AQUAPORIN EXPRESSION AND CHARACTERIZATION IN YEAST

目前已从哺乳动物细胞中克隆了5种水通道蛋白（aquaporins，AQP）。 只有 AQP1蛋白存在于红细胞中， 足以进行详细的生物物理和结构分析。 AQP将 在酵母突变体中表达， 分泌囊泡 这些囊泡可以直接测定水分 和使用停流荧光猝灭或使用 作为蛋白质的丰富来源， 问题研究 因此，水迁移的活化能，溶质 将确定磁导率和单位电导。 高 蛋白质水平将允许重组和结晶 用于结构研究的平面双层，如先前对AQP1所述。 导致肾源性尿崩症（NDI）的AQP2突变形式 将进行定量分析。 通道孔隙的数学模型 基于溶质渗透性的测试。 水通道蛋白同系物 这些不起水道作用的地方将被用来制造嵌合体， 确定透水性的结构决定因素。 总的来说， 对野生型和 以前不可能的突变AQP现在将成为可能。

Five aquaporins (AQPs) have been cloned from mammalian cells. Only the AQP1 protein, found in red blood cells, is available in quantities sufficient for detailed biophysical and structural analyses. AQPs will be expressed in yeast mutants which accumulate plasma membrane-targeted secretory vesicles. These vesicles can be assayed directly for water and solute transport using stopped-flow fluorescence quenching or used as an abundant source of protein for reconstitution and structural studies. Therefore, activation energies for water transport, solute permeabilities, and unitary conductances will be determined. High levels of protein will allow reconstitution and crystallization of planar bilayers for structural studies as previously described for AQP1. Mutant forms of AQP2 responsible for nephrogenic diabetes insipids (NDI) will be quantitatively analyzed. Mathematical models of channel pores based on solute permeabilities will be tested. Aquaporin homologues which do not function as water channels will be used to make chimeras to determine the structural determinants of water permeability. overall, quantitative functional studies and structural studies of wild-type and mutant AQPs that were not previously possible will now be possible.