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

dc.contributor.authorHenderson, S.W.
dc.contributor.authorNakayama, Y.
dc.contributor.authorWhitelaw, M.L.
dc.contributor.authorBruning, J.B.
dc.contributor.authorAnderson, P.A.
dc.contributor.authorTyerman, S.D.
dc.contributor.authorRamesh, S.A.
dc.contributor.authorMartinac, B.
dc.contributor.authorYool, A.J.
dc.date.issued2023
dc.description.abstractHuman 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.
dc.description.statementofresponsibilitySam W. Henderson, Yoshitaka Nakayama, Murray L. Whitelaw, John B. Bruning, Peter A. Anderson, Stephen D. Tyerman, Sunita A. Ramesh, Boris Martinac, and Andrea J. Yool
dc.identifier.citationBiophysical Reports, 2023; 3(1):100100-1-100100-15
dc.identifier.doi10.1016/j.bpr.2023.100100
dc.identifier.issn2667-0747
dc.identifier.issn2667-0747
dc.identifier.orcidHenderson, S.W. [0000-0003-3019-1891]
dc.identifier.orcidBruning, J.B. [0000-0002-6919-1824]
dc.identifier.orcidTyerman, S.D. [0000-0003-2455-1643]
dc.identifier.orcidRamesh, S.A. [0000-0003-2230-4737]
dc.identifier.orcidYool, A.J. [0000-0003-1283-585X]
dc.identifier.urihttps://hdl.handle.net/2440/140364
dc.language.isoen
dc.publisherElsevier BV
dc.relation.granthttp://purl.org/au-research/grants/arc/DP190101745
dc.relation.granthttp://purl.org/au-research/grants/nhmrc/1135974
dc.rights© 2023 The Author(s). This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
dc.source.urihttps://doi.org/10.1016/j.bpr.2023.100100
dc.subjectHuman aquaporin 1 (hAQP1)
dc.subjectProteoliposomes
dc.titleProteoliposomes reconstituted with human aquaporin-1 reveal novel single ion channel properties
dc.typeJournal article
pubs.publication-statusPublished

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