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

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 Ca 2+ -dependent CaMbinding properties of synthetic and recombinant AQP4 CBD peptides were assessed using fluorescence spectroscopy, isothermal titration calorimetry, and two-dimensional 1 H, 15 N-HSQC NMR with 15 N-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.

Insight into the Mammalian Aquaporin Interactome

“…Given that a structure-based drug design targeting individual AQPs remains challenging despite intense efforts [186][187][188][189][190], switching the focus to the AQP interactome may offer new therapeutics avenues for the treatment of human disease states in which AQPs play an important role. CaM C-term [111,126]; N-term [126] MST [111]; NMR [126] DGC ND IP [193][194][195]…”

“…A recent study using AQP4 N- and C-terminal peptides revealed CaM-binding domains located within both termini, with the N-terminus interacting with the N-lobe of calmodulin (Kd: 3.4 ± 0.8 µM, 1:1 binding ratio) and the C-terminus interacting with both the C- and N-lobes of CaM (Kd1: 3.6 ± 1.3 µM; Kd2: 113.6 ± 8.0 µM, 2:1 binding ratio). Moreover, both peptides could interact with CaM simultaneously, forming a ternary complex [ 126 ]; however, it remains to be established if this is also possible in the full-length context. It has been debated whether the AQP4–CaM interaction is strengthened [ 111 ] or not [ 126 ] upon AQP4 phosphorylation at Ser276, a PKA site that has been shown to be involved in AQP4 plasma membrane localization [ 111 ].…”

“…Moreover, both peptides could interact with CaM simultaneously, forming a ternary complex [ 126 ]; however, it remains to be established if this is also possible in the full-length context. It has been debated whether the AQP4–CaM interaction is strengthened [ 111 ] or not [ 126 ] upon AQP4 phosphorylation at Ser276, a PKA site that has been shown to be involved in AQP4 plasma membrane localization [ 111 ]. As this apparent discrepancy might be related to the use of different methodologies, further studies will be required to clarify this.…”

CWLP and PRP940 form plasma-membrane nanodomain complexes with aquaporins, interact with PP2A and contribute to dehydration tolerance

“…From the viewpoint of aquaporin regulation, it is intriguing that all CWLP-interacting PP2A-B’’ subunits contain EF-hand motifs similar to those of calmodulins (CaMs). Whereas the 8CM domain of Arabidopsis snLTP1 is found to bind calmodulin (CaM) in a Ca 2+ -dependent manner (169), CaM-binding to human aquaporins inhibits their activity, which is counteracted by phosphorylation of C-terminal Ser residues (170). Therefore, additional studies of DRM complexes are required to determine whether calmodulins, other cargos, protein kinases, known scaffold proteins, signaling factors, and AF network components are associated with CWLP- and PRP940-marked nanodomains.…”

Mechanisms of aquaporin‐4 vesicular trafficking in mammalian cells

“…The most well understood is that of AQP2, which is expressed in the kidney collecting duct and is triggered by vasopressin (Centrone et al., 2022). The trafficking mechanism of AQP4 has been shown to be calmodulin (CaM) and phosphorylation dependent (Ishida et al., 2022; Kitchen, Day, Salman, et al., 2015; Kitchen, Day, Taylor, et al., 2015). Triggered by changes to the extracellular environment, Ca 2+ ions enter the cell via TRPV4 channels and activate calmodulin.…”