Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties
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 activi...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2023-03-01
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| Series: | Biophysical Reports |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2667074723000010 |
| _version_ | 1797936338240012288 |
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| author | 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 |
| author_facet | 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 |
| author_sort | Sam W. Henderson |
| collection | DOAJ |
| description | 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-His6D48A/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. |
| first_indexed | 2024-04-10T18:29:15Z |
| format | Article |
| id | doaj.art-d44623090ff3405f88a8a25dc3d87cef |
| institution | Directory Open Access Journal |
| issn | 2667-0747 |
| language | English |
| last_indexed | 2024-04-10T18:29:15Z |
| publishDate | 2023-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biophysical Reports |
| spelling | doaj.art-d44623090ff3405f88a8a25dc3d87cef2023-02-02T04:50:49ZengElsevierBiophysical Reports2667-07472023-03-0131100100Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel propertiesSam W. Henderson0Yoshitaka Nakayama1Murray L. Whitelaw2John B. Bruning3Peter A. Anderson4Stephen D. Tyerman5Sunita A. Ramesh6Boris Martinac7Andrea J. Yool8School of Biomedicine, University of Adelaide, Adelaide, SA 5005, AustraliaVictor 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 AustraliaInstitute of Photonics and Advanced Sensing, The School of Biological Sciences, University of Adelaide, Adelaide, SA 5005, AustraliaInstitute of Photonics and Advanced Sensing, The School of Biological Sciences, University of Adelaide, Adelaide, SA 5005, AustraliaSchool of Biological Sciences, Faculty of Science and Engineering, Flinders University, Adelaide, SA 5001, AustraliaARC Centre of Excellence in Plant Energy Biology, School of Agriculture, Food and Wine & Waite Research Institute, University of Adelaide, Glen Osmond, SA 5064, AustraliaSchool of Biological Sciences, Faculty of Science and Engineering, Flinders University, Adelaide, SA 5001, AustraliaVictor 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 AustraliaSchool of Biomedicine, University of Adelaide, Adelaide, SA 5005, Australia; Corresponding authorHuman 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-His6D48A/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.http://www.sciencedirect.com/science/article/pii/S2667074723000010 |
| spellingShingle | 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 Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties Biophysical Reports |
| title | Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties |
| title_full | Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties |
| title_fullStr | Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties |
| title_full_unstemmed | Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties |
| title_short | Proteoliposomes reconstituted with human aquaporin-1 reveal novel single-ion-channel properties |
| title_sort | proteoliposomes reconstituted with human aquaporin 1 reveal novel single ion channel properties |
| url | http://www.sciencedirect.com/science/article/pii/S2667074723000010 |
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