ABSTRACT
Membrane transporters, in addition to their major role as specific carriers for ions and small molecules, can also behave as water channels. However, neither the location of the water pathway in the protein nor their functional importance is known. Here, we map the pathway for water and urea through the intestinal sodium/glucose cotransporter SGLT1. Molecular dynamics simulations using the atomic structure of the bacterial transporter vSGLT suggest that water permeates the same path as Na+ and sugar. On a structural model of SGLT1, based on the homology structure of vSGLT, we identified and mutated residues lining the sugar transport pathway to cysteine. The mutants were expressed in Xenopus oocytes, and the unitary water and urea permeabilities were determined before and after modifying the cysteine side chain with reversible methanethiosulfonate reagents. The results demonstrate that water and urea follow the sugar transport pathway through SGLT1. The changes in permeability, increases or decreases, with side-chain modifications depend on the location of the mutation in the region of external or internal gates, or the sugar binding site. These changes in permeability are hypothesized to be due to alterations in steric hindrance to water and urea, and/or changes in protein folding caused by mismatching of side chains in the water pathway. Water permeation through SGLT1 and other transporters bears directly on the structural mechanism for the transport of polar solutes through these proteins. Finally, in vitro experiments on mouse small intestine show that SGLT1 accounts for two-thirds of the passive water flow across the gut.
Subject(s)
Membrane Transport Proteins/physiology , Sodium-Glucose Transporter 1/metabolism , Water/chemistry , Water/metabolism , Animals , Aquaporins/metabolism , Biological Transport/physiology , Carrier Proteins/metabolism , Glucose/metabolism , Intestinal Mucosa/metabolism , Ion Transport/physiology , Membrane Proteins/metabolism , Mice , Models, Biological , Molecular Dynamics Simulation , Mutation , Oocytes/metabolism , Osmotic Pressure , Permeability , Sodium/metabolism , Sodium-Glucose Transporter 1/chemistry , Urea/metabolism , Xenopus/metabolismABSTRACT
Abstract We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient σ, while the solute permeability, P(S), is proportional to 1 - σ. The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mM of a range of different-sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured optically at high resolution and compared to the solute permeability obtained from short-term uptake of radio-labelled solute under isotonic conditions. For each type of aquaporin there was a linear relationship between solute permeability and reflection coefficient, in accordance with the model. We found no evidence for coupling between water and solute fluxes in the pore. In confirmation of molecular dynamic simulations, we conclude that the magnitude of the osmotic water permeability and the reflection coefficient are determined by processes at the arginine selectivity filter located at the outward-facing end of the pore.
Subject(s)
Aquaporin 1/metabolism , Aquaporins/metabolism , Osmosis , Water/metabolism , Amino Acid Sequence , Animals , Aquaporin 1/chemistry , Aquaporins/chemistry , Cell Membrane/metabolism , Cell Membrane Permeability , Models, Biological , Molecular Sequence Data , Protein Structure, Tertiary , Rats , Stochastic Processes , XenopusABSTRACT
Aquaporin (AQP) facilitated water transport is common to virtually all cell membranes and is marked by almost perfect specificity and high flux rates. Simultaneously, protons and cations are strictly excluded to maintain ionic transmembrane gradients. Yet, the AQP cation filters have not been identified experimentally. We report that three point mutations turned the water-specific AQP1 into a proton/alkali cation channel with reduced water permeability and the permeability sequence: H(+) >>K(+) >Rb(+) >Na(+) >Cs(+) >Li(+). Contrary to theoretical models, we found that electrostatic repulsion at the central asn-pro-ala (NPA) region does not suffice to exclude protons. Full proton exclusion is reached only in conjunction with the aromatic/arginine (ar/R) constriction at the pore mouth. In contrast, alkali cations are blocked by the NPA region but leak through the ar/R constriction. Expression of alkali-leaking AQPs depolarized membrane potentials and compromised cell survival. Our results hint at the alkali-tight but solute-unselective NPA region as a feature of primordial channels and the proton-tight and solute-selective ar/R constriction variants as later adaptations within the AQP superfamily.
Subject(s)
Aquaporins/metabolism , Cations/metabolism , Water/metabolism , Amino Acid Substitution/genetics , Animals , Aquaporin 1/chemistry , Aquaporin 1/genetics , Aquaporin 1/metabolism , Aquaporins/chemistry , Models, Chemical , Models, Molecular , Mutagenesis, Site-Directed , Oocytes , Protons , Substrate Specificity , XenopusABSTRACT
The NKCC1 and NKCC2 isoforms of the mammalian NaâºKâº2Clâ» cotransporter were expressed in Xenopus oocytes and the relation between external ion concentration and water fluxes determined.Water fluxes were determined from changes in the oocytes volume and ion fluxes from 86Rb+ uptake. Isotonic increases in external K⺠concentration elicited abrupt inward water fluxes in NKCC1; the K⺠dependence obeyed one-site kinetics with a K0.5 of 7.5 mM. The water fluxes were blocked by bumetanide, had steep temperature dependence and could proceed uphill against an osmotic gradient of 20 mosmol l⻹. A comparison between ion and water fluxes indicates that 460 water molecules are cotransported for each turnover of the protein. In contrast, NKCC2 did not support water fluxes.Water transport in NKCC1 induced by increases in the external osmolarity had high activation energy and was blocked by bumetanide. The osmotic effects of NaCl were smaller than those of urea and mannitol. This supports the notion of interaction between ions and water in NKCC1 and allows for an estimate of around 600 water molecules transported per turnover of the protein. Osmotic gradients did not induce water transport in NKCC2. We conclude that NKCC1 plays a direct role for water balance in most cell types, while NKCC2 fulfils its role in the kidney of transporting ions but not water. The different behaviour of NKCC1 and NKCC2 is discussed on the basis of recent molecular models based on studies of structural and molecular dynamics.
Subject(s)
Sodium-Potassium-Chloride Symporters/physiology , Water/physiology , Animals , Oocytes/physiology , Solute Carrier Family 12, Member 1 , Solute Carrier Family 12, Member 2 , XenopusABSTRACT
An important feature of neuronal signalling is the increased concentration of K(+) in the extracellular space. The K(+) concentration is restored to its original basal level primarily by uptake into nearby glial cells. The molecular mechanisms by which K(+) is transferred from the extracellular space into the glial cell are debated. Although spatial buffer currents may occur, their quantitative contribution to K(+) clearance is uncertain. The concept of spatial buffering of K(+) precludes intracellular K(+) accumulation and is therefore (i) difficult to reconcile with the K(+) accumulation repeatedly observed in glial cells during K(+) clearance and (ii) incompatible with K(+)-dependent glial cell swelling. K(+) uptake into non-voltage clamped cultured glial cells is carried out by the Na(+)/K(+)-ATPase and the Na(+)/K(+)/Cl(-) cotransporter in combination. In brain slices and intact optic nerve, however, only the Na(+)/K(+)-ATPase has been demonstrated to be involved in stimulus-evoked K(+) clearance. The glial cell swelling associated with K(+) clearance is prevented under conditions that block the activity of the Na(+)/K(+)/Cl(-) cotransporter. The Na(+)/K(+)/Cl(-) cotransporter is activated by increased K(+) concentration and cotransports water along with its substrates. It thereby serves as a K(+)-dependent molecular water pump under conditions of increased extracellular K(+) load.
Subject(s)
Membrane Transport Proteins/physiology , Neuroglia/metabolism , Potassium/metabolism , Animals , Homeostasis , HumansABSTRACT
Cerebrospinal fluid (CSF) envelops the brain and fills the central ventricles. This fluid is continuously replenished by net fluid extraction from the vasculature by the secretory action of the choroid plexus epithelium residing in each of the four ventricles. We have known about these processes for more than a century, and yet the molecular mechanisms supporting this fluid secretion remain unresolved. The choroid plexus epithelium secretes its fluid in the absence of a trans-epithelial osmotic gradient, and, in addition, has an inherent ability to secrete CSF against an osmotic gradient. This paradoxical feature is shared with other 'leaky' epithelia. The assumptions underlying the classical standing gradient hypothesis await experimental support and appear to not suffice as an explanation of CSF secretion. Here, we suggest that the elusive local hyperosmotic compartment resides within the membrane transport proteins themselves. In this manner, the battery of plasma membrane transporters expressed in choroid plexus are proposed to sustain the choroidal CSF secretion independently of the prevailing bulk osmotic gradient.
Subject(s)
Choroid Plexus , Membrane Transport Proteins , Biological Transport , Choroid Plexus/metabolism , Epithelium/metabolism , Membrane Transport Proteins/metabolismABSTRACT
BACKGROUND: Disturbances in the brain fluid balance can lead to life-threatening elevation in the intracranial pressure (ICP), which represents a vast clinical challenge. Nevertheless, the details underlying the molecular mechanisms governing cerebrospinal fluid (CSF) secretion are largely unresolved, thus preventing targeted and efficient pharmaceutical therapy of cerebral pathologies involving elevated ICP. METHODS: Experimental rats were employed for in vivo determinations of CSF secretion rates, ICP, blood pressure and ex vivo excised choroid plexus for morphological analysis and quantification of expression and activity of various transport proteins. CSF and blood extractions from rats, pigs, and humans were employed for osmolality determinations and a mathematical model employed to determine a contribution from potential local gradients at the surface of choroid plexus. RESULTS: We demonstrate that CSF secretion can occur independently of conventional osmosis and that local osmotic gradients do not suffice to support CSF secretion. Instead, the CSF secretion across the luminal membrane of choroid plexus relies approximately equally on the Na+/K+/2Cl- cotransporter NKCC1, the Na+/HCO3- cotransporter NBCe2, and the Na+/K+-ATPase, but not on the Na+/H+ exchanger NHE1. We demonstrate that pharmacological modulation of CSF secretion directly affects the ICP. CONCLUSIONS: CSF secretion appears to not rely on conventional osmosis, but rather occur by a concerted effort of different choroidal transporters, possibly via a molecular mode of water transport inherent in the proteins themselves. Therapeutic modulation of the rate of CSF secretion may be employed as a strategy to modulate ICP. These insights identify new promising therapeutic targets against brain pathologies associated with elevated ICP.
Subject(s)
Intracranial Pressure , Membrane Transport Proteins , Animals , Cerebrospinal Fluid/metabolism , Choroid Plexus/metabolism , Humans , Intracranial Pressure/physiology , Membrane Transport Proteins/metabolism , Osmosis , Rats , Sodium/metabolism , SwineABSTRACT
Water transport by the Na+-K+-2Cl(-) cotransporter (NKCC1) was studied in confluent cultures of pigmented epithelial (PE) cells from the ciliary body of the fetal human eye. Interdependence among water, Na+ and Cl(-) fluxes mediated by NKCC1 was inferred from changes in cell water volume, monitored by intracellular self-quenching of the fluorescent dye calcein. Isosmotic removal of external Cl(-) or Na+ caused a rapid efflux of water from the cells, which was inhibited by bumetanide (10 µm). When returned to the control solution there was a rapid water influx that required the simultaneous presence of external Na+ and Cl(-). The water influx could proceed uphill, against a transmembrane osmotic gradient, suggesting that energy contained in the ion fluxes can be transferred to the water flux. The influx of water induced by changes in external [Cl(-)] saturated in a sigmoidal fashion with a Km of 60 mm, while that induced by changes in external [Na+] followed first order kinetics with a Km of about 40 mm. These parameters are consistent with ion transport mediated by NKCC1. Our findings support a previous investigation, in which we showed water transport by NKCC1 to be a result of a balance between ionic and osmotic gradients. The coupling between salt and water transport in NKCC1 represents a novel aspect of cellular water homeostasis where cells can change their volume independently of the direction of an osmotic gradient across the membrane. This has relevance for both epithelial and symmetrical cells.
Subject(s)
Cell Membrane Permeability/physiology , Ciliary Body/physiology , Epithelial Cells/physiology , Sodium-Potassium-Chloride Symporters/physiology , Water/metabolism , Biological Transport/physiology , Bumetanide/pharmacology , Cell Membrane Permeability/drug effects , Cells, Cultured , Chlorides/pharmacokinetics , Ciliary Body/cytology , Epithelial Cells/cytology , Humans , Osmosis/physiology , Protein Isoforms/physiology , Sodium/pharmacokinetics , Sodium Potassium Chloride Symporter Inhibitors/pharmacology , Solute Carrier Family 12, Member 2ABSTRACT
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.
Subject(s)
Carrier Proteins/metabolism , Water/metabolism , Animals , Aquaporins/physiology , Biological Transport/physiology , Diffusion , Epithelium/metabolism , Glucose Transporter Type 2/physiology , Humans , Membrane Transport Proteins/metabolism , Osmosis , Sodium-Glucose Transporter 1/physiology , Sodium-Potassium-Chloride Symporters/physiology , Solute Carrier Family 12, Member 2 , Symporters/physiologyABSTRACT
Neuronal stimulation causes approximately 30% shrinkage of the extracellular space (ECS) between neurons and surrounding astrocytes in grey and white matter under experimental conditions. Despite its possible implications for a proper understanding of basic aspects of potassium clearance and astrocyte function, the phenomenon remains unexplained. Here we present a dynamic model that accounts for current experimental data related to the shrinkage phenomenon in wild-type as well as in gene knockout individuals. We find that neuronal release of potassium and uptake of sodium during stimulation, astrocyte uptake of potassium, sodium, and chloride in passive channels, action of the Na/K/ATPase pump, and osmotically driven transport of water through the astrocyte membrane together seem sufficient for generating ECS shrinkage as such. However, when taking into account ECS and astrocyte ion concentrations observed in connection with neuronal stimulation, the actions of the Na(+)/K(+)/Cl(-) (NKCC1) and the Na(+)/HCO(3) (-) (NBC) cotransporters appear to be critical determinants for achieving observed quantitative levels of ECS shrinkage. Considering the current state of knowledge, the model framework appears sufficiently detailed and constrained to guide future key experiments and pave the way for more comprehensive astroglia-neuron interaction models for normal as well as pathophysiological situations.
Subject(s)
Astrocytes/metabolism , Extracellular Space/metabolism , Ion Transport/physiology , Models, Biological , Neurons/metabolism , Animals , Bicarbonates/metabolism , Chlorides/metabolism , Extracellular Space/chemistry , Humans , Membrane Potentials/physiology , Osmosis/physiology , Paracrine Communication/physiology , Potassium/metabolism , Sodium/metabolism , Sodium-Potassium-Exchanging ATPase/metabolism , Systems BiologyABSTRACT
It is unclear how ammonia is transported by proteins from the Amt/Mep/Rh superfamily. We investigated this for the ammonium transporter TaAMT1;1 from wheat expressed in Xenopus oocytes by two-electrode voltage clamp and radio-labeled uptakes. Inward currents were activated by NH (4) (+) or methylammonium ions (MeA(+)). Importantly, currents increased fivefold when the external pH was decreased from 7.4 to 5.5; this type of pH dependence is unique and is a strong indication of NH (4) (+) or MeA(+) transport. This was confirmed by the close correlation between the uptake of radio-labeled MeA(+) and MeA(+)-induced currents. Homology models of members of the Amt/Mep/Rh superfamily exhibited major divergences in their cytoplasmic regions. A point mutation in this region of TaAMT1;1 abolished the pH sensitivity and decreased the apparent affinities for NH (4) (+) and MeA(+). We suggest a model where NH (4) (+) is transported as NH(3) and H(+) via separate pathways but the latter two recombine before leaving the protein.
Subject(s)
Cation Transport Proteins/metabolism , Cell Membrane/physiology , Ion Channel Gating/physiology , Membrane Potentials/physiology , Oocytes/physiology , Plant Proteins/metabolism , Quaternary Ammonium Compounds/metabolism , Triticum/metabolism , Animals , Biological Transport, Active/physiology , Cation Transport Proteins/chemistry , Cell Membrane/chemistry , Cells, Cultured , Hydrogen-Ion Concentration , Plant Proteins/chemistry , Quaternary Ammonium Compounds/chemistry , Triticum/genetics , Xenopus laevisABSTRACT
AQP9 is an aquaglyceroporin that serves important functions in peripheral organs, including the liver. Reflecting the lack of AQP9 knockout mice, uncertainties still prevail regarding the localization and roles of AQP9 in the central nervous system. Here we present a comprehensive analysis of AQP9 gene expression in brain, based on a quantitative and multipronged approach that includes the use of animals with targeted deletion of the AQP9 gene. We show by real-time PCR that AQP9 mRNA concentration in rat and mouse brain is approximately 3% and approximately 0.5%, respectively, of that in rat and mouse liver, the organ with the highest level of AQP9. By blue native gel analysis it could be demonstrated that the brain contains tetrameric AQP9, corresponding to the functional form of AQP9. The band corresponding to the AQP9 tetramer was absent in AQP9 knockout brain and liver. Immunocytochemistry and in situ hybridization analyses with AQP9 knockout controls show that subpopulations of nigral neurons express AQP9 both at the mRNA and at the protein levels and that populations of cortical cells (including hilar neurons in the hippocampus) contain AQP9 mRNA but no detectable AQP9 immunosignal. The present data provide conclusive evidence for the presence of tetrameric AQP9 in brain and for the expression of AQP9 in neurons.
Subject(s)
Aquaporins/metabolism , Brain/metabolism , Neurons/metabolism , Animals , Aquaporins/genetics , Brain/ultrastructure , Gene Expression , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Protein Structure, Quaternary , RNA, Messenger/metabolism , Rats , Rats, Wistar , Tyrosine 3-Monooxygenase/metabolismABSTRACT
The human aquaporins,AQP3,AQP7, AQP8,AQP9, and possibly AQP10, are permeable to ammonia, and AQP7, AQP9, and possibly AQP3, are permeable to urea. In humans, these aquaporins supplement the ammonia transport of the Rhesus (Rh) proteins and the urea transporters (UTs). The mechanism by which ammonium is transported by aquaporins is not fully resolved. A comparison of transport equations, models, and experimental data shows that ammonia is transported in its neutral form, NH(3). In the presence of NH(3), the aquaporin stimulates H(+) transport. Consequently, this transport of H(+) is only significant at alkaline pH. It is debated whether the H(+) ion passes via the aquaporin or by some external route; the investigation of this problem requires the aquaporin-expressing cell to be voltage-clamped. The ammonia-permeable aquaporins differ from other aquaporins by having a less restrictive aromatic/arginine region, and an exclusively water-permeable aquaporin can be transformed into an ammonia-permeable aquaporin by single point mutations in this region. The ammonia-permeable aquaporins fall into two groups: those that are permeable (AQP3, 7, 9, 10) and those that are impermeable (AQP8) to glycerol. The two groups differ in the amino acid composition of their aromatic/arginine regions. The location of the ammonia-permeable aquaporins in the body parallels that of the Rh proteins. This applies to erythrocytes and to cells associated with nitrogen homeostasis and high rates of anabolism. In the liver, AQPs 8 and 9 are found together with Rh proteins in cells exposed to portal blood coming from the intestine. In the kidney, AQP3 might participate in the excretion of NH(4) (+) in the collecting duct. The interplay between the ammonia-permeable aquaporins and the other types of ammonia- and urea-permeable proteins is not well understood.
Subject(s)
Ammonia/metabolism , Aquaporins/metabolism , Urea/metabolism , Animals , Aquaporins/chemistry , Aquaporins/genetics , Humans , Hydrogen-Ion Concentration , Membrane Transport Proteins/metabolism , Models, Biological , Models, Molecular , Mutation , Permeability , Protein Conformation , Rh-Hr Blood-Group System/metabolism , Structure-Activity Relationship , Urea TransportersABSTRACT
Aquaporin-4 (AQP4) is a brain aquaporin implicated in the pathophysiology of numerous clinical conditions including brain edema. Here we show that rat AQP4 has six cDNA isoforms, formed by alternative splicing. These are named AQP4a-f, where AQP4a and AQP4c correspond to the two classical M1 and M23 isoforms, respectively. The various isoforms are differentially expressed in kidney and brain, and their prevalence does not correspond to the level of the respective mRNAs, pointing to posttranscriptional regulation. The three isoforms lacking exon 2, AQP4b, AQP4d, and AQP4f, have an intracellular localization when expressed in cell lines and do not transport water when expressed in Xenopus oocytes. In contrast, the largest of the new isoforms, AQP4e, which contains a novel N-terminal domain, is localized at the plasma membrane in cell lines and functions as a water transporter in Xenopus oocytes.
Subject(s)
Aquaporin 4/genetics , Protein Isoforms/genetics , Alternative Splicing , Animals , Aquaporin 4/metabolism , Base Sequence , Cell Line , DNA Primers , DNA, Complementary , HeLa Cells , Humans , Protein Isoforms/metabolism , RNA, Messenger/genetics , Rats , Subcellular Fractions/metabolismABSTRACT
The group of leaky epithelia, such as proximal tubule and small intestine, have several common properties in regard to salt and water transport. The fluid transport is isotonic, the transport rate increases in dilute solutions, and water can be transported uphill. Yet, it is difficult to find common features that could form the basis for a general transport model. The direction of transepithelial water transport does not correlate with the direction of the primary active Na+ transport, or with the ultrastucture as defined by the location of apical and basolateral membranes, of the junctional complex and the lateral intercellular spaces. The presence of specific water channels, aquaporins, increases the water permeability of the epithelial cell membranes, i.e., the kidney proximal tubule. Yet other leaky epithelia, for example, the retinal pigment epithelium, have no known aquaporins. There is, however, a general correlation between the direction of transepithelial transport and the direction of transport via cotransporters of the symport type. A simple epithelial model based on water permeabilities, a hyperosmolar compartment and restricted salt diffusion, is unable to explain epithelial transport phenomena, in particular the ability for uphill water transport. The inclusion of cotransporters as molecular water pumps in these models alleviates this problem.
Subject(s)
Body Water/metabolism , Cell Membrane/metabolism , Epithelial Cells/metabolism , Membrane Transport Proteins/metabolism , Water-Electrolyte Balance/physiology , Animals , Aquaporins/metabolism , Cell Membrane/ultrastructure , Cell Membrane Permeability/physiology , Epithelial Cells/ultrastructure , Humans , Membrane Transport Proteins/ultrastructure , Models, BiologicalABSTRACT
Three kinds of membrane proteins have been shown to have water channels properties: the aquaporins, the cotransporters, and the uniports. A molecular-kinetic description of water transport in pores is compared to analytical models based on macroscopic parameters such as pore diameter and length. The use and limitations of irreversible thermodynamics is discussed. Experimental data on water and solute permeability in aquaporins are reviewed. No unifying transport model based on macroscopic parameters can be set up; for example, there is no correlation between solute diameter and permeability. Instead, the influence of hydrogen bonds between solute and pore, and the pH dependence of permeability, point toward a model based upon chemical interactions. The atomic model for AQP1 based on electron crystallographic data defines the dimensions and chemical nature of the aqueous pore. These structural data combined with quantum mechanical modeling and computer simulation might result in a realistic description of water transport. Data on water and solute permeability in cotransporters and uniports are reviewed. The function of these proteins as substrate transporters involves a series of conformational changes. The role of conformational equilibria on the water permeability will be discussed.
Subject(s)
Aquaporins/metabolism , Body Water/metabolism , Cell Membrane/metabolism , Eukaryotic Cells/metabolism , Water-Electrolyte Balance/physiology , Animals , Cell Membrane Permeability/physiology , Diffusion , Eukaryotic Cells/ultrastructure , Humans , Models, Biological , Molecular Structure , ThermodynamicsABSTRACT
Molecular water pumps are membrane proteins of the cotransport type in which a flux of water is coupled to substrate fluxes by a mechanism within the protein. Free energy can be exchanged between the fluxes. Accordingly, the flux of water may be relatively independent of the external water chemical potential and can even proceed uphill. In short, water is being cotransported. The evidence for water cotransport is reviewed with particular emphasis on electrogenic cotransporters expressed in Xenopus oocytes under voltage clamped conditions. Phenomena such as uphill water transport, tight coupling between water transport and clamp current, cotransport of small hydrophilic molecules, and shifts in reversal potentials with osmolarity are discussed with examples from the Na+/glutamate and Na+/glucose cotransporters. Unstirred layers and electrode artifacts as alternative explanations for such cotransport can be ruled out for both experimental and theoretical reasons. Indeed, substrate fluxes mediated by channels or ionophores generate much smaller water fluxes than those observed with cotransporters. Theoretical models, using reasonable values for the intracellular diffusion coefficient, indicate the presence of only small unstirred layers in the membranes studied.
Subject(s)
Body Water/physiology , Cell Membrane/metabolism , Eukaryotic Cells/metabolism , Membrane Transport Proteins/metabolism , Water-Electrolyte Balance/physiology , Animals , Cell Membrane/ultrastructure , Energy Metabolism/physiology , Eukaryotic Cells/ultrastructure , Humans , Ions/metabolism , Membrane Potentials/physiology , Osmotic PressureABSTRACT
Using functional complementation and a yeast mutant deficient in ammonium (NH4+) transport (Deltamep1-3), three wheat (Triticum aestivum) TIP2 aquaporin homologues were isolated that restored the ability of the mutant to grow when 2 mM NH4+ was supplied as the sole nitrogen source. When expressed in Xenopus oocytes, TaTIP2;1 increased the uptake of NH4+ analogues methylammonium and formamide. Furthermore, expression of TaTIP2;1 increased acidification of the oocyte-bathing medium containing NH4+ in accordance with NH3 diffusion through the aquaporin. Homology modeling of TaTIP2;1 in combination with site directed mutagenesis suggested a new subgroup of NH3-transporting aquaporins here called aquaammoniaporins. Mammalian AQP8 sharing the aquaammoniaporin signature also complemented NH4+ transport deficiency in yeast.
Subject(s)
Ammonia/metabolism , Aquaporins/metabolism , Plants/metabolism , Animals , Aquaporins/chemistry , Aquaporins/genetics , Base Sequence , Biological Transport , Cloning, Molecular , DNA Primers , Genetic Complementation Test , Mutagenesis, Site-DirectedABSTRACT
Two highly conserved NPA motifs are a hallmark of the aquaporin (AQP) family. The NPA triplets form N-terminal helix capping structures with the Asn side chains located in the centre of the water or solute-conducting channel, and are considered to play an important role in AQP selectivity. Although another AQP selectivity filter site, the aromatic/Arg (ar/R) constriction, has been well characterized by mutational analysis, experimental data concerning the NPA region--in particular, the Asn position--is missing. Here, we report on the cloning and mutational analysis of a novel aquaglyceroporin carrying one SPA motif instead of the NPA motif from Burkholderia cenocepacia, an epidemic pathogen of cystic fibrosis patients. Of 1357 AQP sequences deposited in RefSeq, we identified only 15 with an Asn exchange. Using direct and phenotypic permeability assays, we found that Asn and Ser are freely interchangeable at both NPA sites without affecting protein expression or water, glycerol and methylamine permeability. However, other mutations in the NPA region led to reduced permeability (S186C and S186D), to nonfunctional channels (N64D), or even to lack of protein expression (S186A and S186T). Using electrophysiology, we found that an analogous mammalian AQP1 N76S mutant excluded protons and potassium ions, but leaked sodium ions, providing an argument for the overwhelming prevalence of Asn over other amino acids. We conclude that, at the first position in the NPA motifs, only Asn provides efficient helix cap stabilization and cation exclusion, whereas other small residues compromise structural stability or cation exclusion but not necessarily water and solute permeability.
Subject(s)
Aquaporins/chemistry , Aquaporins/metabolism , Asparagine/chemistry , Amino Acid Motifs/genetics , Amino Acid Motifs/physiology , Animals , Aquaglyceroporins/chemistry , Aquaglyceroporins/genetics , Aquaglyceroporins/metabolism , Aquaporins/genetics , Blotting, Western , Burkholderia cenocepacia/metabolism , Cations/metabolism , Electrophysiology , Glycerol/metabolism , Methylamines/metabolism , Potassium/metabolism , Structure-Activity Relationship , Water/metabolism , Xenopus/genetics , Xenopus/metabolismABSTRACT
Prevention of cation permeation in wild-type aquaporin-1 (AQP1) is believed to be associated with the Asn-Pro-Ala (NPA) region and the aromatic/arginine selectivity filter (SF) domain. Previous work has suggested that the NPA region helps to impede proton permeation due to the protein backbone collective macrodipoles that create an environment favoring a directionally discontinuous channel hydrogen-bonded water chain and a large electrostatic barrier. The SF domain contributes to the proton permeation barrier by a spatial restriction mechanism and direct electrostatic interactions. To further explore these various effects, the free-energy barriers and the maximum cation conductance for the permeation of various cations through the AQP1-R195V and AQP1-R195S mutants are predicted computationally. The cations studied included the hydrated excess proton that utilizes the Grotthuss shuttling mechanism, a model "classical" charge localized hydronium cation that exhibits no Grotthuss shuttling, and a sodium cation. The hydrated excess proton was simulated using a specialized multi-state molecular dynamics method including a proper physical treatment of the proton shuttling and charge defect delocalization. Both AQP1 mutants exhibit a surprising cooperative effect leading to a reduction in the free-energy barrier for proton permeation around the NPA region due to altered water configurations in the SF region, with AQP1-R195S having a higher conductance than AQP1-R195V. The theoretical predictions are experimentally confirmed in wild-type AQP1 and the mutants expressed in Xenopus oocytes. The combined results suggest that the SF domain is a specialized structure that has evolved to impede proton permeation in aquaporins.