School of Biomedicine, Faculty of Health and Medical Sciences, and the Institute for Photonics and Advanced Sensing, University of Adelaide, Adelaide, SA 5005, Australia.
Biological Sciences, College of Science and Engineering, Flinders University, Bedford Park, SA 5042, Australia.
Biosci Rep. 2024 Aug 28;44(8). doi: 10.1042/BSR20240542.
Aquaporin (AQP) channels found in all domains of life are transmembrane proteins which mediate passive transport of water, glycerol, signaling molecules, metabolites, and charged solutes. Discovery of new classes of ion-conducting AQP channels has been slow, likely reflecting time- and labor-intensive methods required for traditional electrophysiology. Work here defines a sensitive mass-throughput system for detecting AQP ion channels, identified by rescue of cell growth in the K+-transport-defective yeast strain CY162 following genetic complementation with heterologously expressed cation-permeable channels, using the well characterized human AQP1 channel for proof of concept. Results showed AQP1 conferred transmembrane permeability to cations which rescued survival in CY162 yeast. Comprehensive testing showed that growth response properties fully recapitulated AQP1 pharmacological agonist and antagonist profiles for activation, inhibition, dose-dependence, and structure-function relationships, demonstrating validity of the yeast screening tool for AQP channel identification and drug discovery efforts. This method also provided new information on divalent cation blockers of AQP1, pH sensitivity of antagonists, and ion permeability of human AQP6. Site-directed mutagenesis of AQP1 channel regulatory domains confirmed that yeast growth rescue was mediated by the introduced channels. Optical monitoring with a lithium-sensitive photoswitchable probe in living cells independently demonstrated monovalent cation permeability of AQP1 channels in yeast plasma membrane. Ion channel properties of AQP1 expressed in yeast were consistent with those of AQP1 expressed in Xenopus laevis oocyte and K+-transport defective Escherichia coli. Outcomes here establish a powerful new approach for efficient screening of phylogenetically diverse AQPs for yet untested functions as cation channels.
水通道蛋白（AQP）通道存在于所有生命领域，是介导水、甘油、信号分子、代谢物和带电溶质被动运输的跨膜蛋白。新的离子导电 AQP 通道类别的发现一直很缓慢，这可能反映了传统电生理学所需的耗时和劳动密集型方法。这里的工作定义了一种用于检测 AQP 离子通道的敏感高通量系统，该系统通过用经过充分表征的人源 AQP1 通道作为概念验证，用异源表达的阳离子渗透性通道对 K+转运缺陷型酵母菌株 CY162 进行遗传互补来鉴定，该系统通过遗传互补鉴定了 K+转运缺陷型酵母菌株 CY162 的细胞生长得到挽救。结果表明，AQP1 赋予阳离子跨膜通透性，挽救了 CY162 酵母的存活。综合测试表明，生长反应特性完全再现了 AQP1 药理学激动剂和拮抗剂的激活、抑制、剂量依赖性和结构-功能关系，证明了酵母筛选工具用于 AQP 通道鉴定和药物发现工作的有效性。该方法还提供了关于 AQP1 的二价阳离子阻断剂、拮抗剂的 pH 敏感性以及人源 AQP6 的离子通透性的新信息。AQP1 通道调节域的定点突变证实，酵母生长挽救是由引入的通道介导的。用活细胞中的锂敏光开关探针进行光学监测，独立证明了 AQP1 通道在酵母质膜中的单价阳离子通透性。在酵母中表达的 AQP1 的离子通道特性与在非洲爪蟾卵母细胞中表达的 AQP1 和 K+转运缺陷型大肠杆菌中的 AQP1 的特性一致。这里的结果建立了一种强大的新方法，用于有效地筛选尚未作为阳离子通道进行测试的进化上多样化的 AQP。
