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. 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 K_d of 3.4 μM. The C-terminal AQP4 peptide interacted with both the C- and N-lobes of CaM (2:1 binding ratio; K_d1: 3.6 μM, K_d2: 113.6 μM, respectively). A recombinant AQP4 protein domain (recAQP4^CT, containing the entire cytosolic C-terminal sequence) bound CaM in a 1:1 binding mode with a K_d 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.

Keywords: AQP4; Aquaporin; CaM; Calmodulin; Calmodulin-binding domain; NMR; Nuclear magnetic resonance spectroscopy.