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bioRxiv · 10.1101/2021.06.29.450403

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

Ishida, H.·Vogel, H. J.·Conner, A. C.·Kitchen, P.·Bill, R. M.·MacDonald, J. A.
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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 M. CaM bound two C-terminal AQP4 peptides with interactions observed for both the C- and N-lobes of CaM (Kd1: 3.6 M, Kd2: 113.6 M, 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 M. 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.

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Ishida, H., Vogel, H. J., Conner, A. C., Kitchen, P., Bill, R. M., MacDonald, J. A.. 2021-06-29. Simultaneous Binding of the N- and C-terminal Cytoplasmic Domains of Aquaporin 4 to Calmodulin May Contribute to Vesicular Trafficking. https://doi.org/10.1101/2021.06.29.450403

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