Simultaneous binding of the N- and C-terminal cytoplasmic domains of aquaporin 4 to calmodulin


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. 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 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 of AQP4 predominantly interacted with the N-lobe of CaM with a 1:1 binding ratio and a Kd of 3.4 μM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; Kd1: 3.6 μM, Kd2: 113.6 μM, respectively). A recombinant AQP4 protein domain (recAQP4CT, containing the entire cytosolic C-terminal 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 support a unique adapter protein binding mode for CaM with AQP4.

Publication DOI:
Divisions: College of Health & Life Sciences > School of Biosciences
College of Health & Life Sciences > School of Biosciences > Cellular and Molecular Biomedicine
College of Health & Life Sciences
Additional Information: CC-BY-ND 4.0 International license. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
Publication ISSN: 1879-2642
Full Text Link:
Related URLs: https://linking ... 005273621002856 (Publisher URL)
PURE Output Type: Article
Published Date: 2022-02-01
Published Online Date: 2021-12-07
Accepted Date: 2021-11-30
Authors: Ishida, Hiroaki
Vogel, Hans J.
Conner, Alex C.
Kitchen, Philip
Bill, Roslyn M. (ORCID Profile 0000-0003-1331-0852)
Macdonald, Justin A.


Export / Share Citation


Additional statistics for this record