Identification and Molecular Mechanisms of the Rapid Tonicity-induced Relocalization of the Aquaporin 4 Channel

Philip Kitchen,Rebecca E Day,Luke H.J Taylor,Mootaz M Salman,Roslyn M Bill,Matthew T Conner,Alex C Conner

doi:10.1074/jbc.m115.646034

Abstract

The aquaporin family of integral membrane proteins is composed of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves PKA activation, influx of extracellular calcium, and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain edema.

Highlights

The water channel protein aquaporin 4 (AQP4) controls water permeability of the blood-brain barrier
The translocation response was not due to a reduction in extracellular potassium concentration as isotonic reduction of [Kϩ]o had no effect on Relative membrane expression (RME) (Fig. 2F, left), whereas hypotonicity in the presence of constant [Kϩ]o had the same effect as hypotonicity induced by dilution of all solutes (Fig. 2F, center)
AQP4-GFP fusion proteins in HEK293 cells and endogenous AQP4 in primary rat astrocytes rapidly relocalize to the plasma membrane in response to a reduction in local tonicity

Summary

Background

The water channel protein aquaporin 4 (AQP4) controls water permeability of the blood-brain barrier. We have identified a novel regulatory pathway of endogenous AQP4 translocation in primary cortical astrocytes This study describes this mechanism in a model HEK293 cell line and shows how tonicity-dependent changes in the environment lead to a calcium-dependent, calmodulin-dependent, and PKA-specific, reversible translocation of AQP4 to the cell surface. We identify a known kinase site and putative PKA target site, serine 276, in the C-terminal tail of AQP4 that is necessary for the translocation response. This site has been shown to be phosphorylated in vivo, and we show that the mechanism requires PKA activity in primary cortical astrocytes. Our data represent a novel mechanism that could be a new avenue for therapeutic discovery in the treatment of cytotoxic brain edema

Experimental Procedures

Results

Discussion