Lack of Aquaporin 3 in bovine erythrocyte membranes correlates with low glycerol permeation (original) (raw)
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Single-channel permeability and glycerol affinity of human aquaglyceroporin AQP3
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2019
For its fundamental relevance, transport of water and glycerol across the erythrocyte membrane has long been investigated before and after the discovery of aquaporins (AQPs), the membrane proteins responsible for water and glycerol transport. AQP1 is abundantly expressed in the human erythrocyte for maintaining its hydrohomeostasis where AQP3 is also expressed (at a level ~30folds lower than AQP1) facilitating glycerol transport. This research is focused on two of the remaining questions: How permeable is AQP3 to water? What is the glycerol-AQP3 affinity under near-physiological conditions? Through atomistic modelling and large-scale simulations, we found that AQP3 is two to three times more permeable to water than AQP1 and that the glycerol-AQP3 affinity is approximately 500/M. Using these computed values along with the data from the latest literature on AQP1 and on erythrocyte proteomics, we estimated the water and glycerol transport rates across the membrane of an entire erythrocyte. We used these rates to predict the time courses of erythrocyte swelling-shrinking in response to inward and outward osmotic gradients. Experimentally, we monitored the time course of human erythrocytes when subject to an osmotic or glycerol gradient with light scattering in a stopped-flow spectrometer. We observed close agreement between the experimentally measured and the computationally predicted time courses of erythrocytes, which corroborated our computational conclusions on the AQP3 waterpermeability and the glycerol-AQP3 affinity.
Erythroid Expression and Oligomeric State of the AQP3 Protein
Journal of Biological Chemistry, 2002
Biochemical and biophysical studies have shown that the strictly water-permeable aquaporins have a tetrameric structure, whereas results concerning the oligomeric state of GlpF, the glycerol facilitator of Escherichia coli, are dependent upon the analytical technique used. Here, we analyzed the oligomerization of the AQP3 aquaglyceroporin, which presents a mixed selectivity for water, glycerol, and urea. At first, based on transcript detection by reverse transcription-PCR from human erythroid tissues and membrane expression detected by flow cytometry analysis, we demonstrated that AQP3 is expressed on human and rat but not on mouse red blood cells. Then, the quaternary structure of AQP3 was determined using as models human red blood cell membranes, which carry both AQP1 and AQP3, and two heterologous expression systems: Xenopus laevis oocyte, for density and size estimation of aquaporins, and Saccharomyces cerevisiae yeast, which expressed a non-glycosylated form of AQP3. By velocity sedimentation in sucrose gradient after non-denaturing detergent solubilization, AQP3 was essentially found as mono-and dimeric species in conditions under which AQP1 preserved its tetrameric structure. Freeze-fracture studies on oocyte plasma membranes gave a size of AQP3 particles in favor of a dimeric or trimeric structure. Finally, by cross-linking experiments with red blood cell membranes, AQP3 is visible as different oligomeric structures, including a tetrameric one. Since the discovery of the first water channel, aquaporin-1 (AQP1), 1 which selectively transports water, others members of the major intrinsic protein (MIP) family have been identified. Among them, some are also permeable to small solutes such as urea and glycerol, and these were named aquaglyceroporins. In mammals, the first one to be characterized was AQP3 (1, 2), and more recently, AQP7 (3) and AQP9 (4, 5) were discovered. These proteins have a higher sequence identity with GlpF, the bacterial glycerol facilitator of Escherichia coli, than with strict aquaporins. The three-dimensional structure of AQP1 has been extensively studied and determined recently at high resolution (6). These analyses have confirmed the proposed hourglass model (7) in which each monomer is formed of six tilted helices
Aquaporin 6 is permeable to glycerol and urea
Pfl�gers Archiv European Journal of Physiology, 2004
The passive water permeability (L p) of AQP6 is activated by Hg 2+. Our aim was to test if L p was associated with a permeability to small hydrophilic molecules such as glycerol and urea. AQP6 was expressed in Xenopus laevis oocytes and activated by 0.3 mM of HgCl 2 in the bathing solution. HgCl 2 caused the oocytes to swell at a rate of about 0.3% min −1. The L p of AQP6 was determined from brief additions or removals of mannitol from the bathing solution, and compared to the L p obtained from adding glycerol or urea. In paired experiments, L p (mannitol) was 2.4±0.1, L p (glycerol) 1.5±0.2, and L p (urea) 0.7±0.1 (units: 10 −5 cm s −1 osmol −1 ; 14 oocytes); the latter was not different from L p s obtained for native oocytes. The L p s were independent of the Hg 2+-induced swelling and of the magnitude of the osmotic challenge (−75 to +100 mosmol). Hg 2+ stimulated the uptake of [ 14 C] glycerol fivefold and [ 14 C]urea twofold in AQP6-expressing oocytes. There were no significant uptakes in native oocytes nor in AQP1-expressing oocytes whether these were treated with HgCl 2 or not. We conclude that water, glycerol and urea share an aqueous pathway in AQP6.
Channel-Dependent Permeation of Water and Glycerol in Mouse Morulae1
Biology of Reproduction, 2006
The cryosensitivity of mammalian embryos depends on the stage of development. Because permeability to water and cryoprotectants plays an important role in cryopreservation, it is plausible that the permeability is involved in the difference in the tolerance to cryopreservation among embryos at different developmental stages. In this study, we examined the permeability to water and glycerol of mouse oocytes and embryos, and tried to deduce the pathway for the movement of water and glycerol. The water permeability (L P , lm min À1 atm À1) of oocytes and four-cell embryos at 258C was low (0.63-0.70) and its Arrhenius activation energy (E a , kcal/mol) was high (11.6-12.3), which implies that the water permeates through the plasma membrane by simple diffusion. On the other hand, the L p of morulae and blastocysts was quite high (3.6-4.5) and its E a was quite low (5.1-6.3), which implies that the water moves through water channels. Aquaporin inhibitors, phloretin and p-(chloromercuri) benzene-sulfonate, reduced the L p of morulae significantly but not that of oocytes. By immunocytochemical analysis, aquaporin 3, which transports not only water but also glycerol, was detected in the morulae but not in the oocytes. Accordingly, the glycerol permeability (P GLY , 3 10 À3 cm/min) of oocytes was also low (0.01) and its E a was remarkably high (41.6), whereas P GLY of morulae was quite high (4.63) and its E a was low (10.0). Aquaporin inhibitors reduced the P GLY of morulae significantly. In conclusion, water and glycerol appear to move across the plasma membrane mainly by simple diffusion in oocytes but by facilitated diffusion through water channel(s) including aquaporin 3 in morulae. embryo, developmental biology, ovum 1 Supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Effect of Media with Different Glycerol Concentrations on Sheep Red Blood Cells’ Viability In Vitro
Animals, 2021
The use of high doses of glycerol as a livestock feed supplement is followed by a rapid increase in plasma concentrations and consequently in plasma osmolality. Moreover, glycerol is a highly diffusible molecule that can readily permeate the red blood cell (RBC) membrane following a concentration gradient. A rise in glycerol plasma concentrations can thus alter RBC homeostasis. The present study aimed at investigating both glycerol osmotic effects on sheep RBCs and their oxidative response under in vitro conditions. Sheep blood samples were suspended in media supplemented with increasing glycerol concentrations (0, 25, 50, 100, 150, 200, 250, 300, 350, 400 mg/dL), which reflected those found in vivo in previous studies, and incubated at 37 °C for 4h. Thereafter, osmolality and hemolysis were determined in spent media, while cell extracts were used to assay intracellular concentration of glycerol, ATP, Ca2+ ions, oxidative stress markers and reactive oxygen species (ROS).The study co...
Proceedings of the National Academy of Sciences, 2007
Human and rodent erythrocytes are known to be highly permeable to glycerol. Aquaglyceroporin aquaporin (AQP)3 is the major glycerol channel in human and rat erythrocytes. However, AQP3 expression has not been observed in mouse erythrocytes. Here we report the presence of an aquaglyceroporin, AQP9, in mouse erythrocytes. AQP9 levels rise as reticulocytes mature into erythrocytes and as neonatal pups develop into adult mice. Mice bearing targeted disruption of both alleles encoding AQP9 have erythrocytes that appear morphologically normal. Compared with WT cells, erythrocytes from AQP9-null mice are defective in rapid glycerol transport across the cell membrane when measured by osmotic lysis, [ 14 C]glycerol uptake, or stopped-flow light scattering. In contrast, the water and urea permeabilities are intact. Although the physiological role of glycerol in the normal function of erythrocytes is not clear, plasma glycerol is an important substrate for lipid biosynthesis of intraerythrocyt...
Biophysical Assessment of Human Aquaporin-7 as a Water and Glycerol Channel in 3T3-L1 Adipocytes
PLoS ONE, 2013
The plasma membrane aquaporin-7 (AQP7) has been shown to be expressed in adipose tissue and its role in glycerol release/uptake in adipocytes has been postulated and correlated with obesity onset. However, some studies have contradicted this view. Based on this situation, we have re-assessed the precise localization of AQP7 in adipose tissue and analyzed its function as a water and/or glycerol channel in adipose cells. Fractionation of mice adipose tissue revealed that AQP7 is located in both adipose and stromal vascular fractions. Moreover, AQP7 was the only aquaglyceroporin expressed in adipose tissue and in 3T3-L1 adipocytes. By overexpressing the human AQP7 in 3T3-L1 adipocytes it was possible to ascertain its role as a water and glycerol channel in a gain-of-function scenario. AQP7 expression had no effect in equilibrium cell volume but AQP7 loss of function correlated with higher triglyceride content. Furthermore it is also reported for the first time a negative correlation between water permeability and the cell non-osmotic volume supporting the observation that AQP7 depleted cells are more prone to lipid accumulation. Additionally, the strong positive correlation between the rates of water and glycerol transport highlights the role of AQP7 as both a water and a glycerol channel and reflects its expression levels in cells. In all, our results clearly document a direct involvement of AQP7 in water and glycerol transport, as well as in triglyceride content in adipocytes.
Effects of Lens Major Intrinsic Protein on Glycerol Permeability and Metabolism
Journal of Membrane Biology, 1998
Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (p Gly ) was 2.3 ± 0.23 × 10 −6 cm sec −1 with MIP vs. 0.92 ± 0.086 × 10 −6 cm sec −1 in control oocytes. The p Gly of MIP was independent of concentration from 5 × 10 −5 to 5 × 10 −2 M, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mM Hg ++ , 0.2 mM DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mM Cu ++ , respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 ± 0.03 vs. 0.06 ± 0.01 pmol min −1 cell −1 ) without changing the binding of glycerol to the kinase (K M ∼ 10 M).