The first water channel protein (later called aquaporin 1) was first discovered in Cluj-Napoca, Romania.

This invited review briefly outlines the importance of membrane water permeability, highlights the landmarks leading to the discovery of water channels. After a decade of systematic studies on water channels in human RBC Benga's group discovered in 1985 the presence and location of the water channel protein among the polypeptides migrating in the region of 35-60 kDa on the electrophoretogram of RBC membrane proteins. The work was extended and reviewed in several articles. In 1988, Agre and coworkers isolated a new protein from the RBC membrane, nick-named CHIP28 (channel-forming integral membrane protein of 28 kDa). However, in addition to the 28 kDa component, this protein had a 35-60 kDa glycosylated component, the one detected by the Benga's group. Only in 1992 Agre's group suggested that "it is likely that CHIP28 is a functional unit of membrane water channels". Half of the 2003 Nobel Prize in Chemistry was awarded to Peter Agre (Johns Hopkins University, Baltimore, USA) "for the discovery of water channels", actually the first water channel protein from the human red blood cell (RBC) membrane, known today as aquaporin 1 (AQP1). The seminal contributions from 1986 of the Benga's group were grossly overlooked by Peter Agre and by the Nobel Prize Committee. Thousands of science-related professionals from hundreds of academic and research units, as well as participants in several international scientific events, have signed as supporters of Benga; his priority is also mentioned in several comments on the 2003 Nobel Prize.     

Birth of water channel proteins-the aquaporins

If we compare aquaporin (as a proteic pathway for water permeation across biological membranes) with a child we can say that he had a very long gestation period. His possible existence was predicted for a long time (Overton in 1985, Stein and Danielli in 1956), some of his features (transport of water and its reversible inhibition) were assigned by Macey and Farmer in 1970, however this child was first detected by Benga and coworkers in 1986. We clearly demonstrated for the first time the presence and location of a water channel at the human RBC membrane among the polypeptides migrating in the region having 35-60 kDa on the electrophoretogram of RBC membranes, labeled with 203Hg-PCMBS in the conditions of specific inhibition of water diffusion; I suggested that a minor membrane protein that binds PCMBS is involved in water transport and also indicated the way in which the specific protein could be further characterized: by purification and reconstitution in liposomes. Our landmark papers in 1986 can be compared with the first detection of a child "in utero" by ultrasonography, since we discovered one of the essential components of the "aquaporin child" (a molecular weight of 35-60 kDa for the glycosylated component); we have also indicated the way to recognize him after birth (among other children of his group!): placing the isolated children in a certain environment and asking them to perform the same task (one should read: reconstitution studies in liposomes and measurement of water permeability), like aligning athletes for a running test. This was the only certain way to know that the child is really the fastest runner and not just one that is helping (by various means) another child to be fastest runner. A "new child" was observed in 1988 by Agre and coworkers, who identified a novel integral membrane protein in human RBCs having a non-glycosylated component of 28 kDa and a glycosylated component migrating as a diffuse band of 35-60 kDa; they suggested that the new protein (nick-named CHIP28 in 1991) may play a role in linkage of the membrane skeleton to the lipid bilayer. In 1992 Agre and coworkers suggested that CHIP28 is a functional unit of membrane water channels; by reconstitution in liposomes it was demonstrated that CHIP28 is a water channel itself rather than a water channel regulator. In other words the child we first detected was recognized as having the predicted qualities only in 1992. In 1993 CHIP28 was renamed aquaporin 1. Looking in retrospect, asking the crucial question, when was the first water channel protein, aquaporin 1, discovered, a fair and clear cut answer would be: the first water channel protein, now called aquaporin 1, was identified or "seen" in situ in the human RBC membrane by Benga and coworkers in 1986. It was again "seen" when it was by chance purified by Agre and coworkers in 1988 and was again identified when its main feature, the water transport property was found by Agre and coworkers in 1992. If a comparison with the discovery of The New World of America is made, the first man who has "seen" a part, very small indeed, of The New Land was Columbus; later, others, including Amerigo Vespucci (from whom the name derived), have better "seen" a larger part of the new Continent and in the subsequent years many explorers discovered the complexity of the Americas!     

Reconstitution of functional water channels in liposomes containing purified red cell CHIP28 protein.

Water rapidly crosses the plasma membranes of red blood cells (RBCs) and renal tubules through highly specialized channels. CHIP28 is an abundant integral membrane protein in RBCs and renal tubules, and Xenopus laevis oocytes injected with CHIP28 RNA exhibit high osmotic water permeability, Pf [Preston et al. (1992) Science 256, 385-387]. Purified CHIP28 from human RBCs was reconstituted into proteoliposomes in order to establish if CHIP28 is itself the functional unit of water channels and to characterize its physiological behavior. CHIP28 proteoliposomes exhibit Pf which is up to 50-fold above that of control liposomes, but permeability to urea and protons is not increased. Like intact RBC, the Pf of CHIP28 proteoliposomes is reversibly inhibited by mercurial sulfhydryl reagents and exhibits a low Arrhenius activation energy. The magnitude of CHIP28-mediated water flux (11.7 x 10(-14) cm3/s per CHIP28) corresponds to the known Pf of intact RBCs. These results demonstrate that CHIP28 protein functions as a molecular water channel and also indicate that CHIP28 is responsible for most transmembrane water movement in RBCs.     

Water channels in membranes

A systematic programme of comparative nuclear magnetic resonance measurements of the membrane permeability for water (Pd) and of activation energy (E_a,d) of this process in red blood cells of various wild, laboratory and domestic animals was carried out here. The RBC from humans, cow, sheep and kangaroos had P_d values around 5·10^?3 cm/s at 25 °C, 7 · 10^?3 cm/s at 37 °C with E_a,d values around 25 kJ/mol. For RBC from other ten marsupial species and from mouse, rat and rabbit, the P_d values were more than twice as high as for human RBC. For mosr RBC a high value of Pd was associated with a low value of E_a,d (range from 15 to 21 kJ/mol), pointing to specialized channels for water diffusion incorporated in membrane proteins. Recently a channel-forming integral protein of 28 kDa (CHIP 28) was identified as a major water channel protein in the RBC membrane. A procedure for quantitating the purified CHIP 28 by densitometry of silver-stained polyacrylmide gel electrophoreograms was developed. The analysis of a purified fraction of CHIP 28 showed that the 28 kDa component represents approximately two-thirds of the sample with the remainder comprising the glycosylated high-molecular-weight component. A correlation between the content in CHIP 28 and the relative water permeability among RBC from different vertebrate species was attempted.     

Functional reconstitution of the isolated erythrocyte water channel CHIP28.

Measurements of water permeability indicate the existence of a facilitated water transporting pathway in erythrocytes, kidney tubules and amphibian urinary bladder. Two lines of evidence suggest that one type of water channel is an approximately 30-kDa protein: the approximately 30-kDa target size determined by radiation inactivation (van Hoek, A. N., Hom, M. L., Luthjens, L. H., de Jong, M. D., Dempster, J. A., and van Os, C. H. (1991) J. Biol. Chem. 266, 16633-16635) and the increased water permeability in oocytes that express mRNA encoding a 28-kDa erythrocyte protein (CHIP28, Preston, B. M., Carroll, T. P., Guggino, W. B., and Agre, P. (1992) Science 256, 385-387). We report direct evidence that CHIP28 is the erythrocyte water channel. Osmotic water permeability (Pf) remained high (0.029 cm/s, 37 degrees C) when erythrocyte membranes were stripped of nearly all proteins except for CHIP28. N-terminal sequence analysis confirmed that the 28-kDa protein was CHIP28. Pf in proteoliposomes reconstituted with solubilized CHIP28 was high (Pf = 0.03 cm/s, 37 degrees C), the activation energy was low (2.2 kcal/mol), and Pf was decreased by greater than 50-fold by mercurial sulfhydryl reagents and Me2SO. The single-channel water permeability was approximately 10(-13) cm3/s, slightly higher than that of the gramicidin A channel. The water channel excluded the small solute urea. These data establish a procedure to reconstitute functional water channels into liposomes and demonstrate that CHIP28 is the erythrocyte water channel.     

CHIP28 water channels are localized in constitutively water-permeable segments of the nephron

The sites of water transport along the nephron are well characterized, but the molecular basis of renal water transport remains poorly understood. CHIP28 is a 28-kD integral protein which was proposed to mediate transmembrane water movement in red cells and kidney (Preston, G. M., T. P. Carroll, W. B. Guggino, and P. Agre. 1992. Science [Wash. DC]. 256:385-387). To determine whether CHIP28 could account for renal epithelial water transport, we used specific polyclonal antibodies to quantitate and localize CHIP28 at cellular and subcellular levels in rat kidney using light and electron microscopy. CHIP28 comprised 3.8% of isolated proximal tubule brush border protein. Except for the first few cells of the S1 segment, CHIP28 was immunolocalized throughout the convoluted and straight proximal tubules where it was observed in the microvilli of the apical brush border and in basolateral membranes. Very little CHIP28 was detected in endocytic vesicles or other intracellular structures in proximal tubules. Uninterrupted, heavy immunostaining of CHIP28 was also observed over both apical and basolateral membranes of descending thin limbs, including both short and long loops of Henle. These nephron sites have constitutively high osmotic water permeabilities. CHIP28 was not detected in ascending thin limbs, thick ascending limbs, or distal tubules, which are highly impermeable to water. Moreover, CHIP28 was not detected in collecting duct epithelia, where water permeability is regulated by antidiuretic hormone. These determinations of abundance and structural organization provide evidence that the CHIP28 water channel is the predominant pathway for constitutive transepithelial water transport in the proximal tubule and descending limb of Henle's loop.     

Permeability and Reflection Coefficients of Urea and Small Amides in the Human Red Cell

Measurement of the transport parameters that govern the passage of urea and amides across the red cell membrane leads to important questions about transport of water. It had initially been thought that small protein channels, permeable to water and small solutes, traversed the membrane (see Solomon, 1987). Recently, however, very strong evidence has been presented that the 28 kDa protein, CHIP28, found in the red cell membrane, is the locus of the water channel (see Agre et al., 1993). CHIP28 transports water very rapidly but does not transport small nonelectrolytes such as urea. The irreversible thermodynamic parameter, σ _i , the reflection coefficient, is a measure of the relationship between the permeability of the solute and that of water. If a solute permeates by dissolution in the membrane, σ _i = 1.0; if it permeates by passage through an aqueous channel, σ _i < 1.0. For urea, Goldstein and Solomon (1960) found that σ_urea= 0.62 ± 0.03 which meant that urea crosses the red cell membrane in a water-filled channel. This result and many subsequent observations that showed that σ_urea < 1.0 are at variance with the observation that CHIP28 is impermeable to urea. In view of this problem, we have made a new series of measurements of σ _i for urea and other small solutes by a different method, which obviates many of the criticisms Macey and Karan (1993) have made of our earlier method. The new method (Chen et al., 1988), which relies upon fluorescence of the intracellular dye, fluorescein sulfonate, leads to the corrected value, σ_urea,corr= 0.64 ± 0.03 for ghosts, in good agreement with earlier data for red cells. Thus, the conclusion on irreversible thermodynamic and other grounds that urea and water share a common channel is in disagreement with the view that CHIP28 provides the sole channel for water entrance into the cell. Received: 6 February 1996/Revised: 20 May 1996     

Aquaporin-1, nothing but a water channel

Aquaporin-1 (AQP1) is a membrane channel that allows rapid water movement driven by a transmembrane osmotic gradient. It was claimed to have a secondary function as a cyclic nucleotide-gated ion channel. However, upon reconstitution into planar bilayers, the ion channel exhibited a 10-fold lower single channel conductance than in Xenopus oocytes and a 100-fold lower open probability (<10(-6)) of doubtful physiological significance (Saparov, S. M., Kozono, D., Rothe, U., Agre, P., and Pohl, P. (2001) J. Biol. Chem. 276, 31515-31520). Investigating AQP1 expressed in human embryonic kidney cells, we now have shown that the discrepancy is not due to alterations of AQP1 properties upon reconstitution into bilayers but rather to regulatory processes of the oocyte expression system that may have been misinterpreted as AQP1 ion channel activity. As confirmed by laser scanning reflection microscopy, from 0.8 to 1.4 x 10(6) AQP1 copies/cell contributed to osmotic cell swelling. The proper plasma membrane localization was confirmed by observing the fluorescence of the N-terminal yellow fluorescent protein tag. Whole-cell patch clamp experiments of wild type or tagged AQP1-expressing cells revealed that neither cGMP nor cAMP mediated ion channel activity. The lack of significant CNG ion channel activity rules out a secondary role of AQP1 water channels in cellular signal transduction.     

The AQP2 water channel: Effect of vasopressin treatment,microtubule disruption,and distribution in neonatal rats

Aquaporin 2 is a collecting duct water channel that is located in apical vesicles and in the apical plasma membrane of collecting duct principal cells. It shares 42% identity with the proximal tubule/thin descending limb water channel, CHIP28. The present study was aimed at addressing three questions concerning the location and behavior of the AQP2 protein under different conditions. First, does the AQP2 channel relocate to the apical membrane after vasopressin treatment? Our results show that AQP2 is diffusely distributed in cytoplasmic vesicles in collecting duct principal cells of homozygous Brattleboro rats that lack vasopressin. In rats injected with exogenous vasopressin, however, AQP2 became concentrated in the apical plasma membrane of principal cells, as determined by immunofluorescence and immunogold electron microscopy. This behavior is consistent with the idea that AQP2 is the vasopressin-sensitive water channel. Second, is the cellular location of AQP2 modified by microtubule disruption? In normal rats, AQP2 has a mainly apical and subapical location in principal cells, but in colchicine-treated rats, it is distributed on vesicles that are scattered throughout the entire cytoplasm. This is consistent with the dependence on microtubules of apical protein targeting in many cell types, and explains the inhibitory effect of microtubule disruption on the hydroosmotic response to vasopressin in sensitive epithelia, including the collecting duct. Third, is AQP2 present in neonatal rat kidneys? We show that AQP2 is abundant in principal cells from neonatal rats at all days after birth. The detection of AQP2 in early neonatal kidneys indicates that a lack of this protein is not responsible for the relatively weak urinary concentrating response to vasopressin seen in neonatal rats.     

Comparative simulations of aquaporin family: AQP1, AQPZ, AQP0 and GlpF

Molecular dynamics simulations were performed for four members of the aquaporin family (AQP1, AQPZ, AQP0, and GlpF) in the explicit membrane environment. The single-channel water permeability, pf, was evaluated to be GlpF approximately AQPZ > AQP1 > AQP0, while their relative pore sizes were GlpF > AQP1 > AQPZ > AQP0. This relation between pf and pore size indicates that water permeability was determined not only by the channel radius, but also another competing factor. Analysis of water dynamics revealed that this factor was the single-file nature of water transport.     

Water and ion permeation of aquaporin-1 in planar lipid bilayers. Major differences in structural determinants and stoichiometry.

The aquaporin-1 (AQP1) water channel protein is known to facilitate the rapid movement of water across cell membranes, but a proposed secondary role as an ion channel is still unsettled. Here we describe a method to simultaneously measure water permeability and ion conductance of purified human AQP1 after reconstitution into planar lipid bilayers. Water permeability was determined by measuring Na(+) concentrations adjacent to the membrane. Comparisons with the known single channel water permeability of AQP1 indicate that the planar lipid bilayers contain from 10(6) to 10(7) water channels. Addition of cGMP induced ion conductance in planar bilayers containing AQP1, whereas cAMP was without effect. The number of water channels exceeded the number of active ion channels by approximately 1 million-fold, yet p-chloromethylbenzenesulfonate inhibited the water permeability but not ion conductance. Identical ion channel parameters were achieved with AQP1 purified from human red blood cells or AQP1 heterologously expressed in Saccharomyces cerevisae and affinity purified with either N- or C-terminal poly-histidine tags. Rp-8-Br-cGMP inhibited all of the observed conductance levels of the cation selective channel (2, 6, and 10 pS in 100 mm Na(+) or K(+)). Deletion of the putative cGMP binding motif at the C terminus by introduction of a stop codon at position 237 yielded a truncated AQP1 protein that was still permeated by water but not by ions. Our studies demonstrate a method for simultaneously measuring water permeability and ion conductance of AQP1 reconstituted into planar lipid bilayers. The ion conductance occurs (i) through a pathway distinct from the aqueous pathway, (ii) when stimulated directly by cGMP, and (iii) in only an exceedingly small fraction of AQP1 molecules.     

Tetrameric assembly of CHIP28 water channels in liposomes and cell membranes: a freeze-fracture study

Channel forming integral protein of 28 kD (CHIP28) functions as a water channel in erythrocytes, kidney proximal tubule and thin descending limb of Henle. CHIP28 morphology was examined by freeze-fracture EM in proteoliposomes reconstituted with purified CHIP28, CHO cells stably transfected with CHIP28k cDNA, and rat kidney tubules. Liposomes reconstituted with HPLC-purified CHIP28 from human erythrocytes had a high osmotic water permeability (Pf0.04 cm/s) that was inhibited by HgCl2. Freeze-fracture replicas showed a fairly uniform set of intramembrane particles (IMPs); no IMPs were observed in liposomes without incorporated protein. By rotary shadowing, the IMPs had a diameter of 8.5 +/- 1.3 nm (mean +/- SD); many IMPs consisted of a distinct arrangement of four smaller subunits surrounding a central depression. IMPs of similar size and appearance were seen on the P-face of plasma membranes from CHIP28k-transfected (but not mock-transfected) CHO cells, rat thin descending limb (TDL) of Henle, and S3 segment of proximal straight tubules. A distinctive network of complementary IMP imprints was observed on the E-face of CHIP28-containing plasma membranes. The densities of IMPs in the size range of CHIP28 IMPs, determined by non-linear regression, were (in IMPs/microns 2): 2,494 in CHO cells, 5,785 in TDL, and 1,928 in proximal straight tubules; predicted Pf, based on the CHIP28 single channel water permeability of 3.6 x 10(-14) cm3/S (10 degrees C), was in good agreement with measured Pf of 0.027 cm/S, 0.075 cm/S, and 0.031 cm/S, respectively, in these cell types. Assuming that each CHIP28 monomer is a right cylindrical pore of length 5 nm and density 1.3 g/cm3, the monomer diameter would be 3.2 nm; a symmetrical arrangement of four cylinders would have a greatest diameter of 7.2 nm, which after correction for the thickness of platinum deposit, is similar to the measured IMP diameter of approximately 8.5 nm. These results provide a morphological signature for CHIP28 water channels and evidence for a tetrameric assembly of CHIP28 monomers in reconstituted proteoliposomes and cell membranes.     

Aquaporin-1 in the peritoneal membrane: Implications for water transport across capillaries and peritoneal dialysis

Peritoneal dialysis (PD) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced in the peritoneal cavity. The dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores (radius < 3 A) responsible for the transport of solute-free water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water perfectly fit with the characteristics of the ultrasmall pore. Treatment with corticosteroids induces the expression of AQP1 in peritoneal capillaries and increases water permeability and ultrafiltration in rats, without affecting the osmotic gradient and the permeability for small solutes. Studies in knockout mice provided further evidence that osmotically-driven water transport across the peritoneal membrane is mediated by AQP1. AQP1 and endothelial NO synthase (eNOS) show a distinct regulation within the endothelium lining peritoneal capillaries. In acute peritonitis, the upregulation of eNOS and increased release of NO dissipate the osmotic gradient and result in ultrafiltration failure, despite the unchanged expression of AQP1. These data illustrate the potential of the peritoneal membrane to investigate the role and regulation of AQP1 in the endothelium. They also emphasize the critical role of AQP1 during peritoneal dialysis and suggest that manipulating AQP1 expression may be used to increase water permeability across the peritoneal membrane.     

Lipopolysaccharide changes the subcellular distribution of aquaporin 5 and increases plasma membrane water permeability in mouse lung epithelial cells

Aquaporin-5 (AQP5), a major water channel in lung epithelial cells, plays an important role in maintaining water homeostasis in the lungs. Cell surface expression of AQP5 is regulated by not only mRNA and protein synthesis but also changes in subcellular distribution. We investigated the effect of lipopolysaccharide (LPS) on the subcellular distribution of AQP5 in a mouse lung epithelial cell line (MLE-12). LPS caused significant increases in AQP5 in the plasma membrane at 0.5-2 h. Immunofluorescence and Western blotting strongly suggested that LPS altered AQP5 subcellular distribution from an intracellular vesicular compartment to the plasma membrane. The specific p38 MAP kinase inhibitor SB 203580 apparently prevented LPS-induced changes in AQP5 distribution. Furthermore, LPS increased the osmotic water permeability of MLE-12 cells. These findings demonstrate that LPS increases cell surface AQP5 expression by changing its subcellular distribution and increases membrane osmotic water permeability through activation of p38 MAP kinase.     

水通道蛋白的生理功能 ——水通道基因敲除小鼠表型研究进展

水通道蛋白 (aquaporin, AQP) 是一族细胞膜上选择性高效转运水分子的特异孔道. 自从 Agre 等于 1992 年从红细胞膜发现第一个水通道蛋白 AQP1以来,有关水通道蛋白结构与功能的研究取得了迅速的、系列性的进展 . 已报道的哺乳动物 AQP 家族已有 11 个在蛋白质序列上有同源性成员 (AQP0~AQP10). AQP 在体内各系统组织中广泛表达,除了在与体液分泌和吸收密切相关的多种上皮和内皮细胞高表达外,在一些与体液转运无明显关系的组织细胞如红细胞、白细胞、脂肪细胞和骨骼肌细胞等处也有表达,提示 AQP 可能在多种器官生理和病理中发挥重要作用. 基因打靶技术是研究特定基因在体内生理功能的有力手段. 目前 AQP1、3、4、5 基因敲除和 AQP2 基因点突变的基因敲入小鼠模型 ( 模拟人类常染色体隐性遗传尿崩症 ) 已成功建立并广泛用于表型研究,在 AQP 水通道蛋白生理功能方面获得许多重要进展.     

All-trans retinoic acid increases expression of aquaporin-5 and plasma membrane water permeability via transactivation of Sp1 in mouse lung epithelial cells

Aquaporin-5 (AQP5) is a water-selective channel protein that is expressed in lacrimal glands, salivary glands, and distal lung. Several studies using AQP5 knockout mice have revealed that AQP5 plays an important role in maintaining water homeostasis in the lung. We report here that all-trans retinoic acid (atRA) increases plasma membrane water permeability, AQP5 mRNA and protein expression, and AQP5 promoter activity in MLE-12 cells. The promoter activation induced by atRA was diminished by mutation at the Sp1/Sp3 binding element (SBE), suggesting that the SBE mediates the effects of atRA. In addition, atRA increased the binding of Sp1 to the SBE without changing the levels of Sp1 in the nucleus. Taken together, our data indicate that atRA increases AQP5 expression through transactivation of Sp1, leading to an increase in plasma membrane water permeability.     

Aquaporin-1 in the peritoneal membrane: Implications for water transport across capillaries and peritoneal dialysis

Peritoneal dialysis (PD) is an established mode of renal replacement therapy, based on the exchange of fluid and solutes between blood in peritoneal capillaries and a dialysate that has been introduced in the peritoneal cavity. The dialysis involves diffusive and convective transports and osmosis through the highly vascularized peritoneal membrane. Computer simulations predicted that the membrane contains ultrasmall pores (radius < 3 Å) responsible for the transport of solute-free water across the capillary endothelium during crystalloid osmosis. The distribution of the water channel aquaporin-1 (AQP1), as well as its molecular structure ensuring an exquisite selectivity for water perfectly fit with the characteristics of the ultrasmall pore. Treatment with corticosteroids induces the expression of AQP1 in peritoneal capillaries and increases water permeability and ultrafiltration in rats, without affecting the osmotic gradient and the permeability for small solutes. Studies in knockout mice provided further evidence that osmotically-driven water transport across the peritoneal membrane is mediated by AQP1. AQP1 and endothelial NO synthase (eNOS) show a distinct regulation within the endothelium lining peritoneal capillaries. In acute peritonitis, the upregulation of eNOS and increased release of NO dissipate the osmotic gradient and result in ultrafiltration failure, despite the unchanged expression of AQP1. These data illustrate the potential of the peritoneal membrane to investigate the role and regulation of AQP1 in the endothelium. They also emphasize the critical role of AQP1 during peritoneal dialysis and suggest that manipulating AQP1 expression may be used to increase water permeability across the peritoneal membrane.     

Water permeability and characterization of aquaporin-11

The water permeability of aquaporin-11 (AQP11), which has a cysteine substituted for an alanine at a highly conserved asparagine-proline-alanine (NPA) motif in the water channel family, is controversial. Our previous study, however, showed that AQP11 is water permeable in proteoliposomes in which AQP11 molecules were reconstituted after purification with Fos-choline 10, which is the most suitable detergent available for stable solubilization of AQP11. In our previous study, we were unable to exclude the effect of the detergent on the water conductance. Therefore, in the present study, we measured the water permeability of AQP11 without detergent using vesicles that directly formed from Sf9 cell membranes expressing AQP11 molecules. The water permeability of AQP11 was 8-fold lower than that of AQP1 and 3-fold higher than that of mock-infected cell membrane, and was reversibly inhibited by mercury ions. Considering the slow but constant water permeable functions of AQP11, we performed homology modeling to search for a common structural feature. When comparing our model with those of other AQP structures, we found that Tyr83 facing the channel pore might be a key amino acid residue that decreases the water permeation of AQP11. Our findings indicate that AQP11 could be involved in slow but constant water movement across the membrane.     

Light inactivation of water transport and protein-protein interactions of aquaporin-Killer Red chimeras

Aquaporins (AQPs) have a broad range of cellular and organ functions; however, nontoxic inhibitors of AQP water transport are not available. Here, we applied chromophore-assisted light inactivation (CALI) to inhibit the water permeability of AQP1, and of two AQP4 isoforms (M1 and M23), one of which (M23) forms aggregates at the cell plasma membrane. Chimeras containing Killer Red (KR) and AQPs were generated with linkers of different lengths. Osmotic water permeability of cells expressing KR/AQP chimeras was measured from osmotic swelling-induced dilution of cytoplasmic chloride, which was detected using a genetically encoded chloride-sensing fluorescent protein. KR-AQP1 red fluorescence was bleached rapidly (~10% per second) by wide-field epifluorescence microscopy. After KR bleaching, KR-AQP1 water permeability was reduced by up to 80% for the chimera with the shortest linker. Remarkably, CALI-induced reduction in AQP4-KR water permeability was approximately twice as efficient for the aggregate-forming M23 isoform; this suggests intermolecular CALI, which was confirmed by native gel electrophoresis on cells coexpressing M23-AQP4-KR and myc-tagged M23-AQP4. CALI also disrupted the interaction of AQP4 with a neuromyelitis optica autoantibody directed against an extracellular epitope on AQP4. CALI thus permits rapid, spatially targeted and irreversible reduction in AQP water permeability and interactions in live cells. Our data also support the utility of CALI to study protein-protein interactions as well as other membrane transporters and receptors.