Aquaporin-4 Water Permeability Research Articles

The Water Permeability and Pore Entrance Structure of Aquaporin-4 Depend on Lipid Bilayer Thickness

Aquaporin-4 (AQP4), the primary water channel in glial cells of the mammalian brain, plays a critical role in water transport in the central nervous system. Previous experiments have shown that the water permeability of AQP4 depends on the cholesterol content in the lipid bilayer, but it was not clear whether changes in permeability were due to direct cholesterol-AQP4 interactions or to indirect effects caused by cholesterol-induced changes in bilayer elasticity or bilayer thickness. To determine the effects resulting only from bilayer thickness, here we use a combination of experiments and simulations to analyze AQP4 in cholesterol-free phospholipid bilayers with similar elastic properties but different hydrocarbon core thicknesses previously determined by x-ray diffraction. The channel (unit) water permeabilities of AQP4 measured by osmotic-gradient experiments were 3.5 ± 0.2 × 10−13 cm3/s (mean ± SE), 3.0 ± 0.3 × 10−13 cm3/s, 2.5 ± 0.2 × 10−13 cm3/s, and 0.9 ± 0.1 × 10−13 cm3/s in bilayers containing (C22:1)(C22:1)PC, (C20:1)(C20:1)PC, (C16:0)(C18:1)PC, and (C13:0)(C13:0)PC, respectively. Channel permeabilities obtained by molecular dynamics (MD) simulations were 3.3 ± 0.1 × 10−13 cm3/s and 2.5 ± 0.1 × 10−13 cm3/s in (C22:1)(C22:1)PC and (C14:0)(C14:0)PC bilayers, respectively. Both the osmotic-gradient and MD-simulation results indicated that AQP4 channel permeability decreased with decreasing bilayer hydrocarbon thickness. The MD simulations also suggested structural modifications in AQP4 in response to changes in bilayer thickness. Although the simulations showed no appreciable changes to the radius of the pore located in the hydrocarbon region of the bilayers, the simulations indicated that there were changes in both pore length and α-helix organization near the cytoplasmic vestibule of the channel. These structural changes, caused by mismatch between the hydrophobic length of AQP4 and the bilayer hydrocarbon thickness, could explain the observed differences in water permeability with changes in bilayer thickness.

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.

A new gating site in human aquaporin-4: Insights from molecular dynamics simulations

Aquaporin-4 (AQP4) is the predominant water channel in different organs and tissues. An alteration of its physiological functioning is responsible for several disorders of water regulation and, thus, is considered an attractive target with a promising therapeutic and diagnostic potential. Molecular dynamics (MD) simulations performed on the AQP4 tetramer embedded in a bilayer of lipid molecules allowed us to analyze the role of spontaneous fluctuations occurring inside the pore. Following the approach by Hashido et al. [Hashido M, Kidera A, Ikeguchi M (2007) Biophys J 93: 373–385], our analysis on 200ns trajectory discloses three domains inside the pore as key elements for water permeation. Herein, we describe the gating mechanism associated with the well-known selectivity filter on the extracellular side of the pore and the crucial regulation ensured by the NPA motifs (asparagine, proline, alanine). Notably, on the cytoplasmic side, we find a putative gate formed by two residues, namely, a cysteine belonging to the loop D (C178) and a histidine from loop B (H95). We observed that the spontaneous reorientation of the imidazole ring of H95 acts as a molecular switch enabling H-bond interaction with C178. The occurrence of such local interaction seems to be responsible for the narrowing of the pore and thus of a remarkable decrease in water flux rate. Our results are in agreement with recent experimental observations and may represent a promising starting point to pave the way for the discovery of chemical modulators of AQP4 water permeability.

Inhibition of aquaporin-1 but not aquaporin-4 water permeability by bacopaside I derived from Bacopa monnieri

Inhibition of aquaporin-1 but not aquaporin-4 water permeability by bacopaside I derived from Bacopa monnieri

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.

Water Permeability of Aquaporin-4 Channel Depends on Bilayer Composition, Thickness, and Elasticity

Aquaporin-4 (AQP4) is the primary water channel in the mammalian brain, particularly abundant in astrocytes, whose plasma membranes normally contain high concentrations of cholesterol. Here we test the hypothesis that the water permeabilities of two naturally occurring isoforms (AQP4-M1 and AQP4-M23) depend on bilayer mechanical/structural properties modulated by cholesterol and phospholipid composition. Osmotic stress measurements were performed with proteoliposomes containing AQP4 and three different lipid mixtures: 1), phosphatidylcholine (PC) and phosphatidylglycerol (PG); 2), PC, PG, with 40 mol % cholesterol; and 3), sphingomyelin (SM), PG, with 40 mol % cholesterol. The unit permeabilities of AQP4-M1 were 3.3 ± 0.4 × 10−13 cm3/s (mean ± SE), 1.2 ± 0.1 × 10−13 cm3/s, and 0.4 ± 0.1 × 10−13 cm3/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. The unit permeabilities of AQP4-M23 were 2.1 ± 0.2 × 10−13 cm3/s, 0.8 ± 0.1 × 10−13 cm3/s, and 0.3 ± 0.1 × 10−13 cm3/s in PC:PG, PC:PG:cholesterol, and SM:PG:cholesterol, respectively. Thus, for each isoform the unit permeabilities strongly depended on bilayer composition and systematically decreased with increasing bilayer compressibility modulus and bilayer thickness. These observations suggest that altering lipid environment provides a means of regulating water channel permeability. Such permeability changes could have physiological consequences, because AQP4 water permeability would be reduced by its sequestration into SM:cholesterol-enriched raft microdomains. Conversely, under ischemic conditions astrocyte membrane cholesterol content decreases, which could increase AQP4 permeability.

Neuromyelitis optica IgG does not alter aquaporin‐4 water permeability, plasma membrane M1/M23 isoform content, or supramolecular assembly

Neuromyelitis optica (NMO) is thought to be caused by immunoglobulin G autoantibodies (NMO-IgG) against astrocyte water channel aquaporin-4 (AQP4). A recent study (Hinson et al. (2012) Proc Natl Acad Sci USA 109:1245-1250) reported that NMO-IgG inhibits AQP4 water permeability directly and causes rapid cellular internalization of the M1 but not M23 isoform of AQP4, resulting in AQP4 clustering, enhanced complement-dependent cytotoxicity, and tissue swelling. Here, we report evidence challenging this proposed mechanism of NMO-IgG-mediated pathology. We measured osmotic water permeability by stopped-flow light scattering on plasma membrane vesicles isolated from AQP4-expressing CHO cells, an approach that can detect changes in water permeability as small as 5% and is not confounded by internalization effects. We found similar single-molecule water permeability for M1-AQP4 tetramers and M23-AQP4 clusters (orthogonal arrays of particles, OAPs). Exposure of AQP4 to high concentrations of NMO-IgG from six seropositive NMO patients, and to high-affinity recombinant monoclonal NMO antibodies, did not reduce AQP4 water permeability. Also, NMO-IgG did not reduce water permeability in AQP4-reconstituted proteoliposomes. In transfected cells expressing M1- or M23-AQP4 individually, NMO-IgG caused more rapid internalization of M23- than M1-AQP4. In cells coexpressing both isoforms, M1- and M23-AQP4 comingled in OAPs that were internalized together in response to NMO-IgG. Super-resolution imaging and native gel electrophoresis showed that the size of AQP4 OAPs was not altered by NMO sera or recombinant NMO antibodies. We conclude that NMO-IgG does not: (i) inhibit AQP4 water permeability, (ii) cause preferential internalization of M1-AQP4, or (iii) cause intramembrane AQP4 clustering.

Consequences of NMO-IgG binding to aquaporin-4 in neuromyelitis optica

Hinson et al. (1) suggested a unique mechanism for the pathogenesis of neuromyelitis optica (NMO), a neuroinflammatory disease caused by binding of an autoantibody (NMO-IgG) to aquaporin-4 (AQP4) on astrocyte plasma membranes. AQP4 is expressed as long (called M1) and short (called M23) isoforms, the latter forming orthogonal arrays of particles (OAPs). Hinson et al. (1) reported that M1-AQP4 is rapidly internalized in cells whereas M23-AQP4 resists internalization, and that NMO-IgG inhibits AQP4 water permeability. They propose that NMO pathogenesis involves NMO-IgG–induced internalization of M1-AQP4 and direct inhibition of AQP4 water permeability, resulting in increased OAP size, enhanced complement-dependent cytotoxicity, and tissue swelling. Although their mechanism is unique and interesting, data from our laboratory and others challenge each of their major findings and hence the validity of their model.

Rapid and Reversible Inhibition of Aquaporin-4 by Zinc

Aquaporin-4 (AQP4) is the predominant water channel in the brain. Although AQP4 plays an important role in brain water homeostasis, the molecular mechanisms of AQP4 regulation are not fully understood. In this report, we show how Zn(2+) rapidly and reversibly decreases the water permeability of AQP4 when it is reconstituted into proteoliposomes. Mutagenesis analysis identified Cys178, located in cytoplasmic loop D, as a target residue of ZnCl(2) inhibition. Moreover, treatment with diamide enhanced the inhibitory effects of ZnCl(2). These results suggest that the water permeability of AQP4 may be regulated by dynamic changes in intracellular Zn(2+) concentration linked to the cellular redox state.

Inhibition of Aquaporin-1 and Aquaporin-4 Water Permeability by a Derivative of the Loop Diuretic Bumetanide Acting at an Internal Pore-Occluding Binding Site

Aquaporin (AQP) water channels, essential for fluid homeostasis, are expressed in perivascular brain end-feet regions of astroglia (AQP4) and in choroid plexus (AQP1). At a high concentration, the loop diuretic bumetanide has been shown to reduce rat brain edema after ischemic stroke by blocking Na(+)-K(+)-2Cl(-) cotransport. We hypothesized that an additional inhibition of AQP contributes to the protection. We show that osmotic water flux in AQP4-expressing Xenopus laevis oocytes is reduced by extracellular bumetanide (> or =100 microM). The efficacy of block by bumetanide is increased by injection intracellularly. Forty-five synthesized bumetanide derivatives were tested on oocytes expressing human AQP1 and rat AQP4. Of these, one of the most effective was the 4-aminopyridine carboxamide analog, AqB013, which inhibits AQP1 and AQP4 (IC(50) approximately 20 microM, applied extracellularly). The efficacy of block was enhanced by mutagenesis of intracellular AQP4 valine-189 to alanine (V189A, IC(50) approximately 8 microM), confirming the aquaporin as the molecular target of block. In silico docking of AqB013 supported an intracellular candidate binding site in rat AQP4 and suggested that the block involves occlusion of the AQP water pore at the cytoplasmic side. AqB013 at 2 microM had no effect, and 20 microM caused 20% block of human Na(+)-K(+)-2Cl(-) cotransporter activity, in contrast to >90% block of the transporter by bumetanide. AqB013 did not affect X. laevis oocyte Cl(-) currents and did not alter rhythmic electrical conduction in an ex vivo gastric muscle preparation. The identification of AQP-selective pharmacological agents opens opportunities for breakthrough strategies in the treatment of edema and other fluid imbalance disorders.

Lack of aquaporin-4 water transport inhibition by antiepileptics and arylsulfonamides

Inhibitors of brain glial water channel aquaporin-4 (AQP4) are of potential clinical utility, as they are predicted to modulate brain edema, neuroexcitation and glial scarring. Recently, Huber et al. ( Bioorg. Med. Chem. 2007, 17, 1270–1273; in press) reported that a series of arylsulfonamides, antiepileptics, and related small molecules strongly inhibited AQP4 water transport with IC 50s down to 1 μM. We retested the compounds with greatest reported potencies, including acetylsulfanilamide, acetazolamide, 6-ethoxy-benzothiazole-2-sulfonamide, topiramate, zonisamide, phenytoin, lamotrigine, and sumatriptan, in AQP4-transfected mammalian cells and primary cultures of brain glial cells, using several sensitive assays of osmotic water permeability. Contrary to the findings of Huber et al., in our studies we found no significant inhibition of AQP4 water permeability by any of the compounds at concentrations up to 100 μM.

Aquaporin-4 independent Kir4.1 K + channel function in brain glial cells

Functional interaction of glial water channel aquaporin-4 (AQP4) and inwardly rectifying K + channel Kir4.1 has been suggested from their apparent colocalization and biochemical interaction, and from the slowed glial cell K + uptake in AQP4-deficient brain. Here, we report multiple lines of evidence against functionally significant AQP4–Kir4.1 interactions. Whole-cell patch-clamp of freshly isolated glial cells from brains of wild-type and AQP4 null mice showed no significant differences in membrane potential, barium-sensitive Kir4.1 K + current or current–voltage curves. Single-channel patch-clamp showed no differences in Kir4.1 unitary conductance, voltage-dependent open probability or current–voltage relationship. Also, Kir4.1 protein expression and distribution were similar in wild-type and AQP4 null mouse brain and in the freshly isolated glial cells. Functional inhibition of Kir4.1 by barium or RNAi knock-down in primary glial cell cultures from mouse brain did not significantly alter AQP4 water permeability, as assayed by calcein fluorescence quenching following osmotic challenge. These studies provide direct evidence against functionally significant AQP4–Kir4.1 interactions in mouse glial cells, indicating the need to identify new mechanism(s) to account for altered seizure dynamics and extracellular space K + buffering in AQP4 deficiency.

Lead induces increased water permeability in astrocytes expressing aquaporin 4

The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we report that lead increases AQP4 water permeability (P f) in astrocytes. A rat astrocyte cell line that does not express aquaporin 4 was transiently transfected with aquaporin 4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability in these cells and in AQP4-negative cells located on the same plate. AQP4-expressing astrocytes had a three-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 40%, increase in water permeability in astrocytes expressing AQP4, but had no effect on P f in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout, while treatment with a lead chelator, meso-2,3-dimercaptosuccinic acid, abolished the lead-induced increase in P f. The effect of lead was attenuated in the presence of a calcium (Ca 2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, but not in the presence of a protein kinase C inhibitor. In cells expressing AQP4 where the consensus site for CaMKII phosphorylation was mutated, lead failed to increase water permeability. Lead exposure also increased P f in rat astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on P f in astrocytes transfected with aquaporin 3. In situ hybridization studies on rat brain after oral lead intake for three days showed no change in distribution of AQP4 mRNA. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may contribute to the pathology of acute lead intoxication.

101 Water Permeability in Astrocytes and The Effect of Lead Intoxication

Background: The water channel aquaporin 4 (AQP4) is abundantly expressed in astrocytes. There is now compelling evidence that AQP4 may contribute to an unfavorable course in brain edema. Acute lead intoxication is a condition that causes brain damage preceded by brain edema. Here we have investigated the effect of lead on AQP4 water permeability in astrocytes and AQP4 mRNA expression in brain. Methods: An astrocyte cell line that did not express AQP4 was transiently transfected with AQP4 tagged with green fluorescent protein (GFP). Using confocal laser scanning microscopy we measured water permeability (Pf) in these cells and in AQP4-negative cells located on the same plate. Pf in astroglial cells in primary culture was also measured after lead exposure. The effect of lead on AQP4 mRNA distribution in the brain was studied by in situ hybridization. Results: AQP4-expressing astrocytes had more than 3-fold higher water permeability than astrocytes not expressing AQP4. Lead exposure induced a significant, 30%, increase in Pf in astrocytes expressing AQP4, but had no effect on Pf in astrocytes not expressing AQP4. The increase in water permeability persisted after lead washout. Lead exposure also increased Pf in astroglial cells in primary culture, which express endogenous AQP4. Lead had no effect on Pf in astrocytes transfected with another aquaporin, AQP3. In situ hybridization studies on adult rat brain showed no change in distribution of AQP4 mRNA after oral lead intake for three days. Conclusion: We here show that lead specifically increases water permeability in astrocytes expressing the water channel AQP4. Lead is so far the only heavy metal shown to modulate the effect of AQP4. It is suggested that lead-triggered stimulation of water transport in AQP4-expressing astrocytes may be a contributing factor to brain edema in acute lead intoxication.

Water permeability of aquaporin-4 is decreased by protein kinase C and dopamine.

Aquaporin-4 (AQP4) plays an important role in the basolateral movement of water in the collecting duct. Here we show that this water channel can be dynamically regulated. Water permeability (P(f)) was measured in individual LLC-PK1 cells that were transiently transfected with AQP4. To identify which cells were transfected, AQP4 was tagged at the NH2 terminus with green fluorescent protein. Transfected cells showed a strong fluorescent signal in basolateral membrane and a low-to-negligible signal in the cytosol and apical membrane. Activation of protein kinase C (PKC) with phorbol 12,13-dibutyrate (PDBu) significantly decreased P(f) of cells expressing AQP4 but had no effect on neighboring untransfected cells. No redistribution of AQP4 in response to PDBu was detected. Dopamine also decreased the P(f) in transfected cells. The effect was abolished by the PKC inhibitor Ro 31-8220. Reduction of AQP4 water permeability by PDBu and dopamine was abolished by point mutation of Ser(180), a consensus site for PKC phosphorylation. We conclude that PKC and dopamine decrease AQP4 water permeability via phosphorylation at Ser180 and that the effect is likely mediated by gating of the channel.