학술논문
Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties.
Document Type
Academic Journal
Author
Henderson SW; School of Biomedicine, University of Adelaide, Adelaide, SA 5005, Australia.; Nakayama Y; Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, NSW 2010, Australia.; School of Clinical Medicine, UNSW Medicine & Health, St Vincent's Healthcare Clinical Campus, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW Australia.; Whitelaw ML; Institute of Photonics and Advanced Sensing, The School of Biological Sciences, University of Adelaide, Adelaide, SA 5005, Australia.; Bruning JB; Institute of Photonics and Advanced Sensing, The School of Biological Sciences, University of Adelaide, Adelaide, SA 5005, Australia.; Anderson PA; School of Biological Sciences, Faculty of Science and Engineering, Flinders University, Adelaide, SA 5001, Australia.; Tyerman SD; ARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine & Waite Research Institute, University of Adelaide, Glen Osmond, SA 5064, Australia.; Ramesh SA; School of Biological Sciences, Faculty of Science and Engineering, Flinders University, Adelaide, SA 5001, Australia.; Martinac B; Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, NSW 2010, Australia.; School of Clinical Medicine, UNSW Medicine & Health, St Vincent's Healthcare Clinical Campus, Faculty of Medicine and Health, University of New South Wales, Sydney, NSW Australia.; Yool AJ; School of Biomedicine, University of Adelaide, Adelaide, SA 5005, Australia.
Source
Publisher: Cell Press Elsevier Country of Publication: United States NLM ID: 9918266001106676 Publication Model: eCollection Cited Medium: Internet ISSN: 2667-0747 (Electronic) Linking ISSN: 26670747 NLM ISO Abbreviation: Biophys Rep (N Y) Subsets: PubMed not MEDLINE
Subject
Language
English
Abstract
Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris , and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His 6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His 6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His 6 D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities.
Competing Interests: The authors declare no competing interests.
(© 2023 The Author(s).)
Competing Interests: The authors declare no competing interests.
(© 2023 The Author(s).)