Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties

Sam W Henderson; Yoshitaka Nakayama; Murray L Whitelaw; John B Bruning; Peter A Anderson; Stephen D Tyerman; Sunita A Ramesh; Boris Martinac; Andrea J Yool

doi:10.1016/j.bpr.2023.100100

Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties

Biophys Rep (N Y). 2023 Jan 14;3(1):100100. doi: 10.1016/j.bpr.2023.100100. eCollection 2023 Mar 8.

Authors

Sam W Henderson¹, Yoshitaka Nakayama^{2

3}, Murray L Whitelaw⁴, John B Bruning⁴, Peter A Anderson⁵, Stephen D Tyerman⁶, Sunita A Ramesh⁵, Boris Martinac^{2

3}, Andrea J Yool¹

Affiliations

¹ School of Biomedicine, University of Adelaide, Adelaide, SA 5005, Australia.
² Victor Chang Cardiac Research Institute, Lowy Packer Building, Darlinghurst, NSW 2010, Australia.
³ 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.
⁴ Institute of Photonics and Advanced Sensing, The School of Biological Sciences, University of Adelaide, Adelaide, SA 5005, Australia.
⁵ School of Biological Sciences, Faculty of Science and Engineering, Flinders University, Adelaide, SA 5001, Australia.
⁶ 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.

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-His₆ was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His₆ 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-His₆ ^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.