HIGH RESOLUTION ELECTRON MICROSCOPY OF WATER CHANNEL

本研究的长期目标是了解水跨膜通道的功能机制。水通道蛋白（AQP）是一类存在于细菌、酵母、昆虫、植物、哺乳动物和两栖动物组织中的水通道蛋白家族，属于MIP （Major Intrinsic Protein）超家族。水通道蛋白对正常细胞功能所需的水平衡的主动调节至关重要；例如，AQP2的缺陷与肾源性尿崩症等疾病有关。AQP1是水通道蛋白的一个子集，存在于肾脏、胆囊、脾脏、肺、肠、内耳和眼睛等器官的多种组织中。这些通道被认为是水特异性的，在液体吸收和分泌过程中将水输送到许多上皮细胞和内皮细胞层。我们建议继续努力确定AQP1的原子模型。在本资助年度，我们已取得AQP1的投影图，分辨率约为3.5埃，以及3D图，分辨率约为6埃。利用电子晶体学在3.5埃单位下获得三维结构的集中努力面临着几个技术问题：高度倾斜样品的图像和衍射图显示非常有限的垂直于倾斜轴的结构信息。此外，目前使用电子晶体学确定的结构分辨率不足以清晰地观察水分子，这对于了解水通道蛋白的功能机制及其对水的特异性至关重要。然而，获得膜蛋白三维晶体用于x射线晶体学研究的巨大困难一直是一个主要障碍。在过去的一年里，我们把精力集中在3D结晶上，并成功地获得了适合x射线晶体学研究的晶体。原生数据集的分辨率约为3埃，尽管晶体显示的衍射斑分辨率高于2.5埃。我们现在把主要精力集中在获得重原子衍生物上。大约3埃或更高分辨率的水通道结构将为理解MIP超家族对水跨膜运输的调节提供一个范例。原子模型将揭示通道的分子细节，包括调节其水运输特异性的机制。它还将提供在AQPcic双点突变体中观察到的特异性变化的结构原理，从水到甘油和蛋白质低聚状态的改变，从四聚体到单体形式。了解水通道的分子机制可以深入了解导致肾源性尿崩症等疾病的蛋白质缺陷的结构基础。

The long term objective of this research is to understand the functional mechanism of water transport across membrane channels. The aquaporins (AQP) are a family of water channel proteins found in bacteria, yeast, insect, plant, mammalian and amphibian tissues and belong to the MIP (Major Intrinsic Protein) superfamily. Aquaporins are critical for the active regulation of water balance required for normal cell function; defects in AQP2, for example, have been related to diseases such as nephrogenic diabetes insipidus. AQP1 is a subset of the aquaporins and can be found to exist in a variety of tissues from organs such as the kidney, gall bladder, spleen, lung, intestine, inner ear and eyes. These channels are believed to be water specific, transporting water across a number of epithelial and endothelial cell layers during fluid absorption and secretion. We propose to continue our efforts in determining the atomic model of AQP1. In the current grant years, we have obtained the projection map of AQP1 at a resolution of about 3.5 Angstrom units resolution and a 3D map at approximately 6 Angstrom units. Concentrated efforts to obtain the 3D structure at 3.5 Angstrom units using electron crystallography were faced with several technical problems: images and diffraction patterns of highly tilted samples show very limited structural information normal to the tilt axis. In addition, the current resolution of structures determined using electron crystallography is not adequate to clearly observe water molecules which is crucial for understanding the aquaporin functional mechanisms and their specificity for water. However, the great difficulty in obtaining 3D crystals of membrane proteins for x-ray crystallographic studies has been a major stumbling block. In the last year, we have focused our efforts on 3D crystallization and were successful in obtaining crystals suitable for x-ray crystallographic studies. Native date sets have been collected to approximately 3 Angstrom units resolution, although the crystals show diffraction spots to better than 2.5 Angstrom units resolution. We are now focusing our major efforts on obtaining heavy atom derivatives. The structure of the water channel at approximately 3 Angstrom units resolution or higher would provide a paradigm for understanding the regulation of the transport of water across the membrane by the MIP superfamily. The atomic model will reveal the molecular details of the channel including the mechanism that regulates its specificity for water transport. It will also provide a structural rationale for the change in specificity, from water to glycerol and the alteration of the protein oligomeric state, from tetramer to monomeric form, observed in a double point mutant of AQPcic. The knowledge of the molecular mechanisms of water channels could provide insights into the structural basis of protein defects resulting in diseases such as nephrogenic diabetes insipidus.