A generic high-throughput assay to detect aquaporin functional mutants: Potential application to discovery of aquaporin inhibitors

• Rapid detection of functional mutants is challenging for water channel aquaporins.
• Available methods are time consuming and only applicable to specific aquaporins.
• We report a generic high-throughput assay based on yeast response to rapid freezing.
• The assay can potentially be of value in the screen for aquaporin inhibitors.

BackgroundThe discovery of stable, yet functional, protein mutants is a limiting factor in the development of biotechnological applications, structural studies or in drug discovery. Rapid detection of functional mutants is especially challenging for water channel aquaporins, as they do not have a directly measurable enzymatic or binding activity. Current methods available are time consuming and only applicable to specific aquaporins.MethodsHerein we describe an assay based on the protective effect of aquaporins on yeast S. cerevisiae in response to rapid freezing.ResultsYeast overexpressing a functional water-permeable aquaporin of choice are rescued after the challenge, while inactive or blocked aquaporins confer no protection and lead to cell death. The potential of this assay is shown by screening a small number of E. coli aquaporin Z (AQPZ) mutants. Additionally, a library of ~ 10,000 drug-like compounds was tested against human AQP1 (hAQP1).ConclusionsSince rescue is only dependent on transmembrane water flux, the assay is applicable to water-permeable aquaporins of any origin.General significanceMapping of permissive mutations on the aquaporin structure can help delineate the minimal requirements for effective water transport. Alternatively, the assay can be potentially used to discover compounds that inhibit aquaporin water transport. When additionally screened for thermostability, functional aquaporin mutants can be useful in the development of biomimetic membranes for water purification, or to improve the likelihood of producing well-diffracting crystals, enabling rational design of much needed aquaporin inhibitors.

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