the Pf or oligomerization in red cell membranes. CHIP exhibited a similar conductance for water when reconstituted into membranes of varied lipid compositions. The sensitivities of CHIP-mediated Pf to specific sulfhydryl reagents were identical to known sensitivities of red cell Pf, including a delayed response to p-(chloromercuri)benzenesulfonate. CHIP did not increase the permeability of the proteoliposome membranes to H+/OH- or NH3. These studies demonstrate that CHIP proteoliposomes exhibit all known characteristics of water channels in native red cells and therefore provide a defined system for biophysical analysis of transmembrane water movements.