Phosphorylation of rat aquaporin‐4 at Ser¹¹¹ is not required for channel gating

Abstract

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser(111) has been reported to increase the water permeability of AQP4 expressed in an astrocytic cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser(111) was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range of activators and inhibitors of PKG and PKA. Mutation of Ser(111) to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations of a phosphorylation of AQP4.Ser(111) recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser(111) , failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. Thus, our data indicate a lack of phosphorylation of Ser(111) and of phosphorylation-dependent gating of AQP4.

Contributions of the Na+/K+‐ATPase, NKCC1, and Kir4.1 to hippocampal K+ clearance and volume responses

“…These cultures have previously been used for volume measurements in (Assentoft et al 2013; Ateya et al 2005). In brief, adult astrocytes were isolated from gelatin-sponge implants left in the animal for three days.…”

“…Astrocytes for volume measurements: adult male Sprague-Dawley rats (250-300 g) were used for the astrocytic preparation as described in detail in (Langan et al, 1995). These cultures have previously been used for volume measurements in (Assentoft et al, 2013;Ateya et al, 2005). In brief, adult astrocytes were isolated from gelatin-sponge implants left in the animal for 3 days.…”

Turning down the volume: Astrocyte volume change in the generation and termination of epileptic seizures

“…The NKA was recently shown to be capable of forming a macromolecular complex with AQP4 alongside the metabotropic glutamate receptor mGluR5, which may also regulate AQP4 permeability (Illarionova et al 2010). Group I mGluRs (such as mGluR5) or K ir 4.1 channels may indirectly increase or decrease astrocyte water permeability through phosphorylation or dephosphorylation, respectively, of AQP4 channels (Gunnarson et al 2008; Song and Gunnarson 2012), although these data are controversial (Assentoft et al 2013; Assentoft et al 2014). Lack of a direct functional interaction between NKA or K ir 4.1 and AQP4 could explain why Larsen and colleagues found neither K + uptake pathway to be directly responsible for stimulus-induced shrinkage of hippocampal tissue (Larsen et al 2014).…”

A novel human aquaporin-4 splice variant exhibits a dominant-negative activity: a new mechanism to regulate water permeability

“…However, in rat astrocyte primary cultures, which retain a high level of AQP4 protein, phosphorylation appears not involved in modulating its activity (Nicchia et al. , 2008), and Ser-111 appears not involved in AQP4 channel gating (Assentoft et al. , 2013).…”