Development of membrane permeation inhibitors which are specific for kidney collecting duct, based on protein structure-function analysis.

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In this research, intended to clarify structure-function relationship of kidney collecting duct water channel, AQP2, by using yeast expression system. The newly developved system used a yeast strain BJ3505 which lacks endogenous protease. Yeast cells were incubated at 30 degree, a-id intracellular vesicle fration was obtained by sucrose density gradient. Western blot analysis confirmed the selective expression of AQP2 proteins in this fraction. Osmotic water permeability (Pf) of these vesicles was determined by stoped flow method. Expression of AQP2 induced 22-fold increase of Pf, demonstrating the establishment of the suitable system for analysis of structure-function relationship of AQP2.Using this system, we measured Pf of mutant AQP2s ; mutations were introduced along the aminoacid sequence. The results showed that when mutations were introduced between Loop C and Loop E, Pf was severely impaired. The amount AQP2 protein in the vesicles were not different among the mutants. Thus, we could determine the domain which is critically important for aquaous route of AQP2. We are currently narrowing this region 1r isolate critical residues, this information would be directly applicable in the development of inhibitors of membrane transport.