Role of Pore-Lining Residues in Defining the Rate of Water Conduction by Aquaporin-0 (original) (raw)
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Investigative Ophthalmology & Visual Science, 2003
PURPOSE. To first assess the distribution of posttranslationally truncated products of aquaporin 0 (AQP0) in dissected sections of a normal human lens and to determine the effect of backbone cleavage on the water permeability of AQP0. METHODS. A 27-year-old lens was concentrically dissected into six sections. Membrane protein was isolated from each section and cleaved with cyanogen bromide, and the peptides were separated and analyzed by reverse-phase (RP)-HPLC-mass spectrometry (MS). The sites of posttranslational AQP0 C-terminal truncation were determined by mass spectrometry. Truncated forms of AQP0 were expressed in a Xenopus laevis oocyte system, and the effect of truncation on AQP0 water permeability was assessed in an oocyte osmotic swelling assay. RESULTS. The extent of truncation at many sites within the C terminus increased with fiber cell age, and the effects of truncations after residues 234, 238, and 243 on AQP0 water permeability were examined. Truncation after residue 243 resulted in an approximate 15% decrease in permeability compared with the full-length protein, AQP0 1-263. However, rather than a direct effect on water transport, analysis of surface protein expression indicated that the decrease in permeability was a result of less efficient protein trafficking to the oocyte surface and that the permeabilities of full-length and 1-243 AQP0 were indistinguishable. Further, C-terminal truncation of AQP0 to 1-234 and 1-238, completely impaired trafficking into the plasma membrane, precluding the measurement of permeability. CONCLUSIONS. These data provide evidence that loss of 20 amino acids from the C terminus may not directly affect the ability of AQP0 to transport water.
2011
To study the interaction between the lens-specific water channel protein, aquaporin 0 (AQP0) and the lensspecific intermediate filament protein, filensin, and the effect of this interaction on the water permeability of AQP0. The effect of other factors on the interaction was also investigated. Methods: Expression plasmids were constructed in which glutathione-S-transferase (GST) was fused to the AQP0 COOHterminal region (GST-AQP0-C), which contains the major phosphorylation sites of the protein. Plasmids for AQP0 COOHterminal mutants were also constructed in which one, three or five sites were pseudophosphorylated, and the proteins expressed from these GST-fusion plasmids were assayed for their interaction with lens proteins. Expressed recombinant GST-fusion proteins were purified using glutathione beads and incubated with rat lens extract. Western blotting was used to identify the lens proteins that interacted with the GST-fusion proteins. Filensin tail and rod domains were also expressed as GST-fusion proteins and their interactions with AQPO were analyzed. Additionally, the water permeability of AQP0 was calculated by expressing AQP0 with or without the filensin peptide on the cell membrane of Xenopus oocytes by injecting cRNAs for AQP0 and filensin. Results: The GST-AQP0-C construct interacted with the tail region of lens filensin and the GST-filensin-tail construct interacted with lens AQP0, but the GST-filensin-rod construct did not interact with AQP0. GST-AQP0-C also interacted with a purified recombinant filensin-tail peptide after cleavage from GST. The AQP0/filensin-tail interaction was not affected by pseudophosphorylation of the AQP0 COOH-terminal tail, nor was it affected by changes in pH. Xenopus oocytes expressing AQP0 on the plasma membrane showed increased water permeability, which was lowered when the filensin COOH-terminal peptide cRNA was coinjected with the cRNA for AQP0. Conclusions: The filensin COOH-terminal tail region interacted with the AQP0 COOH-terminal region and the results strongly suggested that the interaction was direct. It appears that interactions between AQP0 and filensin helps to regulate the water permeability of AQP0 and to organize the structure of lens fiber cells, and may also help to maintain the transparency of the lens. Aquaporins are a family of ubiquitous membrane proteins that form channels allowing the permeation of water and small, neutral molecules, such as glycerol, across cell membranes [1,2]. Aquaporin 0 (AQP0), also known as major intrinsic peptide (MIP) 26, is the most abundant fiber cell membrane protein in lens. AQP0 is also expressed in retinal amacrine cells, retinal ganglion cells and liver cells [3,4]. AQP0 constitutes more than 60% of the total membrane protein content of fiber cells [5,6] and consists of six transmembrane helices with both the NH2-and COOH-termini localized to the cell cytoplasm. AQP0 exists as a tetramer and each subunit contains an individual aqueous pore [7]. Compared with other aquaporins, AQP0 has special properties, including a very limited ability to transport water [2,8]. There may be a specific reason for the low permeability of this lens water channel but it is not yet known.
Regulation of Aquaporin Water Permeability in the Lens
Investigative Ophthalmology & Visual Science, 2005
To examine Ca 2ϩ -and pH-mediated regulation of water permeability of endogenously expressed aquaporin (AQP)0 in lens fiber cells and AQP1 in lens epithelial cells. METHODS. Large, right-side-out membrane vesicles were formed from freshly isolated groups of lens fiber cells. Osmotic shrinking or swelling of these vesicles was used to determine the water permeability of endogenously expressed AQP0. The results were compared with those in similar studies of freshly isolated lens epithelial cells, which endogenously expressed AQP1, and of oocytes, which exogenously expressed AQP0. RESULTS. In the lens or in oocytes, decreasing external pH from 7.5 to 6.5 caused a two-to fourfold increase in the water permeability of mammalian AQP0. Several lines of evidence suggest that this effect is mediated by the binding of H ϩ to a histidine in the first extracellular loop (His40). Lens AQP1 lacks His40 and also lacks pH sensitivity. Increasing Ca 2ϩ caused a two-to fourfold increase in the water permeability of endogenous AQP0. The Ca 2ϩ effect on mouse AQP0 was a 2.5-fold increase in the lens, whereas in oocytes, it was a 4-fold decrease. In either environment, the effect was mediated through calmodulin, most likely through its binding to the proximal domain of the C terminus. Lens AQP1 does not have a similar domain and does not have calcium sensitivity. CONCLUSIONS. In either the lens or oocytes, Ca 2ϩ and H ϩ appear to affect the same mechanism, probably either the open probability of the water channel, or open-channel permeability. The difference between calcium's effects in lens versus oocytes was remarkable and is not understood. However, in the lens, Ca 2ϩ and H ϩ are both increased in inner fiber cells, and so in the physiologically relevant environment, both may act to increase the water permeability of AQP0. (Invest Ophthalmol Vis Sci.
Cellular and Molecular Biology of the Aquaporin Water Channels
Annual Review of Biochemistry, 1999
The high water permeability characteristic of mammalian red cell membranes is now known to be caused by the protein AQP1. This channel freely permits movement of water across the cell membrane, but it is not permeated by other small, uncharged molecules or charged solutes. AQP1 is a tetramer with each subunit containing an aqueous pore likened to an hourglass formed by obversely arranged tandem repeats. Cryoelectron microscopy of reconstituted AQP1 membrane crystals has revealed the three-dimensional structure at 3-6 Å. AQP1 is distributed in apical and basolateral membranes of renal proximal tubules and descending thin limbs as well as capillary endothelia. Ten mammalian aquaporins have been identified in water-permeable tissues and fall into two groupings. Orthodox aquaporins are waterselective and include AQP2, a vasopressin-regulated water channel in renal collecting duct, in addition to AQP0, AQP4, and AQP5. Multifunctional aquaglyceroporins AQP3, AQP7, and AQP9 are permeated by water, glycerol, and some other solutes. Aquaporins are being defined in numerous other species including amphibia, insects, plants, and microbials. Members of the aquaporin family are implicated in numerous physiological processes as well as the pathophysiology of a wide range of clinical disorders.
Molecular Aspects of Medicine, 2012
After a decade of work on the water permeability of red blood cells (RBC) Benga group in Cluj-Napoca, Romania, discovered in 1985 the first water channel protein in the RBC membrane. The discovery was reported in publications in 1986 and reviewed in subsequent years. The same protein was purified by chance by Agre group in Baltimore, USA, in 1988, who called in 1991 the protein CHIP28 (CHannel forming Integral membrane Protein of 28 kDa), suggesting that it may play a role in linkage of the membrane skeleton to the lipid bilayer. In 1992 the Agre group identified CHIP28's water transport property. One year later CHIP28 was named aquaporin 1, abbreviated as AQP1. In this review the molecular structure-function relationships of AQP1 are presented. In the natural or model membranes AQP1 is in the form of a homotetramer, however, each monomer has an independent water channel (pore). The three-dimensional structure of AQP1 is described, with a detailed description of the channel (pore), the molecular mechanisms of permeation through the channel of water molecules and exclusion of protons. The permeability of the pore to gases (CO 2 , NH 3 , NO, O 2) and ions is also mentioned. I have also reviewed the functional roles and medical implications of AQP1 expressed in various organs and cells (microvascular endothelial cells, kidney, central nervous system, eye, lacrimal and salivary glands, respiratory apparatus, gastrointestinal tract, hepatobiliary compartments, female and male reproductive system, inner ear, skin). The role of AQP1 in cell migration and angiogenesis in relation with cancer, the genetics of AQP1 and mutations in human subjects are also mentioned. The role of AQP1 in red blood cells is discussed based on our comparative studies of water permeability in over 30 species.
Differential water permeability and regulation of three aquaporin 4 isoforms
Cellular and Molecular Life Sciences, 2010
Aquaporin 4 (AQP4) is expressed in the perivascular glial endfeet and is an important pathway for water during formation and resolution of brain edema. In this study, we examined the functional properties and relative unit water permeability of three functional isoforms of AQP4 expressed in the brain (M1, M23, Mz). The M23 isoform gave rise to square arrays when expressed in Xenopus laevis oocytes. The relative unit water permeability differed significantly between the isoforms in the order of M1 [ Mz [ M23. None of the three isoforms were permeable to small osmolytes nor were they affected by changes in external K ? concentration. Upon protein kinase C (PKC) activation, oocytes expressing the three isoforms demonstrated rapid reduction of water permeability, which correlated with AQP4 internalization. The M23 isoform was more sensitive to PKC regulation than the longer isoforms and was internalized significantly faster. Our results suggest a specific role for square array formation.