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Simultaneous Binding of the N- and C-terminal Cytoplasmic Domains of Aquaporin 4 to Calmodulin May Contribute to Vesicular Trafficking

Hiroaki Ishida, Hans J. Vogel, Alex C. Conner, Philip Kitchen, Roslyn M. Bill, View ORCID ProfileJustin A. MacDonald
doi: https://doi.org/10.1101/2021.06.29.450403
Hiroaki Ishida
1Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada
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Hans J. Vogel
1Biochemistry Research Group, Department of Biological Sciences, University of Calgary, Calgary, AB, T2N 1N4, Canada
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Alex C. Conner
2College of Medical and Dental Sciences, University of Birmingham, Birmingham, B15 2TT, UK
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Philip Kitchen
3College of Health and Life Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK
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Roslyn M. Bill
3College of Health and Life Sciences, Aston University, Aston Triangle, Birmingham, B4 7ET, UK
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Justin A. MacDonald
4Department of Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, Calgary, AB, T2N 4Z6, Canada
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  • ORCID record for Justin A. MacDonald
  • For correspondence: jmacdo@ucalgary.ca
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ABSTRACT

Aquaporin 4 (AQP4) is a water transporting, transmembrane channel protein that has important regulatory roles in maintaining cellular water homeostasis. Several other AQP proteins exhibit calmodulin (CaM)-binding properties, and CaM has recently been implicated in the cell surface localization of AQP4 that occurs in response to osmotically-driven changes in cell swelling in the central nervous system. The objective of the present study was to assess the CaM-binding properties of AQP4 in detail. Inspection of AQP4 revealed two putative CaM-binding domains (CBDs) in the cytoplasmic N- and C-terminal regions, respectively. The Ca2+-dependent CaM-binding properties of synthetic and recombinant AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1H, 15N-HSQC NMR with 15N-labeled CaM. The N-terminal CBD peptide of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 μM. CaM bound two C-terminal AQP4 peptides with interactions observed for both the C- and N-lobes of CaM (Kd1: 3.6 μM, Kd2: 113.6 μM, respectively). A recombinant AQP4 protein domain (rAQP4ct, containing the entire cytosolic C-terminal domain sequence) bound CaM in a 1:1 binding mode with a Kd of 6.1 μM. A ternary bridging complex could be generated with the N- and C-lobes of CaM interacting simultaneously with the N- and C-terminal CBD peptides. These data suggest that this unique adapter protein binding mode of CaM and AQP4 may be an important regulatory mechanism for the vesicular trafficking of AQP4.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-ND 4.0 International license.
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Posted June 29, 2021.
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Simultaneous Binding of the N- and C-terminal Cytoplasmic Domains of Aquaporin 4 to Calmodulin May Contribute to Vesicular Trafficking
Hiroaki Ishida, Hans J. Vogel, Alex C. Conner, Philip Kitchen, Roslyn M. Bill, Justin A. MacDonald
bioRxiv 2021.06.29.450403; doi: https://doi.org/10.1101/2021.06.29.450403
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Simultaneous Binding of the N- and C-terminal Cytoplasmic Domains of Aquaporin 4 to Calmodulin May Contribute to Vesicular Trafficking
Hiroaki Ishida, Hans J. Vogel, Alex C. Conner, Philip Kitchen, Roslyn M. Bill, Justin A. MacDonald
bioRxiv 2021.06.29.450403; doi: https://doi.org/10.1101/2021.06.29.450403

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