Mette Assentoft

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

Network activity in the brain is associated with a transient increase in extracellular K+ concentration. The excess K+ is removed from the extracellular space by mechanisms proposed to involve Kir4.1‐mediated spatial buffering, the Na+/K+/2Cl− cotransporter 1 (NKCC1), and/or Na+/K+‐ATPase activity. Their individual contribution to [K+]o management has been of extended controversy. This study aimed, by several complementary approaches, to delineate the transport characteristics of Kir4.1, NKCC1, and Na+/K+‐ATPase and to resolve their involvement in clearance of extracellular K+ transients. Primary cultures of rat astrocytes displayed robust NKCC1 activity with [K+]o increases above basal levels. Increased [K+]o produced NKCC1‐mediated swelling of cultured astrocytes and NKCC1 could thereby potentially act as a mechanism of K+ clearance while concomitantly mediate the associated shrinkage of the extracellular space. In rat hippocampal slices, inhibition of NKCC1 failed to affect the rate of K+ removal from the extracellular space while Kir4.1 enacted its spatial buffering only during a local [K+]o increase. In contrast, inhibition of the different isoforms of Na+/K+‐ATPase reduced post‐stimulus clearance of K+ transients. The astrocyte‐characteristic α2β2 subunit composition of Na+/K+‐ATPase, when expressed in Xenopus oocytes, displayed a K+ affinity and voltage‐sensitivity that would render this subunit composition specifically geared for controlling [K+]o during neuronal activity. In rat hippocampal slices, simultaneous measurements of the extracellular space volume revealed that neither Kir4.1, NKCC1, nor Na+/K+‐ATPase accounted for the stimulus‐induced shrinkage of the extracellular space. Thus, NKCC1 plays no role in activity‐induced extracellular K+ recovery in native hippocampal tissue while Kir4.1 and Na+/K+‐ATPase serve temporally distinct roles. GLIA 2014;62:608–622

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

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 Ser111 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 Ser111 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 Ser111 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.Ser111 recorded no phosphorylation‐induced change in water permeability. A phospho‐specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser111, 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 Ser111 and of phosphorylation‐dependent gating of AQP4.

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

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 Ser111 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 Ser111 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 Ser111 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.Ser111 recorded no phosphorylation‐induced change in water permeability. A phospho‐specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser111, 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 Ser111 and of phosphorylation‐dependent gating of AQP4.

H95 Is a pH-Dependent Gate in Aquaporin 4

Aquaporin 4 (AQP4) is a transmembrane protein from the aquaporin family and is the predominant water channel in the mammalian brain. The regulation of permeability of this protein could be of potential therapeutic use to treat various forms of damage to the nervous tissue. In this work, based on data obtained from in silico and in vitro studies, a pH sensitivity that regulates the osmotic water permeability of AQP4 is demonstrated. The results indicate that AQP4 has increased water permeability at conditions of low pH in atomistic computer simulations and experiments carried out on Xenopus oocytes expressing AQP4. With molecular dynamics simulations, this effect was traced to a histidine residue (H95) located in the cytoplasmic lumen of AQP4. A mutant form of AQP4, in which H95 was replaced with an alanine (H95A), loses sensitivity to cytoplasmic pH changes in in vitro osmotic water permeability, thereby substantiating the in silico work.

Characterization of a novel phosphorylation site in the sodium–chloride cotransporter, NCC

•  The sodium–chloride cotransporter, NCC, is essential for renal electrolyte balance and its function can be regulated by protein phosphorylation •  Here we report the role and regulation of a novel phosphorylation site in NCC at Ser124 •  Ser124 phosphorylation plays a role in mediating full NCC transport activity, but does not seem to be involved in NCC trafficking •  Various physiological stimuli such as vasopressin and aldosterone regulate the abundance of the Ser124 phosphorylation status and other phosphorylation sites in NCC •  Unlike other known phosphorylation sites in NCC, the STE20/SPS1‐related proline–alanine‐rich kinase and oxidative stress‐response kinases (SPAK and OSR1) were not able to phosphorylate NCC at Ser124 •  The results demonstrate that phosphorylation of NCC is a major factor in determining the function of NCC under various physiological conditions

Regulation and Function of AQP4 in the Central Nervous System.

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. Based on studies on AQP4−/− mice, AQP4 has been assigned physiological roles in stimulus-induced K+ clearance, paravascular fluid flow, and brain edema formation. Conflicting data have been presented on the role of AQP4 in K+ clearance and associated extracellular space shrinkage and on the stroke-induced alterations of AQP4 expression levels during edema formation, raising questions about the functional importance of AQP4 in these (patho)physiological aspects. Phosphorylation-dependent gating of AQP4 has been proposed as a regulatory mechanism for AQP4-mediated osmotic water transport. This paradigm was, however, recently challenged by experimental evidence and molecular dynamics simulations. Regulatory patterns and physiological roles for AQP4 thus remain to be fully explored.

AQP4 plasma membrane trafficking or channel gating is not significantly modulated by phosphorylation at COOH-terminal serine residues

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 serves as a water entry site during brain edema formation, and regulation of AQP4 may therefore be of therapeutic interest. Phosphorylation of aquaporins can regulate plasma membrane localization and, possibly, the unit water permeability via gating of the AQP channel itself. In vivo phosphorylation of six serine residues in the COOH terminus of AQP4 has been detected by mass spectrometry: Ser(276), Ser(285), Ser(315), Ser(316), Ser(321), and Ser(322). To address the role of these phosphorylation sites for AQP4 function, serine-to-alanine mutants were created to abolish the phosphorylation sites. All mutants were detected at the plasma membrane of transfected C6 cells, with the fraction of the total cellular AQP4 expressed at the plasma membrane of transfected C6 cells being similar between the wild-type (WT) and mutant forms of AQP4. Activation of protein kinases A, C, and G in primary astrocytic cultures did not affect the plasma membrane abundance of AQP4. The unit water permeability was determined for the mutant AQP4s upon heterologous expression in Xenopus laevis oocytes (along with serine-to-aspartate mutants of the same residues to mimic a phosphorylation). None of the mutant AQP4 constructs displayed alterations in the unit water permeability. Thus phosphorylation of six different serine residues in the COOH terminus of AQP4 appears not to be required for proper plasma membrane localization of AQP4 or to act as a molecular switch to gate the water channel.

Regulation of the Water Channel Aquaporin-2 via 14-3-3θ and -ζ.

The 14-3-3 family of proteins are multifunctional proteins that interact with many of their cellular targets in a phosphorylation-dependent manner. Here, we determined that 14-3-3 proteins interact with phosphorylated forms of the water channel aquaporin-2 (AQP2) and modulate its function. With the exception of σ, all 14-3-3 isoforms were abundantly expressed in mouse kidney and mouse kidney collecting duct cells (mpkCCD14). Long-term treatment of mpkCCD14 cells with the type 2 vasopressin receptor agonist dDAVP increased mRNA and protein levels of AQP2 alongside 14-3-3β and -ζ, whereas levels of 14-3-3η and -θ were decreased. Co-immunoprecipitation (co-IP) studies in mpkCCD14 cells uncovered an AQP2/14-3-3 interaction that was modulated by acute dDAVP treatment. Additional co-IP studies in HEK293 cells determined that AQP2 interacts selectively with 14-3-3ζ and -θ. Use of phosphatase inhibitors in mpkCCD14 cells, co-IP with phosphorylation deficient forms of AQP2 expressed in HEK293 cells, or surface plasmon resonance studies determined that the AQP2/14-3-3 interaction was modulated by phosphorylation of AQP2 at various sites in its carboxyl terminus, with Ser-256 phosphorylation critical for the interactions. shRNA-mediated knockdown of 14-3-3ζ in mpkCCD14 cells resulted in increased AQP2 ubiquitylation, decreased AQP2 protein half-life, and reduced AQP2 levels. In contrast, knockdown of 14-3-3θ resulted in increased AQP2 half-life and increased AQP2 levels. In conclusion, this study demonstrates phosphorylation-dependent interactions of AQP2 with 14-3-3θ and -ζ. These interactions play divergent roles in modulating AQP2 trafficking, phosphorylation, ubiquitylation, and degradation.

The Vasopressin Type-2 Receptor and Prostaglandin Receptors EP2 and EP4 can Increase Aquaporin-2 Plasma Membrane Targeting Through a cAMP Independent Pathway

Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be cAMP dependent. However, on the basis of recent reports, it was hypothesized in the current study that increased cAMP levels are not necessary for AQP2 membrane targeting. The role and dynamics of cAMP signaling in AQP2 membrane targeting in Madin-Darby canine kidney and mouse cortical collecting duct (mpkCCD14) cells was examined using selective agonists against the V2R (dDAVP), EP2 (butaprost), and EP4 (CAY10580). During EP2 stimulation, AQP2 membrane targeting continually increased during 80 min of stimulation; whereas cAMP levels reached a plateau after 10 min. EP4 stimulation caused a rapid and transient increase in AQP2 membrane targeting, but did not significantly increase cAMP levels. After washout of the EP2 agonist or dDAVP, AQP2 membrane abundance remained elevated for at least 80 min, whereas cAMP levels rapidly decreased. Similar effects of the EP2 agonist were also observed for AQP2 constitutively nonphosphorylated at ser-269. The adenylyl cyclase inhibitor SQ22536 did not prevent AQP2 targeting during stimulation of each receptor, nor after dDAVP washout. In conclusion, this study demonstrates that although direct stimulation with cAMP causes AQP2 membrane targeting, cAMP is not necessary for receptor-mediated AQP2 membrane targeting and Gs-coupled receptors can also signal through an alternative pathway that increases AQP2 membrane targeting.

The α2β2 isoform combination dominates the astrocytic Na+/K+-ATPase activity and is rendered nonfunctional by the α2.G301R familial hemiplegic migraine type 2-associated mutation

Synaptic activity results in transient elevations in extracellular K+, clearance of which is critical for sustained function of the nervous system. The K+ clearance is, in part, accomplished by the neighboring astrocytes by mechanisms involving the Na+/K+‐ATPase. The Na+/K+‐ATPase consists of an α and a β subunit, each with several isoforms present in the central nervous system, of which the α2β2 and α2β1 isoform combinations are kinetically geared for astrocytic K+ clearance. While transcript analysis data designate α2β2 as predominantly astrocytic, the relative quantitative protein distribution and isoform pairing remain unknown. As cultured astrocytes altered their isoform expression in vitro, we isolated a pure astrocytic fraction from rat brain by a novel immunomagnetic separation approach in order to determine the expression levels of α and β isoforms by immunoblotting. In order to compare the abundance of isoforms in astrocytic samples, semi‐quantification was carried out with polyhistidine‐tagged Na+/K+‐ATPase subunit isoforms expressed in Xenopus laevis oocytes as standards to obtain an efficiency factor for each antibody. Proximity ligation assay illustrated that α2 paired efficiently with both β1 and β2 and the semi‐quantification of the astrocytic fraction indicated that the astrocytic Na+/K+‐ATPase is dominated by α2, paired with β1 or β2 (in a 1:9 ratio). We demonstrate that while the familial hemiplegic migraine‐associated α2.G301R mutant was not functionally expressed at the plasma membrane in a heterologous expression system, α2+/G301R mice displayed normal protein levels of α2 and glutamate transporters and that the one functional allele suffices to manage the general K+ dynamics.