Water-Transporting Proteins

Transport through lipids and aquaporins is osmotic and entirely driven by the difference in osmotic pressure. Water transport in cotransporters and uniporters is different: Water can be cotransported, energized by coupling to the substrate flux by a mechanism closely associated with protein. In the K⁺/Cl⁻ and the Na⁺/K⁺/2Cl⁻ cotransporters, water is entirely cotransported, while water transport in glucose uniporters and Na⁺-coupled transporters of nutrients and neurotransmitters takes place by both osmosis and cotransport. The molecular mechanism behind cotransport of water is not clear. It is associated with the substrate movements in aqueous pathways within the protein; a conventional unstirred layer mechanism can be ruled out, due to high rates of diffusion in the cytoplasm. The physiological roles of the various modes of water transport are reviewed in relation to epithelial transport. Epithelial water transport is energized by the movements of ions, but how the coupling takes place is uncertain. All epithelia can transport water uphill against an osmotic gradient, which is hard to explain by simple osmosis. Furthermore, genetic removal of aquaporins has not given support to osmosis as the exclusive mode of transport. Water cotransport can explain the coupling between ion and water transport, a major fraction of transepithelial water transport and uphill water transport. Aquaporins enhance water transport by utilizing osmotic gradients and cause the osmolarity of the transportate to approach isotonicity.     

Forging the link between structure and function of electrogenic cotransporters: the renal type IIa Na+/Pi cotransporter as a case study

Electrogenic cotransporters are membrane proteins that use the electrochemical gradient across the cell membrane of a cosubstrate ion, for example Na⁺ or H⁺, to mediate uphill cotransport of a substrate specific to the transport protein. The cotransport process involves recognition of both cosubstrate and substrate and translocation of each species according to a defined stoichiometry. Electrogenicity implies net movement of charges across the membrane in response to the transmembrane voltage and therefore, in addition to isotope flux assays, the cotransport kinetics can be studied in real-time using electrophysiological methods. As well as the cotransport mode, many cotransporters also display a uniport or slippage mode, whereby the cosubstrate ions translocate in the absence of substrate. The current challenge is to define structure–function relationships by identifying functionally important elements in the protein that confer the transport properties and thus contribute to the ultimate goal of having a 3-D model of the protein that conveys both structural and functional information. In this review we focus on a functional approach to meet this challenge, based on a combination of real-time electrophysiological assays, together with molecular biological and biochemical methods. This is illustrated, by way of example, using data obtained by heterologous expression of the renal Na⁺-coupled inorganic phosphate cotransporter (NaP_i-IIa) for which structure–function relationships are beginning to emerge.     

Osmotic water transport with glucose in GLUT2 and SGLT

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na⁺-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na⁺-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.     

The existence of molecular water pumps in the nervous system: a review of the evidence

Recently, the presence if both influx and efflux molecular water pumps (MWP's) in vertebrate cells has been reported. These appear to use a common mechanism; the intercompartmental cotransport of water uphill against a gradient as a hydrophylic osmolyte is transported down its own gradient, in a regulated fashion, by a membrane spanning cotransporter protein. In each case, the dwell time of the transported osmolyte is short in that it is metabolically converted and its products either eliminated or recycled, thereby maintaining the required high intercompartmental gradient. An influx water pump osmolyte has been identified as a sodium-glucose complex, and an efflux water pump osmolyte as N-acetylhistidine. These osmolytes may also be archetypal representatives of many other osmolytes with similar functions in a variety of cells. When recycled, the osmolyte metabolites appear to be dewatered during high affinity binding that is associated with their active transport back across the membrane prior to intracellular resynthesis of the osmolyte. Since these cyclical systems result in the pumping of water, they also appear to create a previously unrecognized motive force which results in the establishment of unidirectional transcellular water flows between apical and basolateral cell membranes. As neurons represent highly specialized forms of animal cells, and cells which are also extremely sensitive to changes in osmotic pressure, the presence of these water pumps in the CNS could be significant. There would be connotations with regard to how neurons regulate water balance and transaxonal flow as well as to how these factors affect the integrated function of the nervous system. In this article, evidence of the presence of MWP's in the nervous system, and how they might relate to aspects of both normal and abnormal brain function is reviewed.     

Regulation of Na-K-2Cl cotransport by phosphorylation and protein-protein interactions

The Na-K-2Cl cotransporter plays important roles in cell ion homeostasis and volume control and is particularly important in mediating the movement of ions and thus water across epithelia. In addition to being affected by the concentration of the transported ions, cotransport is affected by cell volume, hormones, growth factors, oxygen tension, and intracellular ionized Mg(2+) concentration. These probably influence transport through three main routes acting in parallel: cotransporter phosphorylation, protein-protein interactions and cell Cl(-) concentration. Many effects are mediated, at least in part, by changes in protein phosphorylation, and are disrupted by kinase and phosphatase inhibitors, and manoeuvres that reduce cell ATP content. In some cases, phosphorylation of the cotransporter itself on serine and threonine (but not tyrosine) is associated with changes in transport rate, in others, phosphorylation of associated proteins has more influence. Analysis of the stimulation of cotransport by calyculin A, arsenite and deoxygenation suggests that the cotransporter is phosphorylated by several kinases and dephosphorylated by several phosphatases. These kinases and phosphatases may themselves be regulated by phosphorylation of residues including tyrosine, with Src kinases possibly playing an important role. Protein-protein interactions also influence cotransport activity. Cotransporter molecules bind to each other to form high molecular weight complexes, they also bind to other members of the cation-chloride cotransport family, to a variety of cytoskeletal proteins, and to enzymes that are part of regulatory cascades. Many of these interactions affect transport and may override the effects of cotransporter phosphorylation. Cell Cl(-) may also directly affect the way the cotransporter functions independently of its role as substrate.     

Structural Determinants of Water Permeation through the Sodium-Galactose Transporter vSGLT

Sodium-glucose transporters (SGLTs) facilitate the movement of water across the cell membrane, playing a central role in cellular homeostasis. Here, we present a detailed analysis of the mechanism of water permeation through the inward-facing state of vSGLT based on nearly 10 μs of molecular dynamics simulations. These simulations reveal the transient formation of a continuous water channel through the transporter that permits water to permeate the protein. Trajectories in which spontaneous release of galactose is observed, as well as those in which galactose remains in the binding site, show that the permeation rate, although modulated by substrate occupancy, is not tightly coupled to substrate release. Using a, to our knowledge, novel channel-detection algorithm, we identify the key residues that control water flow through the transporter and show that solvent gating is regulated by side-chain motions in a small number of residues on the extracellular face. A sequence alignment reveals the presence of two insertion sites in mammalian SGLTs that flank these outer-gate residues. We hypothesize that the absence of these sites in vSGLT may account for the high water permeability values for vSGLT determined via simulation compared to the lower experimental estimates for mammalian SGLT1.     

Intracellular hypertonicity is responsible for water flux associated with Na+/glucose cotransport

Detection of a significant transmembrane water flux immediately after cotransporter stimulation is the experimental basis for the controversial hypothesis of secondary active water transport involving a proposed stoichiometry for the human Na(+)/glucose cotransporter (SGLT1) of two Na(+), one glucose, and 264 water molecules. Volumetric measurements of Xenopus laevis oocytes coexpressing human SGLT1 and aquaporin can be used to detect osmotic gradients with high sensitivity. Adding 2 mM of the substrate alpha-methyl-glucose (alphaMG) created mild extracellular hypertonicity and generated a large cotransport current with minimal cell volume changes. After 20, 40, and 60 s of cotransport, the return to sugar-free, isotonic conditions was accompanied by measurable cell swelling averaging 0.051, 0.061, and 0.077 nl/s, respectively. These water fluxes are consistent with internal hypertonicities of 1.5, 1.7, and 2.2 mOsm for these cotransport periods. In the absence of aquaporin, the measured hypertonicites were 4.6, 5.0, and 5.3 mOsm for the same cotransport periods Cotransport-dependent water fluxes, previously assumed to be water cotransport, could be largely explained by hypertonicities of such amplitudes. Using intracellular Na(+) injection and Na(+)-selective electrode, the intracellular diffusion coefficient for Na(+) was estimated at 0.29 +/- 0.03 x 10(-5) cm(2) s(-1). Using the effect of intracellular alphaMG injection on the SGLT1-mediated outward current, the intracellular diffusion coefficient of alphaMG was estimated at 0.15 +/- 0.01 x 10(-5) cm(2) s(-1). Although these intracellular diffusion coefficients are much lower than in free aqueous solution, a diffusion model for a single solute in an oocyte would require a diffusion coefficient three times lower than estimated to explain the local osmolyte accumulation that was experimentally detected. This suggests that either the diffusion coefficients were overestimated, possibly due to the presence of convection, or the diffusion in cytosol of an oocyte is more complex than depicted by a simple model.     

Response to Zeuthen and Zeuthen's Comment to the Editor: Enough Local Hypertonicity Is Enough

In a Comment to the Editor, Zeuthen and Zeuthen criticize our treatment of the water cotransport hypothesis. In this response, we argue that we calculated water cotransport as if there were no significant local osmotic gradient generated in the first minute of Na/glucose cotransport. It is surprising to receive this type of criticism from Zeuthen and Zeuthen, as the same treatment was used in at least six studies from his laboratory where it is systematically assumed that “intracellular unstirred layers effects” are negligible. Zeuthen and Zeuthen also state that “the cotransport hypothesis predicts the measurements better than the osmotic hypothesis”. We present a quantitative comparison that challenges this contention. We would like to conclude by stating that our article was not about comparing different numerical models but about an experimental measurement of the local osmotic gradient generated after 20, 40, or 60 s of cotransport. Osmotic gradients were indeed detected, and were of appropriate amplitude to explain virtually all water transport observed.     

3H]bumetanide binding in vascular endothelial cells. Quantitation of Na-K-Cl cotransporters

Vascular endothelial cells previously have been shown to possess a prominent Na-K-Cl cotransport system which mediates a K+ influx of approximately 20 mumols/g of protein/min. Endothelial cell cotransport has also been shown to be regulated by a variety of vasoactive agents and their second messengers, suggesting that the transport system may have an important role in endothelial cell function. In the present study we investigated the possibility that the high level of cotransport in these cells is due to a large number of Na-K-Cl cotransporters in the plasma membrane. This was done by evaluating specific saturable binding of [3H]bumetanide to cultured bovine aortic endothelial cells. We found a maximal [3H]bumetanide binding of 0.83 pmol/mg protein with a dissociation constant of 0.13 microM. From these data, the number of [3H]bumetanide binding sites/endothelial cell was determined to be approximately 230,000, and the turnover number for cotransport activity was calculated to be 300 K+ ions/site/s. These findings indicate that endothelial cells do indeed exhibit a large number of Na-K-Cl cotransporters/cell relative to other cell types. We also investigated the effects on [3H]bumetanide binding of agents known to modulate Na-K-Cl cotransport activity. Saturable binding of [3H]bumetanide was found to be reduced significantly by treatment of the cells with 8-bromo-cyclic AMP, 8-bromo-cyclic GMP, phorbol esters, norepinephrine, or rat atriopeptin III, all of which have been shown to inhibit Na-K-Cl cotransport-mediated K+ influx.     

Controversy regarding the secondary active water transport hypothesis.

Historically, water transport across biological membranes has always been considered a passive process, i.e., the net water transport is proportional to the gradients of hydrostatic and osmotic pressure. More recently, this dogma was challenged by the suggestion that secondary active transporters such as the Na/glucose cotransporter (SGLT1) could perform secondary active water transport with a fixed stoichiometry. In the case of SGLT1, the stoichiometry would consist of one glucose molecule to two Na+ ions to 220-400 water molecules. In the present minireview, we summarize and criticize the evidence supporting and opposing this water cotransport hypothesis. Published and unpublished observations from our own laboratory are also presented in support of the idea that transport-dependent osmotic gradients begin to build up immediately after cotransport commences and are fully responsible for the cell swelling observed.     

Uncoupled Active Transport Mechanisms Accounting for Low Selectivity in Multidrug Carriers: P-Glycoprotein and SMR Antiporters

The extraordinarily low substrate specificity of P-glycoprotein conflicts with the notion that specific substrate interactions are required in the control of the reaction path in an active transport system. The difficulty is shown to be overcome by a half-coupled mechanism in which the ATP reaction is linked to carrier transformations, as in a fully coupled system, but in which the transported substrate plays a passive role. The mechanism, which requires no specific interaction with the substrate, brings about uphill transport. A half-coupled mechanism is directly supported by two observations: (i) almost completely uncoupled ATPase activity in purified P-glycoprotein, and (ii) a pattern of substrate specificity like that of passive systems, where maximum rates for different substrates vary little (unlike active systems, where maximum rates vary greatly). The mechanism accommodates other findings: partial inhibition of ATPase activity by an actively transported substrate; simultaneous binding and translocation of more than one substrate molecule; and stimulation or inhibition of the transport of one substrate molecule by another. A half-coupled system associated with an internal competitive inhibitor should behave as if tightly coupled, in agreement with the effects of the synthetic peptide, polytryptophan. The degree of coupling in the intact system is yet to be determined, however. A half-coupled ATPase mechanism could originally have evolved in a flippase, where immersion of the carrier in its substrate, the membrane lipid, precludes uncoupled ATP hydrolysis. These concepts may have wider application. An uncoupled antiport mechanism, driven by a proton gradient rather than ATP, can explain low selectivity in the SMR multidrug carriers of bacteria, and a half-coupled mechanism for the ion-driven cotransport of water (the substrate in which the carrier site is immersed) can explain a recently proposed uphill flow of water. Received: 23 April 1999/Revised: 29 July 1999     

Locating missing water molecules in protein cavities by the three-dimensional reference interaction site model theory of molecular solvation

Water molecules confined in protein cavities are of great importance in understanding the protein structure and functions. However, it is a nontrivial task to locate such water molecules in protein by the ordinary molecular simulation and modeling techniques as well as experimental methods. The present study proves that the three-dimensional reference interaction site model (3D-RISM) theory, a recently developed statistical-mechanical theory of molecular solvation, has an outstanding advantage in locating such water molecules. In this paper, we demonstrate that the 3D-RISM theory is able to reproduce the structure and the number of water molecules in cavities of hen egg-white lysozyme observed commonly in the X-ray structures of different resolutions and conditions. Furthermore, we show that the theory successfully identified a water molecule in a cavity, the existence of which has been ambiguous even from the X-ray results. In contrast, we confirmed that molecular dynamics simulation is helpless at present to find such water molecules because the results substantially depend on the initial coordinates of water molecules. Possible applications of the theory to problems in the fields of biochemistry and biophysics are also discussed.     

Active-site hydration and water diffusion in cytochrome P450cam: a highly dynamic process

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.     

The structural pathway for water permeation through sodium-glucose cotransporters

Although water permeation across cell membranes occurs through several types of membrane proteins, the only permeation mechanism resolved at atomic scale is that through aquaporins. Crystallization of the Vibrio parahaemolyticus sodium-galactose transporter (vSGLT) allows investigation of putative water permeation pathways through both vSGLT and the homologous human Na-glucose cotransporter (hSGLT1) using computational methods. Grand canonical Monte Carlo and molecular dynamics simulations were used to stably insert water molecules in both proteins, showing the presence of a water-filled pathway composed of ∼100 water molecules. This provides a structural basis for passive water permeation that is difficult to reconcile with the water cotransport hypothesis. Potential-of-mean-force calculations of water going through the crystal structure of vSGLT shows a single barrier of 7.7 kCal mol⁻¹, in agreement with previously published experimental data for cotransporters of the SGLT family. Electrophysiological and volumetric experiments performed on hSGLT1-expressing Xenopus oocytes showed that the passive permeation pathway exists in different conformational states. In particular, experimental conditions that aim to mimic the conformation of the crystal structure displayed passive water permeability. These results provide groundwork for understanding the structural basis of cotransporter water permeability.     

Inactivation of photosystems I and II in response to osmotic stress in Synechococcus. Contribution of water channels

The effects of osmotic stress due to sorbitol on the photosynthetic machinery were investigated in the cyanobacterium Synechococcus R-2. Incubation of cells in 1.0 M sorbitol inactivated photosystems I and II and decreased the intracellular solute space by 50%. These effects of sorbitol were reversible: Photosynthetic activity and cytoplasmic volume returned to the original values after removal of the osmotic stress. A blocker of water channels prevented the osmotic-stress-induced inactivation and shrinkage of the intracellular space. It also prevented the recovery of photosynthetic activity and cytoplasmic volume when applied just before release from osmotic stress. Inhibition of protein synthesis by lincomycin had no significant effects on the inactivation and recovery processes, an observation that suggests that protein synthesis was not involved in these processes. Our results suggest that osmotic stress decreased the amount of water in the cytoplasm via the efflux of water through water channels (aquaporins), with resultant increases in intracellular concentrations of ions and a decrease in photosynthetic activity.     

Uptake, intracellular transport, and degradation of polyethylene glycol-modified asialofetuin in hepatocytes.

Polyethylene glycol (PEG) is attached to proteins in order to increase their half-life in the circulation and reduce their immunogenicity in vivo. For many applications involving "targeting" molecules, it is important to know how PEG modification of the molecule affects its interaction with a receptor and the subsequent internalization, intracellular transport, and lysosomal degradation. As a model system, we used asialofetuin, which binds to the galactose receptor of hepatocytes, because removal of sialic acid exposes galactose residues. We modified asialofetuin by attaching various amounts of PEG of molecular weight 1900 or 5000. The preparations were labeled with 125I so that endocytosis and degradation could be followed in suspended hepatocytes. Depending on the number of PEG molecules attached, receptor-mediated uptake was affected to varying degrees. If two-thirds of the exposed amino groups of the asialofetuin molecule were modified, the rate of uptake decreased to less than one-fourth of controls; degradation of endocytosed molecules was 12% of controls. The reduction in endocytic uptake was due to a reduced rate of formation of the receptor-ligand complex. Subcellular frationation in density gradients showed that PEG-modified asialofetuin is transported intracellularly and degraded in the same manner as the native protein, but the rate of proteolysis is reduced. This observation explains the paradoxical result of experiments with injection of modified asialofetuin into rats in vivo: even though the clearance of one preparation of PEG-asialofetuin was much slower than that of the native protein, accumulation of radioactivity in the liver from the modified protein was twice as high. The hepatocytes accounted for 85% of the hepatic accumulation of either PEG-modified or native asialofetuin in vivo.     

Molecular characterization of Vibrio parahaemolyticus vSGLT: a model for sodium-coupled sugar cotransporters

The Na(+)/galactose cotransporter (vSGLT) of Vibrio parahaemolyticus, tagged with C-terminal hexahistidine, has been purified to apparent homogeneity by Ni(2+) affinity chromatography and gel filtration. Resequencing the vSGLT gene identified an important correction: the N terminus constitutes an additional 13 functionally essential residues. The mass of His-tagged vSGLT expressed under its native promoter, as determined by electrospray ionization-mass spectrometry (ESI-MS), verifies these 13 residues in wild-type vSGLT. A fusion protein of vSGLT and green fluorescent protein, comprising a mass of over 90 kDa, was also successfully analyzed by ESI-MS. Reconstitution of purified vSGLT yields proteoliposomes active in Na(+)-dependent galactose uptake, with sugar preferences (galactose > glucose > fucose) reflecting those of wild-type vSGLT in vivo. Substrates are transported with apparent 1:1 stoichiometry and apparent K(m) values of 129 mm (Na(+)) and 158 microm (galactose). Freeze-fracture electron microscopy of functional proteoliposomes shows intramembrane particles of a size consistent with vSGLT existing as a monomer. We conclude that vSGLT is a suitable model for the study of sugar cotransporter mechanisms and structure, with potential applicability to the larger SGLT family of important sodium:solute cotransporters. It is further demonstrated that ESI-MS is a powerful tool for the study of proteomics of membrane transporters.     

Analysis of CDK2 active-site hydration: a method to design new inhibitors

The interactions between the protein and the solvent were analyzed, and protein regions with a high density of water molecules, as well as structural water molecules, were determined by using molecular dynamics (MD) simulations. A number of water molecules that were in contact with the protein for the whole trajectory were determined. Their interaction energies and hydrogen bonds with protein residues were analyzed. Altogether, 39, 27, 49, and 32 water molecules bound to the protein were found for trajectories of the free CDK2, CDK2/ATP, CDK2/roscovitine, and CDK2/isopentenyladenine complexes, respectively. Positions of observed water molecules were compared with X-ray crystallography data. Special attention was paid to water molecules in the active site of the enzyme, and especially to the deep pocket, where the N9 roscovitine side-chain is buried. Exchange of active-site water molecules with bulk water through the tunnel from the pocket was observed. In the CDK2/isopentenyladenine complex simulation, two water molecules that arrange interaction between the inhibitor and the enzyme via an H-bond were observed. Two stable water molecules in the trajectory of the free CDK2 were found that occupy the same position as the nitrogens N3 and N9 of the isopentenyladenine or N1 and N6 nitrogens of the adenosine triphosphate (ATP). The positions of structural water molecules were compared with the positions of substrate polar groups and crystallographic water molecules found in the Brookhaven Protein Data Bank for various CDK2 complexes. It was concluded that tracing tightly bound water molecules may substantially help in designing new inhibitors.     

Molecular sieving of red cell membranes during gradual osmotic hemolysis

Summary Rat red blood corpuscles were held stationary with respect to a continuously flowing solution in either a specially constructed centrifuge or in glass filters. The concentration of the solution was gradually decreased to cause the swelling and subsequent gradual osmotic hemolysis of the cells. The passage of the intracellular molecules —potassium, adenylate kinase, and hemoglobin—across the cell membranes and into the flowing solution was determined as a function of time. Ions and molecules begin passage across the membranes in the order of increasing molecular size. The initial flow of potassium is followed by the initial flows of hemoglobin and adenylate kinase. The flow of hemoglobin has been interpreted as the flows of hemoglobin monomers, dimers, and tetramers such that the time sequence is: potassium; hemoglobin monomer; adenylate kinase/hemoglobin dimer; and finally, hemoglobin tetramer. It is concluded that the stressed cell membrane has molecular sieving properties and that the exclusion limit (effective hole size) increases as a function of time during the initial stages of gradual osmotic hemolysis. The process of gradual osmotic hemolysis is discussed in terms of molecular sieving through stress-induced effective membrane holes. It is suggested that a portion of the membrane protein might form an elastic network which would account for the gradual increase in size and apparent homogeneity of the effective holes.This work was prepared under the auspices of the U.S. Atomic Energy Commission.