Leila V. Virkki

Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane

We report here the application of a previously described method to directly determine the CO2 permeability (PCO2) of the cell membranes of normal human red blood cells (RBCs) vs. those deficient in aquaporin 1 (AQP1), as well as AQP1‐expressing Xenopus laevis oocytes. This method measures the exchange of 18O between CO2, HCO3–, and H2O in cell suspensions. In addition, we measure the alkaline surface pH (pHS) transients caused by the dominant effect of entry of CO2 vs. HCO3– into oocytes exposed to step increases in [CO2]. We report that 1) AQP1 constitutes the major pathway for molecular CO2 in human RBCs; lack of AQP1 reduces PCO2 from the normal value of 0.15 ± 0.08 (SD; n85) cm/s by 60% to 0.06 cm/s. Expression of AQP1 in oocytes increases PCO2 2‐fold and doubles the alkaline pHS gradient. 2) pCMBS, an inhibitor of the AQP1 water channel, reduces PCO2 of RBCs solely by action on AQP1 as it has no effect in AQP1‐deficient RBCs. 3) PCO2 determinations of RBCs and pHS measurements of oocytes indicate that DIDS inhibits the CO2 pathway of AQP1 by half. 4) RBCs have at least one other DIDS‐sensitive pathway for CO2. We conclude that AQP1 is responsible for 60% of the high PCO2 of red cells and that another, so far unidentified, CO2 pathway is present in this membrane that may account for at least 30% of total PCO2.—Endeward, V., Musa‐Aziz, R., Cooper, G. J., Chen, L., Pelletier, M. F., Virkki, L. V., Supuran, C. T., King, L. S., Boron, W. F., Gros, G. Evidence that aquaporin 1 is a major pathway for CO2 transport across the human erythrocyte membrane. FASEB J. 20, 1974–1981 (2006)

Functional Characterization of Two Naturally Occurring Mutations in the Human Sodium-Phosphate Cotransporter Type IIa

Mutations in the gene encoding the human sodium‐phosphate cotransporter (NPT2), causing reduced phosphate affinity and dominant‐negative behavior, were described. (1) We found no evidence of altered kinetics or dominant‐negative effects. Thus, the mutations cannot account for the clinical phenotype.

Voltage Clamp Fluorometric Measurements on a Type II Na+-coupled Pi Cotransporter: Shedding Light on Substrate Binding Order

Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637–651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (ΔF), which suggested that this site lies in an environment that is affected by conformational change in the protein. ΔF was substrate dependent (no ΔF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1–18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.

Cloning and functional characterization of a novel aquaporin from Xenopus laevis oocytes.

We have cloned a novel aquaporin (AQP) fromXenopus laevis oocytes, which we have provisionally named AQPxlo. The predicted protein showed highest homology (39–50%) to aquaglyceroporins. Northern blot analysis showed strong hybridization to an ∼1.4-kb transcript in X. laevis fat body and oocytes, whereas a weaker signal was obtained in kidney. We injected in vitro transcribed cRNA encoding AQPxlo into Xenopus oocytes for functional characterization. AQPxlo expression increased osmotic water permeability (P f ), as well as the uptake of glycerol and urea. However, AQPxlo excluded larger polyols and thiourea. An alkaline extracellular pH (pH o ) increasedP f and to a lesser extent urea uptake but not glycerol uptake. Remarkably, low HgCl2 concentrations (0.3–10 μm) reduced P f and urea uptake, whereas high concentrations (300–1000 μm) reversed the inhibition. We propose that AQPxlo is a new AQP paralogue unknown in mammals.

Green tea extract reduces induction of p53 and apoptosis in UVB-irradiated human skin independent of transcriptional controls

Abstract:  Ultraviolet (UV) irradiation plays a pivotal role in human skin carcinongenesis. Preclinically, systemically and topically applied green tea extract (GTE) has shown reduction of UV‐induced (i) erythema, (ii) DNA damage, (iii) formation of radical oxygen species and (iv) downregulation of numerous factors related to apoptosis, inflammation, differentiation and carcinogenesis. In humans, topical GTE has so far only been tested in limited studies, with usually very high GTE concentrations and over short periods of time. Both chemical stability of GTE and staining properties of highly concentrated green tea polyphenols limit the usability of highly concentrated green tea extracts in cosmetic products. The present study tested the utility of stabilized low‐dose GTE as photochemopreventive agents under everyday conditions. We irradiated with up to 100 mJ/cm2 of UVB light skin patches which were pretreated with either OM24®‐containing lotion or a placebo lotion. Biopsies were taken from both irradiated and un‐irradiated skin for both immunohistochemistry and DNA microarray analysis. We found that while OM24® treatment did not significantly affect UV‐induced erythema and thymidine dimer formation, OM24® treatment significantly reduced UV‐induced p53 expression in keratinocytes. We also found that OM24® treatment significantly reduced the number of apoptotic keratinocytes (sunburn cells and TUNEL‐positive cells). Carefully controlled DNA microarray analyses showed that OM24® treatment does not induce off‐target changes in gene expression, reducing the likelihood of unwanted side‐effects. Topical GTE (OM24®) reduces UVB‐mediated epithelial damage already at low, cosmetically usable concentrations, without tachyphylaxis over 5 weeks, suggesting GTE as suitable everyday photochemopreventive agents.

Mapping conformational changes of a type IIb Na+/Pi cotransporter by voltage clamp fluorometry.

The fluorescence of a fluorophore depends on its environment, and if attached to a protein it may report on conformational changes. We have combined two-electrode voltage clamp with simultaneous fluorescence measurements to detect conformational changes in a type IIb Na+/Pi cotransporter expressed in Xenopus oocytes. Four novel Cys, labeled with a fluorescent probe, yielded voltage- and substrate-dependent changes in fluorescence (F). Neither Cys substitution nor labeling significantly altered the mutant electrogenic properties. Different F responses to voltage and substrate were recorded at the four sites. S155C, located in an intracellular re-entrant loop in the first half of the protein, and E451C, located in an extracellular re-entrant loop in the second half of the protein, both showed Na+, Li+, and Pi-dependent F signals. S226C and Q319C, located at opposite ends of a large extracellular loop in the middle of the protein, mainly responded to changes in Na+ and Li+. Hyperpolarization increased F for S155C and S226C but decreased F for Q319C and E451C. The labeling and F response of S155C, confirmed that the intracellular loop containing Ser-155 is re-entrant as it is accessible from the extracellular milieu. The behavior of S155C and E451C indicates a strong involvement of the two re-entrant loops in conformational changes during the transport cycle. Moreover, the data for S226C and Q319C suggest that also the large extracellular loop is associated with transport function. Finally, the reciprocal voltage dependences of the S155C-E451C and S226C-Q319C pairs suggest reciprocal conformational changes during the transport cycle for their respective local environments.

Functionally Important Residues in the Predicted 3rd Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)

The type IIa Na+/Pi, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na+ and one divalent Pi ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and Pi transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of Pi transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the Pi-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na+-dependent Pi transport. Since electrogenic NaPi-II transports 3 Na+/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na+ binding sites in NaPi-IIa.

β‐Adrenergic Stimulation of Volume‐Sensitive Chloride Transport in Lamprey Erythrocytes

We measured the effects of a &bgr;‐adrenergic agonist, isoproterenol, on chloride transport and volume regulation of lamprey (Lampetra fluviatilis) erythrocytes in isotonic (288 mosm L−1) and hypotonic (192 mosm L−1) medium. Isoproterenol at a high concentration (10−5 M) did not influence chloride transport in isotonic medium but markedly increased chloride fluxes in hypotonic conditions: unidirectional flux increased from 100 mmol kg dcw−1 h−1 in the absence to 350 mmol kg dcw−1 h−1 ( \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape

Functional characterization of human NBC4 as an electrogenic Na+-HCO cotransporter (NBCe2).

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