Epithelial Fluid Transport is Due to Electro-osmosis (80%), Plus Osmosis (20%).

Epithelial fluid transport, an important physiological process shrouded in a long-standing enigma, may finally be moving closer to a solution. We propose that, for the corneal endothelium, relative proportions for the driving forces for fluid transport are 80% of paracellular electro-osmosis, and 20% classical transcellular osmosis. These operate in a cyclical process with a period of 9.2 s, which is dictated by the decrease and exhaustion of cellular Na+. Paracellular electro-osmosis is sketched here, and partially discussed as much as the subject still allows; transcellular osmosis is presented at length.

Chemokine and cytokine levels in osteoarthritis and rheumatoid arthritis synovial fluid.

To develop a method of the assay of chemokine and cytokine signaling in synovial fluid from patients suffering from osteoarthritis (OA) or rheumatoid arthritis (RA) and evaluate the effect of heterophilic antibodies on the reliability of the data. 21 synovial fluid samples from OA and 16 synovial fluid samples from RA patients were analyzed using a unique 2 step dot sandwich ELISA based micro-well protein array designed to detect heterophilic antibody signaling in the presence or absence of an effective heterophilic blocking reagent with assays carried out for Eotaxin, hGROa, interleukin (IL)-8, IP10, MCP-1, MCP-2, MIG, RANTES, TARC and IL-6. Array analysis reveals that the selective presence of heterophilic antibodies interferes with the accurate assay of synovial fluid samples from a minority of RA patients but not OA synovia. Using a commercial blocking diluent OA and RA synovial fluids reveal significant differences in chemokine content (IL-6, Eotaxin, hGROa, MCP-2, MIG, TARC, IL-8, RANTES). Using a two-step assay protocol it is possible to readily detect inappropriate antibody signaling due to heterophilic antibodies and devise a protocol designed to eliminate this problem thereby more accurately quantify cytokines and chemokines specific to both RA and OA fluids.

Pro-inflammatory cytokines in glaucomatous aqueous and encysted Molteno implant blebs and their relationship to pressure.

PURPOSE: To ascertain the presence of additional pro-inflammatory cytokines in glaucomatous aqueous, and their relationship with IOP. METHODS: To quantify the levels of 23 pro-inflammatory cytokines, and correlate levels with IOP, aqueous humor samples were analyzed from 23 eyes with open angle glaucoma (OAG) undergoing glaucoma filtration procedures, and from 24 Molteno blebs during the hypertensive phase. Control aqueous was derived from 13 eyes without glaucoma undergoing cataract removal. RESULTS: A significant difference (P<0.05) was noted between hypertensive bleb aqueous and controls in the amount TGF-ß2, interleukins IL-6, IL-10, and chemokine (C-X-C motif) ligand 1 (CXCL1; GROα). The levels of these cytokines were higher in the glaucomatous aqueous, but not significantly so. A significant difference was noted in levels of chemokine (C-C motif) ligand 2 (CCL2; MCP-1, monocyte chemotactic protein-1) in the glaucoma eye and bleb aqueous compared with controls. Of the 23 cytokines tested for, 19 were found in the bleb group, 14 in the glaucoma group, and 16 in the control group. Compared with controls, all cytokines levels were higher in the glaucoma group and highest in the bleb group. CONCLUSIONS: The study confirms the well documented presence of TGF-ß2 in glaucomatous aqueous. The presence of significant levels of CCL2 in glaucomatous aqueous is a new finding. The finding of higher levels of all the cytokines in the aqueous from the encysted blebs, in which the IOP was the highest, suggests that their levels increase with an increase in IOP, as well as the possibility that encysted blebs form cytokines.

DPOFA, a Cl⁻/HCO3⁻ exchanger antagonist, stimulates fluid absorption across basolateral surface of the retinal pigment epithelium.

BACKGROUND: Retinal detachment is a disorder of the eye in which sensory retina separates from the retinal pigment epithelium (RPE) due to accumulation of fluid in subretinal space. Pharmacological stimulation of fluid reabsorption from subretinal space to choroid across the RPE has been suggested as a treatment strategy for retinal detachment. DPOFA, (R)-(+)-(5,6-dichloro 2,3,9,9a-tetrahydro 3-oxo-9a-propyl-1H-fluoren-7-yl)oxy]acetic acid, is an abandoned drug capable of inhibiting Cl⁻/HCO3⁻ exchanger activity. We hypothesized that DPOFA may increase fluid absorption across basolateral surface of the RPE. METHODS: Reverse transcription polymerase chain reaction (RT-PCR) analysis of mRNA for six different transporters that may act as Cl⁻/HCO3⁻ exchangers was conducted in bovine and human RPE to confirm that RPE from two species expresses the same repertoire of Cl⁻/HCO3⁻ exchanger isoforms. The degree of amino acid homology between orthologous human and bovine RPE-specific isoforms was calculated after performing protein alignments. Transport of fluid across bovine RPE-choroid explants mounted in the Ussing chamber was used to assess the ability of DPOFA to modulate fluid absorption across the RPE. RESULTS: Using RT-PCR we showed that three isoforms (SLC4A2, SLC4A3, and SLC26A6) are strongly expressed in human and bovine RPE preparations. Amino acid comparisons conducted for RPE-specific isoforms support the use of bovine RPE-choroid explants as an adequate experimental system for assessing fluid absorption activity for DPOFA. Our data is consistent with the fact that DPOFA stimulates fluid absorption across the RPE in bovine RPE-choroid explants. CONCLUSIONS: DPOFA seems to stimulate transport of water across the RPE in bovine RPE-choroid explants. Additional experiments are required to establish dose-dependent effect of DPOFA on fluid absorption in the bovine RPE-choroid experimental system.

Comparative permeabilities of the paracellular and transcellular pathways of corneal endothelial layers.

Layers of rabbit corneal endothelial cells were cultured on permeable inserts. We characterized the diffusional permeability of the cell layer to nonelectrolyte and charged molecules and compared the diffusional and filtration permeabilities of the paracellular and transcellular pathways. We determined the rates of diffusion of (3)H- and (14)C-labeled nonelectrolyte test molecules and estimated the equivalent pore radius of the tight junction. Negatively charged molecules permeate slower than neutral molecules, while positively charged molecules permeate faster. Palmitoyl-DL-carnitine, which opens tight junctions, caused an increase of permeability and equivalent pore radius. Diffusional water permeability was determined with (3)H-labeled water; the permeabilities of the tight junction and lateral intercellular space were calculated using tissue geometry and the Renkin equation. The diffusional permeability (P(d)) of the paracellular pathway to water is 0.57 µm s(-1) and that of the transcellular path is 2.52 µm s(-1). From the P(d) data we calculated the filtration permeabilities (P(f)) for the paracellular and transcellular pathways as 41.3 and 30.2 µm s(-1), respectively. In conclusion, the movement of hydrophilic molecules through tight junctions corresponds to diffusion through negatively charged pores (r = 2.1 ± 0.35 nm). The paracellular water permeability represents 58% of the filtration permeability of the layer, which points to that route as the site of sizable water transport. In addition, we calculated for NaCl a reflection coefficient of 0.16 ≤ σ(NaCl) ≤ 0.33, which militates against osmosis through the junctions and, hence, indirectly supports the electro-osmosis hypothesis.

Profile of lipid and protein autacoids in diabetic vitreous correlates with the progression of diabetic retinopathy.

OBJECTIVE: This study was aimed at obtaining a profile of lipids and proteins with a paracrine function in normal and diabetic vitreous and exploring whether the profile correlates with retinal pathology. RESEARCH DESIGN AND METHODS: Vitreous was recovered from 47 individuals undergoing vitreoretinal surgery: 16 had nonproliferative diabetic retinopathy (NPDR), 15 had proliferative diabetic retinopathy, 7 had retinal detachments, and 9 had epiretinal membranes. Protein and lipid autacoid profiles were determined by protein arrays and mass spectrometry-based lipidomics. RESULTS: Vitreous lipids included lipoxygenase (LO)- and cytochrome P450 epoxygenase (CYP)-derived eicosanoids. The most prominent LO-derived eicosanoid was 5-hydroxyeicosate traenoic acid (HETE), which demonstrated a diabetes-specific increase (P = 0.027) with the highest increase in NPDR vitreous. Vitreous also contained CYP-derived epoxyeicosatrienoic acids; their levels were higher in nondiabetic than diabetic vitreous (P < 0.05). Among inflammatory, angiogenic, and angiostatic cytokines and chemokines, only vascular endothelial growth factor (VEGF) showed a significant diabetes-specific profile (P < 0.05), although a similar trend was noted for tumor necrosis factor (TNF)-alpha. Soluble VEGF receptors R1 and R2 were detected in all samples with lowest VEGF-R2 levels (P < 0.05) and higher ratio of VEGF to its receptors in NPDR and PDR vitreous. CONCLUSIONS: This study is the first to demonstrate diabetes-specific changes in vitreous lipid autacoids including arachidonate and docosahexanoate-derived metabolites indicating an increase in inflammatory versus anti-inflammatory lipid mediators that correlated with increased levels of inflammatory and angiogenic proteins, further supporting the notion that inflammation plays a role the pathogenesis of this disease.

Corneal endothelium transports fluid in the absence of net solute transport.

The corneal endothelium transports fluid from the corneal stroma to the aqueous humor, thus maintaining stromal transparency by keeping it relatively dehydrated. This fluid transport mechanism is thought to be driven by the transcellular transports of HCO(3)(-) and Cl(-) in the same direction, from stroma to aqueous. In parallel to these anion movements, for electroneutrality, there are paracellular Na(+) and transcellular K(+) transports in the same direction. The resulting net flow of solute might generate local osmotic gradients that drive fluid transport. However, there are reports that some 50% residual fluid transport remains in nominally HCO(3)(-) free solutions. We have examined the driving force for this residual fluid transport. We confirm that in nominally HCO(3)(-) free solutions, 48% of control fluid transport remains. When in addition Cl(-) channels are inhibited, 30% of control fluid movement still remains. Addition of a carbonic anhydrase inhibitor has no further effect. These manipulations combined inhibit the transcellular transport of all anions, without which there cannot be any net transport of solute and consequently no local osmotic gradients, yet there is residual fluid movement. Only the further addition of benzamil, an inhibitor of epithelial Na(+) channels, abolishes fluid transport completely. Our data are inconsistent with transcellular local osmosis and instead support the paradigm of paracellular fluid transport driven by electro-osmotic coupling.

Functional and molecular characterization of multiple K-Cl cotransporter isoforms in corneal epithelial cells.

The dependence of regulatory volume decrease (RVD) activity on potassium-chloride cotransporter (KCC) isoform expression was characterized in corneal epithelial cells (CEC). During exposure to a 50% hypotonic challenge, the RVD response was larger in SV40-immortalized human CEC (HCEC) than in SV40-immortalized rabbit CEC (RCEC). A KCC inhibitor-[(dihydroindenyl)oxy] alkanoic acid (DIOA)-blocked RVD more in HCEC than RCEC. Under isotonic conditions, N-ethylmaleimide (NEM) produced KCC activation and transient cell shrinkage. Both of these changes were greater in HCEC than in RCEC. Immunoblot analysis of HCEC, RCEC, primary human CEC (pHCEC), and primary bovine CEC (BCEC) plasma membrane enriched fractions revealed KCC1, KCC3, and KCC4 isoform expression, whereas KCC2 was undetectable. During a hypotonic challenge, KCC1 membrane content increased more rapidly in HCEC than in RCEC. Such a challenge induced a larger increase and more transient p44/42MAPK activation in HCEC than RCEC. On the other hand, HCEC and RCEC p38MAPK phosphorylation reached peak activations at 2.5 and 15 min, respectively. Only in HCEC, pharmacological manipulation of KCC activity modified the hypotonicity-induced activation of p44/42MAPK, whereas p38MAPK phosphorylation was insensitive to such procedures in both cell lines. Larger increases in HCEC KCC1 membrane protein content correlated with their ability to undergo faster and more complete RVD. Furthermore, pharmacological activation of KCC increased p44/42MAPK phosphorylation in HCEC but not in RCEC, presumably a reflection of low KCC1 membrane expression in RCEC. These findings suggest that KCC1 plays a role in (i) maintaining isotonic steady-state cell volume homeostasis, (ii) recovery of isotonic cell volume after a hypotonic challenge through RVD, and (iii) regulating hypotonicity-induced activation of the p44/42MAPK signaling pathway required for cell proliferation.

Modulation of tight junction properties relevant to fluid transport across rabbit corneal endothelium.

Paracellular junctions could play an important role in corneal endothelial fluid transport. In this study we explored the effects of different reagents on the tight junctional barrier by assessing the translayer specific electrical resistance (TER) across rabbit corneal endothelial preparations and cultured rabbit corneal endothelial cells' (CRCEC) monolayers, the paracellular permeability (Papp) for fluorescein isothiocyanate (FITC) dextrans across CRCEC, and fluid transport across de-epithelialized rabbit corneal endothelial preparations. Palmitoyl carnitine (PC), poly-L-lysine (PLL), adenosine triphosphate (ATP), and dibutyryl adenosine 3',5'-cyclic monophosphate (dB-cAMP) were used to modulate corneal endothelial fluid transport and tight junctions (TJs). After seeding, the TER across CRCEC reached maximal values (29.2+/-1.0 Omega cm2) only after the 10th day. PC (0.1 mM) caused decreases both in TER (by 40%) and fluid transport (swelling rate: 18.5+/-0.3 microm/h), and an increase in Papp. PLL resulted in increased TER rose and Papp but decreased fluid transport (swelling rate: 10+/-0.3 microm/h). dB-cAMP (0.1 mM) and ATP (0.1 mM) decreased TER by 16% and 6%, increased Papp slightly, and stimulated fluid transport; the rates of de-swelling (in microm/h) were -5.4+/-0.3 and -12.1+/-0.4, respectively. PC might cause the junctions to open up unspecifically and thus increase passive leak. PLL is a known junctional charge modifier that may be adding steric hindrance to the tight junctions. The results with dB-cAMP and ATP are consistent with fluid transport via the paracellular route.

Lack of threshold for anisotonic cell volume regulation.

Most cells possess mechanisms that are able to detect cellular volume shifts and to signal the initiation of appropriate volume regulatory responses. However, the identity and characteristics of the detecting mechanism remain obscure. In this study, we explored the influence of hypertonic and hypotonic challenges of varying magnitude on the characteristics of the ensuing regulatory volume increase (RVI) and regulatory volume decrease (RVD) of cultured bovine corneal endothelial cells (CBCECs). The main question we asked was whether a threshold of stimulation existed that would unleash a regulatory response. CBCECs (passage 1-3) were seeded on rectangular glass coverslips and grown for 1-2 days. We used a procedure based on detection of light scattering to monitor the transient volume changes of such plated cells when subjected to osmotic challenge. The osmometric responses were asymmetric: cells shrank faster than they swelled (by a factor of 3). Complete volume regulatory responses took 10-12 min. Bumetanide (50 microM) resulted in incomplete (50%) RVI. We found no threshold as the cells examined responded to hypertonic and hypotonic stimuli as low as 1%. There was some gradation as stimuli of <4% resulted in incomplete volume regulation. The degree of activation of the volume responses grew as an exponential buildup with the strength of the anisotonic challenge. We discuss how our observations are consistent with volume sensing mechanisms based on both ionic strength and the cytoskeleton.

A single amino acid residue can determine the sensitivity of SERCAs to artemisinins.

Artemisinins are the most important class of antimalarial drugs. They specifically inhibit PfATP6, a SERCA-type ATPase of Plasmodium falciparum. Here we show that a single amino acid in transmembrane segment 3 of SERCAs can determine susceptibility to artemisinin. An L263E replacement of a malarial by a mammalian residue abolishes inhibition by artemisinins. Introducing residues found in other Plasmodium spp. also modulates artemisinin sensitivity, suggesting that artemisinins interact with the thapsigargin-binding cleft of susceptible SERCAs.

Regulation of Na-K-2Cl cotransport in cultured bovine corneal endothelial cells.

We have previously demonstrated the presence of a Na(+)-K(+)-2Cl cotransporter in cultured bovine corneal endothelial cells (CBCEC) and determined that this cotransporter is located in the basolateral membrane. This transporter may contribute to volume regulation and transendothelial fluid transport. We have now investigated factors regulating the activity of the cotransporter. This activity was assessed by measuring the bumetanide-sensitive (86)Rubidium ((86)Rb) uptake in (86)Rb-containing solutions. Data were normalized to protein content determined with a Lowry protein assay. We investigated the regulation by extracellular and intracellular ion concentrations, by osmotic gradients, and by second messengers. Our results indicate that extracellular Na+ and K+ each are required for activation of the cotransporter and activate with first-order kinetics at half-maximally effective concentrations (k(1/2)) of 21.1 and 1.33 mM, respectively. Extracellular Cl- is also required for cotransport activation, but shows higher order kinetics; the k(1/2) for Cl- is 28.1 mM and the Hill coefficient 2.1. HCO(3)(-) exerts a modulating effect on cotransporter activity; at 0 HCO(3)(-) the bumetanide-sensitive K(+) uptake is reduced by 30% compared to that at 26 mm HCO(3)(-). Manipulations of the intracellular [Cl-] by preincubation in Cl- -free solution or inhibition of Cl- efflux resulted in increased uptake at low [Cl-](i) and decreased uptake at high [Cl-](i). To assess the role of protein kinases in the regulation of cotransport, we have determined the effect of protein kinase inhibitors. H-89 and KT5270, inhibitors of PKA, inhibit cotransport almost completely, while calphostin C, an inhibitor of PKC, produces a small activation of cotransport. The tyrosine kinase inhibitor genistein reduced K+ uptake while its inactive analog daidzein was without effect. The calmodulin kinase inhibitor KN-93 was without effect. We also investigated the effects of phosphatase inhibitors. Calyculin A (k(1/2)=21 nM) and okadaic acid (k(1/2)=915 nM) produced approximate doubling of K+ uptake, suggesting that phosphatase 1 is dominant. We also investigated the role of the cytoskeleton and its activation. Reduction of Ca(i)(2+) by preincubation in Ca2+ -free medium as well as by exposure to W-7, an inhibitor of the binding of Ca(2+) to calmodulin, reduced K+ uptake. Consistent with this, ML-7, a relatively specific inhibitor of the Ca2+ -calmodulin activated myosin light chain kinase, inhibited cotransport by 40%. The Ca2+ -calmodulin activated myosin light chain kinase contributes to the modulation of the cytoskeleton by regulating the actin-myosin interaction. Consistent with the above, disruption of the actin polymerization by cytochalasin D led to a decrease in K+ uptake. We conclude that extracellular Na+, K+ and Cl- are requirements for the function of the CBCEC Na(+)-K(+)-2Cl(-) cotransporter, while intracellular Cl- and extracellular HCO(3)(-) modulate its activity. Several protein kinases, including PKA, PKC, tyrosine kinase, and myosin light chain kinase, modulate the K+ uptake. Another modulating pathway for cotransport involves the state of the cytoskeleton.

Predicting the three-dimensional structure of the human facilitative glucose transporter glut1 by a novel evolutionary homology strategy: insights on the molecular mechanism of substrate migration, and binding sites for glucose and inhibitory molecules.

The glucose transporters (GLUT/SLC2A) are members of the major facilitator superfamily. Here, we generated a three-dimensional model for Glut1 using a two-step strategy: 1), GlpT structure as an initial homology template and 2), evolutionary homology using glucose-6-phosphate translocase as a template. The resulting structure (PDB No. 1SUK) exhibits a water-filled passageway communicating the extracellular and intracellular domains, with a funnel-like exofacial vestibule (infundibulum), followed by a 15 A-long x 8 A-wide channel, and a horn-shaped endofacial vestibule. Most residues which, by mutagenesis, are crucial for transport delimit the channel, and putative sugar recognition motifs (QLS, QLG) border both ends of the channel. On the outside of the structure there are two positively charged cavities (one exofacial, one endofacial) delimited by ATP-binding Walker motifs, and an exofacial large side cavity of yet unknown function. Docking sites were found for the glucose substrate and its inhibitors: glucose, forskolin, and phloretin at the exofacial infundibulum; forskolin, and phloretin at an endofacial site next to the channel opening; and cytochalasin B at a positively charged endofacial pocket 3 A away from the channel. Thus, 1SUK accounts for practically all biochemical and mutagenesis evidence, and provides clues for the transport process.

Intracellular [Na+], Na+ pathways, and fluid transport in cultured bovine corneal endothelial cells.

The mechanism of fluid transport across corneal endothelium remains unclear. We examine here the relative contributions of cellular mechanisms of Na+ transport and the homeostasis of intracellular [Na+] in cultured bovine corneal endothelial cells, and the influence of ambient Na+ and HCO3- on the deturgescence of rabbit cornea. Bovine corneal endothelial cells plated on glass coverslips were incubated for 60 min with 10 microm of the fluorescent Na+ indicator SBFI precursor in HCO3- HEPES (BH) Ringer's solution. After loading, cells were placed in a perfusion chamber. Indicator fluorescence (490 nm) was determined with a Chance-Legallais time-sharing fluorometer. Its voltage output was the ratio of the emissions excited at 340 and 380 nm. For calibration, cells were treated with gramicidin D. For fluid transport measurements, rabbit corneas were mounted in a Dikstein-Maurice chamber, and stromal thickness was measured with a specular microscope. The steady-state [Na+]i in BH was 14.36+/-0.38 mM (n = mean+/-s.e.). Upon exposure to Na+ -free BH solution (choline substituted), [Na+]i decreased to 1.81+/-0.20mM (n = 19). When going from Na+ -free plus 100 microm ouabain to BH plus ouabain, [Na+]i increased to 46.17+/-2.50 (n = 6) with a half time of 1.26+/-0.04 min; if 0.1 microm phenamil plus ouabain were present, it reached only 21.78+/-1.50mm. The exponential time constants (min-1) were: 0.56+/-0.04 for the Na+ pump; 0.39+/-0.01 for the phenamil sensitive Na+ channel; and 0.17+/-0.02 for the ouabain-phenamil-insensitive pathways. In HCO3- free medium (gluconate substituted), [Na+]i was 14.03+/-0.11mM; upon changing to BH medium, it increased to 30.77+/-0.74 mm. This last [Na+]i increase was inhibited 66% by 100 microm DIDS. Using BH medium, corneal thickness remained nearly constant, increasing at a rate of only 2.9+/-0.9 microm hr-1 during 3 hr. However, stromal thickness increased drastically (swelling rate 36.1+/-2.6 microm hr-1) in corneas superfused with BH plus 100 microm ouabain. Na+ -free, HCO3- free solution and 100 microm DIDS also led to increased corneal swelling rates (17.7+/-3.6, 14.4+/-1.6 and 14.9+/-1.2 microm hr-1, respectively). The present results are explained by the presence of a DIDS-inhibitable Na+-HCO3- cotransporter and an epithelial Na+ channel, both previously found in these cells. On the other hand, the quantitative picture presented here appears a novelty. The changes we observe are consistent with pump-driven rapid exchange of intracellular Na+, and recirculation of fully 70% of the Na+ pump flux via apical Na+ channels.

Fluid transport across cultured layers of corneal endothelium from aquaporin-1 null mice.

We explored the role of AQP1, the only known aquaporin in corneal endothelium, on active fluid transport and passive osmotic water movements across corneal endothelial layers cultured from AQP1 null mice and wildtype mice. AQP1 null mice had grossly transparent corneas, just as wildtype mice. Endothelial cell layers grown on permeable supports transported fluid at rates of (in microl h(-1) cm(-2), n = 9 mean+/-s.e.): 4.3+/-0.6, wildtype mice (MCE); 3.5+/-0.6, AQP1 null mice (KMCE; difference not significant). The osmotic water flow (also in microl h(-1) cm(-2)) induced by a 100 mOsm sucrose gradient across MCE cell layers (8.7+/-0.6, n = 8) was significantly greater than that across KMCE (5.7+/-0.7, n = 6, p = 0.007). When plated on glass coverslips, plasma membrane osmotic water permeability determined by light scattering was significantly higher for cells from wildtype vs. AQP1 null mice (in microm sec(-1): 74+/-4, n = 19 vs. 44+/-4 microm sec(-1), n = 11, p < 0.001). Unexpectedly, after 10% hypo-osmotic challenge, the extent of the regulatory volume recovery was significantly reduced for AQP1 null mice cells (in%: MCE controls, 99+/-1, n = 19 vs. KMCE: 64+/-5, n = 11, p < 0.001). Thus, as in other 'low rate' fluid transporting epithelia, deletion of AQP1 in mice corneal endothelium reduces osmotic water permeability but not active transendothelial fluid transport. However, that deletion impaired the extent of regulatory volume decrease after a hypo-osmotic challenge, suggesting a novel role for AQP1 in corneal endothelium.

Functional studies of threonine 310 mutations in Glut1: T310I is pathogenic, causing Glut1 deficiency.

We have previously reported on a patient with the Glut1 deficiency syndrome (Online Mendelian Inheritance in Man number 606777) carrying a heterozygous T310I missense mutation in the GLUT1 gene (Klepper, J., Wang, D., Fischbarg, J., Vera, J. C., Jarjour, I. T., O'Driscoll, K. R., and De Vivo, D. C. (1999) Neurochem. Res. 24, 587-594). To investigate the molecular basis for the associated functional deficit, we constructed T310A, T310S, and T310I human GLUT1 mutants for expression in Xenopus laevis oocytes via cRNA injection. For all mutants, glucose transport was decreased, and osmotic water permeability (Pf) was increased. Km values for 3-O-methylglucose (3-OMG) uptake under zero-trans influx and equilibrium exchange influx conditions were, respectively, 13 +/- 1 and 68 +/- 5 mm for wild-type Glut1, 5 +/- 1 and 25 +/- 6 mm for T310A, 6 +/- 3 and 30 +/- 6 mm for T310I, and 5 +/- 1 and 48 +/- 5 mm for T310S. Compared with wild-type Glut1, we determined the following. (a). Zero-trans and equilibrium exchange influx values of 3-OMG were significantly decreased, respectively, 15 and 5% in T310A, 8 and 3% in T310I, and 40 and 34% in T310S mutants. (b). Zero-trans efflux of 3-OMG and dehydroascorbic acid uptake were significantly decreased in mutants. (c). The relative Pf values for T310A, T310I, and T310S were increased 3-, 4.8-, and 3.5-fold compared with wild-type values. We found a very high negative correlation between the rate of glucose uptake and Pf (-0.93), and between hydropathy and uptake (-0.92), a moderate correlation between hydropathy and Pf (0.73), and a minimal correlation between uptake, Pf, and molecular weight. These findings are consistent with a central role for hydropathy rather than size at position 310 of this mutation.

Mutational analysis of the hexose transporter of Plasmodium falciparum and development of a three-dimensional model.

Plasmodium falciparum infection kills more than 1 million children annually. Novel drug targets are urgently being sought as multidrug resistance limits the range of treatment options for this protozoan pathogen. PfHT1, the major hexose transporter of P. falciparum is a promising new target. We report detailed structure-function studies on PfHT1 using site-directed mutagenesis approaches on residues located in helix V (Q169N) and helix VII ((302)SGL --> AGT). Studies with hexose analogues in these mutants have established that hexose recognition and permeation are intimately linked to these helices. A "fructose filter" effect results from the Q169N mutation (abolishing fructose uptake but preserving affinity and transport of glucose, as reported in Woodrow, C. J., Burchmore, R. J. S., and Krishna, S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9931-9936). Associated changes in competition for glucose uptake by C-2, C-3, and C-6 glucose analogues compared with native PfHT1 indicate subtle alterations in substrate interaction in this mutant. The K(m) values for glucose uptake in helix VII mutants are also similar to native PfHT1. Hydrogen bonding to positions C-5 and C-6 in glucose analogues becomes relatively more important in these mutants compared with native PfHT1. To increase understanding of hexose permeation pathways in PfHT1, we have developed the first three-dimensional model for PfHT1. As predicted for GLUT1, the principal mammalian glucose transporter, PfHT1 contains a main and an auxiliary channel. After modeling, the Q169N mutation leads predominantly to local structural changes, including displacement of neighboring helix IV. The (302)SGL position in helix VII lies in the same plane as Gln-169 in helix V but is also adjacent to the main hexose permeation pathway, consistent with results from experiments mutating this triplet motif. Furthermore, there are obvious structural and functional differences between GLUT1 and PfHT1 that can now be explored in detail using the approaches presented here. The development of specific inhibitors for PfHT1 will also be aided by these insights.

Changes in glucose transport and water permeability resulting from the T310I pathogenic mutation in Glut1 are consistent with two transport channels per monomer.

We studied glucose and water passage across wild type (WT) glucose transporter Glut1 and its T310I pathogenic mutant, expressing them in Xenopus laevis oocytes. We found that the T310I mutation produced a 8-fold decrease in glucose transport (zero-trans influx, 13 +/- 2% compared with WT), accompanied by a 2.8-fold increase in the osmotic water permeability (P(f) 280 +/- 40% compared with WT), and no change in the diffusional water permeability (P(d)). The dependence of glucose and water transports on the amounts of mutant cRNA injected was identical exponential buildups (k = 19.7 ng), suggesting that they depend similarly on the quaternary structure. The E(a) values for P(f) were 16 +/- 0.4 (WT) and 11 +/- 1 kcal mol(-1) (T310I). We report for the first time that 10 mm d-glucose and l-glucose inhibit P(f) by approximately 45% in the WT but not in the T310I mutant. In addition, 10 mm maltose reduces P(f) (15-20%) in both cases. However, 5 mm l-glucose increased the P(f) of T310I, consistent with a cooperative effect. These experimental observations and an analysis of our three-dimensional model strongly suggest the presence of two channels per Glut1 monomer, one of which can be blocked by the mutation T310I.