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.
