Thomas Zeuthen

Aquaporin homologues in plants and mammals transport ammonia

Using functional complementation and a yeast mutant deficient in ammonium (NH4 +) transport (Δmep1–3), 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.

Aquaporins in complex tissues: distribution of aquaporins 1–5 in human and rat eye

Multiple physiological fluid movements are involved in vision. Here we define the cellular and subcellular sites of aquaporin (AQP) water transport proteins in human and rat eyes by immunoblotting, high-resolution immunocytochemistry, and immunoelectron microscopy. AQP3 is abundant in bulbar conjunctival epithelium and glands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering the trabecular meshwork and Schlemms canal. Although AQP1 is plentiful in ciliary nonpigmented epithelium, it is not present in ciliary pigmented epithelium. Posterior and anterior epithelium of the iris and anterior lens epithelium also contain significant amounts of AQP1, but AQP0 (major intrinsic protein of the lens) is expressed in lens fiber cells. Retinal Müller cells and astrocytes exhibit notable concentrations of AQP4, whereas neurons and retinal pigment epithelium do not display aquaporin immunolabeling. These studies demonstrate selective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocular epithelia, predicting specific roles for each in the complex network through which water movements occur in the eye.Multiple physiological fluid movements are involved in vision. Here we define the cellular and subcellular sites of aquaporin (AQP) water transport proteins in human and rat eyes by immunoblotting, high-resolution immunocytochemistry, and immunoelectron microscopy. AQP3 is abundant in bulbar conjunctival epithelium and glands but is only weakly present in corneal epithelium. In contrast, AQP5 is prominent in corneal epithelium and apical membranes of lacrimal acini. AQP1 is heavily expressed in scleral fibroblasts, corneal endothelium and keratocytes, and endothelium covering the trabecular meshwork and Schlemms canal. Although AQP1 is plentiful in ciliary nonpigmented epithelium, it is not present in ciliary pigmented epithelium. Posterior and anterior epithelium of the iris and anterior lens epithelium also contain significant amounts of AQP1, but AQP0 (major intrinsic protein of the lens) is expressed in lens fiber cells. Retinal Müller cells and astrocytes exhibit notable concentrations of AQP4, whereas neurons and retinal pigment epithelium do not display aquaporin immunolabeling. These studies demonstrate selective expression of AQP1, AQP3, AQP4, and AQP5 in distinct ocular epithelia, predicting specific roles for each in the complex network through which water movements occur in the eye.

The human Na+–glucose cotransporter is a molecular water pump

1 The human Na+‐glucose cotransporter (hSGLT1) was expressed in Xenopus laevis oocytes. The transport activity, given by the Na+ current, was monitored as a clamp current and the concomitant flux of water followed optically as the change in oocyte volume. 2 When glucose was added to the bathing solution there was an abrupt increase in clamp current and an immediate swelling of the oocyte. The transmembrane transport of two Na+ ions and one sugar molecule was coupled, within the protein itself, to the influx of 210 water molecules. 3 This stoichiometry was constant and independent of the external parameters: Na+ concentrations, sugar concentrations, transmembrane voltages, temperature and osmotic gradients. 4 The cotransport of water occurred in the presence of adverse osmotic gradients. In accordance with the Gibbs equation, energy was transferred within the protein from the downhill fluxes of Na+ and sugar to the uphill transport of water, indicative of secondary active transport of water. 5 Unstirred layer effects were ruled out on the basis of experiments on oocytes treated with gramicidin or other ionophores. Na+ currents maintained by ionophores did not lead to any initial water movements. 6 The finding of a molecular water pump allows for new models of cellular water transport which include coupling between ion and water fluxes at the protein level; the hSGLT1 could account for almost half the daily reuptake of water from the small intestine.

Structure-activity relationships of P-glycoprotein interacting drugs: kinetic characterization of their effects on ATPase activity

We have determined the kinetic parameters for stimulation and inhibition by 34 drugs of the P-glycoprotein ATPase in membranes derived from CR1R12 Chinese hamster ovary cells. The drugs chosen were sets of calmodulin antagonists, steroids, hydrophobic cations, hydrophobic peptides, chemotherapeutic substrates of P-glycoprotein, and some other drugs with lower affinity for P-glycoprotein. We studied how these kinetic parameters correlated with the partition coefficient and the Van der Waals surface area of the drugs. The maximum velocity of ATPase stimulation decreased with surface area and showed a suggestion of a maximum with increasing partition coefficient. The affinity of these drugs for P-glycoprotein showed no significant correlation with partition coefficient but was highly correlated with the surface area suggesting that binding between modulators and P-glycoprotein takes place across a wide interaction surface on the protein.

Transport of water and glycerol in aquaporin 3 is gated by H(

Aquaporins (AQPs) were expressed in Xenopus laevis oocytes in order to study the effects of external pH and solute structure on permeabilities. For AQP3 the osmotic water permeability, L p , was abolished at acid pH values with a pK of 6.4 and a Hill coefficient of 3. TheL p values of AQP0, AQP1, AQP2, AQP4, and AQP5 were independent of pH. For AQP3 the glycerol permeabilityP Gl, obtained from [14C]glycerol uptake, was abolished at acid pH values with a pK of 6.1 and a Hill coefficient of 6. Consequently, AQP3 acts as a glycerol and water channel at physiological pH, but predominantly as a glycerol channel at pH values around 6.1. The pH effects were reversible. The interactions between fluxes of water and straight chain polyols were inferred from reflection coefficients (ς). For AQP3, water and glycerol interacted by competing for titratable site(s): ςGl was 0.15 at neutral pH but doubled at pH 6.4. The ς values were smaller for polyols in which the —OH groups were free to form hydrogen bonds. The activation energy for the transport processes was around 5 kcal mol−1. We suggest that water and polyols permeate AQP3 by forming successive hydrogen bonds with titratable sites.

COMPETITIVE, NON-COMPETITIVE AND COOPERATIVE INTERACTIONS BETWEEN SUBSTRATES OF P-GLYCOPROTEIN AS MEASURED BY ITS ATPASE ACTIVITY

We have studied the interaction between verapamil and other modulators of the P-glycoprotein ATPase from membranes of CR1R12 Chinese hamster ovary cells. Four major categories of interaction were identified. (i) Non-competitive inhibition of verapamils stimulation of enzyme activity was found with vanadate. (ii) Competitive inhibition of the ATPase was found for the pair verapamil and cyclosporin A. (iii) Allosteric inhibition with an increase in the Hill number for verapamil was found in the cases of daunorubicin, epirubicin, gramicidin S and D, vinblastine, amiodarone, and colchicine. (iv) Cooperative stimulation of verapamil-induced ATPase activity was found with progesterone, diltiazem, amitriptyline, and propranolol. At high levels, progesterone and verapamil mutually enhanced each others inhibitory action on the ATPase. Our data show that the substrate binding behavior of P-glycoprotein is complex with more than one binding site being present. This information could form the basis for the development of improved modulators of P-glycoprotein.

Passive water and ion transport by cotransporters.

1 The rabbit Na+‐glucose (SGLT1) and the human Na+‐Cl−‐GABA (GAT1) cotransporters were expressed in Xenopus laevis oocytes, and passive Na+ and water transport were studied using electrical and optical techniques. Passive water permeabilities (Lp) of the cotransporters were determined from the changes in oocyte volume in response to osmotic gradients. The specific SGLT1 and GAT1 Lp values were obtained by measuring Lp in the presence and absence of blockers (phlorizin and SKF89976A). In the presence of the blockers, the Lp values of oocytes expressing SGLT1 and GAT1 were indistinguishable from the Lp of control oocytes. Passive Na+ transport (Na+ leak) was obtained from the blocker‐sensitive Na+ currents in the absence of substrates (glucose and GABA). 3 Passive Na+ and water transport through SGLT1 were blocked by phlorizin with the same sensitivity (inhibitory constant (Ki), 3‐5 μM). When Na+ was replaced with Li+, phlorizin also inhibited Li+ and water transport, but with a lower affinity (Ki, 100 μM). When Na+ was replaced by choline, which is not transported, the SGLT1 Lp was indistinguishable from that in Na+ or Li+, but in this case water transport was less sensitive to phlorizin. 4 The activation energies (Ea) for passive Na+ and water transport through SGLT1 were 21 and 5 kcal mol−1, respectively. The high Ea for Na+ transport is comparable to that of Na+‐glucose cotransport and indicates that the process is dependent on conformational changes of the protein, while the low Ea for water transport is similar to that of water channels (aquaporins). 5 GAT1 also behaved as an SKF89976A‐sensitive water channel. We did not observe passive Na+ transport through GAT1. 6 We conclude that passive water and Na+ transport through cotransporters depend on different mechanisms: Na+ transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel. In the case of SGLT1, we suggest that both the water channel and water cotransport could contribute to isotonic fluid transport across the intestinal brush border membrane.

Maxi K+ channels in leaky epithelia are regulated by intracellular Ca2+, pH and membrane potential

We have studied a Ca2+-activated K+ channel in the ventricular membrane of the epithelium of choroid plexus by means of the patch-clamp technique, using excised inside-out patches. The channel was highly K+ selective. It had a conductance of ∼200 pS with 112 mM KCl on both sides of the membrane. The probability for the channel being open increased with intracellular Ca2+, pH and with membrane potential. The channel shows two gating modes. The primary gating mode has open and closed times which depend strongly on membrane potential, intracellular Ca2+ and pH. It accounts for the variation of the channel open probability. Lowering intracellular pH from 7.4 to 6.4 reduced the channel open probability mainly by increasing the channel closed time. It appears, that H+ can compete with Ca2+ in binding to the same site, thereby preventing channel opening. A second gating mode consisted of short-lived closures, or flickers. The open and closed time for this process were largely independent of membrane potential, intracellular Ca2+ and pH. The channel density was ∼0.4 μm−2 corresponding to a K+-permeability of 2.2 10−5 cm s−1 if the channels were fully open. In cell-attached patches we measured the open probability of the channel in the intact cell membrane. The channel is almost totally closed under normal cellular conditions. This type of channel is therefore not the membrane component that forms the electrodiffusive pathway for K+-ions.

Water transport between CNS compartments: contributions of aquaporins and cotransporters.

Large water fluxes continuously take place between the different compartments of the brain as well as between the brain parenchyma and the blood or cerebrospinal fluid. This water flux is tightly regulated but may be disturbed under pathological conditions that lead to brain edema formation or hydrocephalus. The molecular pathways by which water molecules cross the cell membranes of the brain are not well-understood, although the discovery of aquaporin 4 (AQP4) in the brain improved our understanding of some of these transport processes, particularly under pathological conditions. In the present review we introduce another family of transport proteins as water transporters, namely the cotransporters and the glucose uniport GLUT1. In direct contrast to the aquaporins, these proteins have an inherent ability to transport water against an osmotic gradient. Some of them may also function as water pores in analogy to the aquaporins. The putative role of cotransport proteins and uniports for the water flux into the glial cells, through the choroid plexus and across the endothelial cells of the blood-brain-barrier will be discussed and compared to the contribution of the aquaporins.

Measurement of Cell Volume Changes by Fluorescence Self-Quenching

At high concentrations, certain fluorophores undergo self-quenching, i.e., fluorescence intensity decreases with increasing fluorophore concentration. Accordingly, the self-quenching properties can be used for measuring water volume changes in lipid vesicles. In cells, quantitative determination of water transport using fluorescence self-quenching has been complicated by the requirement of relatively high (mM) and often toxic loading concentrations. Here we report a simple method that uses low (μM) loading concentrations of calcein-acetoxymethyl ester (calcein-AM) to obtain intracellular concentrations of the fluorophore calcein suitable for measurement of changes in cell water volume by self-quenching. The relationship between calcein fluorescence intensity, when excited at 490 nm (its excitation maximum), and calcein concentration was investigated in vitro and in various cultured cell types. The relationship was bell-shaped, with the negative slope in the concentration range where the fluorophore undergoes fluorescence self-quenching. In cultured retinal pigment epithelial cells, calcein fluorescence and extracellular osmolarity were linearly related. A 25-mOsm hypertonic challenge corresponded to a decrease in calcein fluorescence with high signal-to-noise ratio (>15). Similar results were obtained with the fluorophore BCECF when excited at its isosbestic wavelength (436 nm). The present results demonstrate the usefulness of fluorescence self-quenching to measure rapid changes in cell water volume.