Calmodulin Gates Aquaporin 0 Permeability through a Positively Charged Cytoplasmic Loop

James B Fields,Karin L Németh-Cahalan,J Alfredo Freites,Irene Vorontsova,James E Hall,Douglas J Tobias

doi:10.1074/jbc.m116.743724

James B Fields, Karin L Németh-Cahalan + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m116.743724

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Abstract

Aquaporin 0 (AQP0), the major intrinsic protein of the eye lens, plays a vital role in maintaining lens clarity by facilitating the transport of water across lens fiber cell membranes. AQP0 reduces its osmotic water permeability constant (Pf) in response to increases in the external calcium concentration, an effect that is mediated by an interaction with the calcium-binding messenger protein, calmodulin (CaM), and phosphorylation of the CaM-binding site abolishes calcium sensitivity. Despite recent structural characterization of the AQP0-CaM complex, the mechanism by which CaM modulates AQP0 remains poorly understood. By combining atomistic molecular dynamics simulations and oocyte permeability assays, we conclude that serine phosphorylation of AQP0 does not inhibit CaM binding to the whole AQP0 protein. Instead, AQP0 phosphorylation alters calcium sensitivity by modifying the AQP0-CaM interaction interface, particularly at an arginine-rich loop that connects the fourth and fifth transmembrane helices. This previously unexplored loop, which sits outside of the canonical CaM-binding site on the AQP0 cytosolic face, mechanically couples CaM to the pore-gating residues of the second constriction site. We show that this allosteric loop is vital for CaM regulation of the channels, facilitating cooperativity between adjacent subunits and regulating factors such as serine phosphorylation. Similar allosteric interactions may also mediate CaM modulation of the properties of other CaM-regulated proteins.

Highlights

Aquaporin 0 (AQP0), the major intrinsic protein of the eye lens, plays a vital role in maintaining lens clarity by facilitating the transport of water across lens fiber cell membranes
We found that the electrostatic interface between the two proteins interacts with the more conventional CaM binding site to adjust the position of tyrosine 149 (Tyr149) at constriction site II and modulate the Pf of AQP0
Our Brownian dynamics (BD) trajectories were able to reproduce the correct AQP0-CaM binding orientation but only when Molecular dynamics (MD)-equilibrated input configurations of AQP0 and CaM, taken from the simulated AQP0CaM complex reported in Reichow et al [4], were used (Fig. 2 and supplemental Movie 1)

Summary

Results

Electrostatic Interactions between AQP0 and CaM Occur Outside of the AQP0 CaM Binding Domain—We performed BD simulations of the association of AQP0 with CaM using the atomistic model reported by Reichow et al [4] as the initial configuration by separating the CaM proteins from AQP0. Our BD trajectories were able to reproduce the correct AQP0-CaM binding orientation but only when MD-equilibrated input configurations of AQP0 and CaM, taken from the simulated AQP0CaM complex reported in Reichow et al [4], were used (Fig. 2 and supplemental Movie 1). The MD interaction potential energies of a single CaM monomer (with bound calcium ions) interacting with four variants of the AQP0 tetramer. The values are averaged over the two CaM monomers over the course of an ϳ500-ns equilibrated

Binding fraction

Discussion

Experimental Procedures