Fotiadis Dimitrios, Suda Kitaru, Tittmann Peter, Jenö Paul, Philippsen Ansgar, Müller Daniel J, Gross Heinz, Engel Andreas
M.E. Müller-Institute for Microscopy, Biozentrum of the University of Basel, Switzerland.
J Mol Biol. 2002 May 17;318(5):1381-94. doi: 10.1016/s0022-2836(02)00143-2.
Aquaporin-1 (AQP1) is the first functionally identified aquaporin of a growing family of membrane water channels found in all forms of life. Recently, a possible secondary function as a cyclic guanosine monophosphate (cGMP) gated ion channel was attributed to AQP1. We have reconstituted purified protein from bovine and human red blood cell membranes into highly ordered 2D crystals. The topography of both AQP1s was determined by electron microscopy from freeze-dried, unidirectionally metal-shadowed 2D crystals as well as from surface topographs of native crystals recorded in buffer solution with the atomic force microscope (AFM). In spite of the high level of sequence homology between bovine and human AQP1, the surfaces showed distinct differences. Alignment of both sequences and comparison of the acquired surface topographies with the atomic model of human AQP1 revealed the topographic changes on the surface of bovine AQP1 to be induced by a few amino acid substitutions. A striking degree of sequence homology was found between the carboxyl-terminal domains of AQP1s from different organisms and EF-hands from Ca2+-binding proteins belonging to the calmodulin superfamily, suggesting the existence of a Ca2+-binding site at the C terminus of AQP1 instead of the putative cGMP-binding site reported previously. To unveil its position on the acquired surface topographies, 2D crystals of AQP1 were digested with carboxypeptidase Y, which cleaves off the intracellular C terminus. Difference maps of AFM topographs between the native and the peptidase-treated AQP1s showed the carboxylic tail to be close to the 4-fold symmetry axis of the tetramer. SDS-PAGE and matrix-assisted laser desorption/ionisation mass spectrometry of native and decarboxylated bovine and human AQP1 revealed that the EF-hand motif found at the C terminus of AQP1 was partially resistant to peptidase digestion. The importance of the C-terminal domain is implicated by structural instability of decarboxylated AQP1. A possible role of the C terminus and calcium in translocation of AQP1 in cholangiocytes from intracellular vesicles to the plasma membrane and in triggering its fusion is discussed. Functional studies are now required to identify the physiological role of the Ca2+-binding site.
水通道蛋白-1（AQP1）是在所有生命形式中发现的不断增加的膜水通道蛋白家族中第一个在功能上被鉴定的成员。最近，一种作为环磷酸鸟苷（cGMP）门控离子通道的可能的次要功能被归因于AQP1。我们已经将从牛和人红细胞膜中纯化的蛋白质重构成高度有序的二维晶体。通过电子显微镜从冻干的、单向金属阴影的二维晶体以及用原子力显微镜（AFM）在缓冲溶液中记录的天然晶体的表面形貌图来确定两种AQP1的形貌。尽管牛和人AQP1之间的序列同源性水平很高，但表面显示出明显的差异。两种序列的比对以及所获得的表面形貌与人类AQP1原子模型的比较揭示了牛AQP1表面的形貌变化是由少数氨基酸取代引起的。在来自不同生物体的AQP1的羧基末端结构域与属于钙调蛋白超家族的Ca2+结合蛋白的EF手之间发现了惊人程度的序列同源性，这表明在AQP1的C末端存在一个Ca2+结合位点，而不是先前报道的假定的cGMP结合位点。为了揭示其在获得的表面形貌上的位置，用羧肽酶Y消化AQP1的二维晶体，该酶可切割掉细胞内的C末端。天然和经肽酶处理的AQP1的AFM形貌图的差异图显示羧基尾部靠近四聚体的四重对称轴。天然和脱羧的牛和人AQP1的SDS-PAGE和基质辅助激光解吸/电离质谱分析表明，在AQP1的C末端发现的EF手基序对肽酶消化具有部分抗性。脱羧AQP1的结构不稳定性暗示了C末端结构域的重要性。讨论了C末端和钙在胆管细胞中AQP1从细胞内囊泡向质膜转运以及触发其融合中的可能作用。现在需要进行功能研究来确定Ca2+结合位点的生理作用。
