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

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/cm(2) 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.

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 (DeltaF), which suggested that this site lies in an environment that is affected by conformational change in the protein. DeltaF was substrate dependent (no DeltaF 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.

Functional characterization of two naturally occurring mutations in the human sodium-phosphate cotransporter type IIa.

UNLABELLED: Mutations in the gene encoding the human sodium-phosphate cotransporter (NPT2), causing reduced phosphate affinity and dominant-negative behavior, were described. We found no evidence of altered kinetics or dominant-negative effects. Thus, the mutations cannot account for the clinical phenotype. INTRODUCTION: Mutations in NPT22a, the gene encoding the sodium-phosphate cotransporter NaPi-IIa, were for the first time linked to human disease by Priè and colleagues. Two patients are described with renal phosphate wasting who were heterozygous for either the A48F or V147M mutation. Expressed in Xenopus oocytes, both mutants showed reduced phosphate affinity. Furthermore, coexpression of mutants with wildtype (WT) NaPi-IIa resulted in reduced cotransport function, explaining the mutants' dominant-negative effect in the patients. Intrigued by the implications of these findings on transporter kinetics, we decided to examine the transport characteristics of the two mutants in more detail. MATERIALS AND METHODS: We recreated the two mutants, expressed them in Xenopus oocytes, and analyzed their kinetic behavior by two-electrode voltage clamp. We also performed coexpression experiments where we injected mRNA for WT and mutants containing an additional S462C mutation, enabling complete inhibition of cotransport function with cysteine-modifying reagents. Finally, we expressed WT and mutant NaPi-IIa as C-terminal fusions to green fluorescent protein (GFP) in opossum kidney (OK) cells. RESULTS AND CONCLUSIONS: We found in our oocyte expression experiments that P(i)-induced currents were reduced in both mutants, whereas P(i) and Na affinities and other transport characteristics were not affected. The amount of cotransport activity remaining after cysteine modification, corresponding to WT activity, was not affected by coexpression of either mutant. Finally, GFP-tagged WT and mutants were expressed at the apical membrane in OK cells, showing that both mutants are correctly targeted in a mammalian cell. In conclusion, our data from oocyte and OK cell expression studies suggest that the heterozygous A48F and V 147M mutations cannot explain the pathological phenotype observed by Priè and colleagues.

Phosphate transporters: a tale of two solute carrier families.

Phosphate is an essential component of life and must be actively transported into cells against its electrochemical gradient. In vertebrates, two unrelated families of Na+ -dependent P(i) transporters carry out this task. Remarkably, the two families transport different P(i) species: whereas type II Na+/P(i) cotransporters (SCL34) prefer divalent HPO(4)(2-), type III Na(+)/P(i) cotransporters (SLC20) transport monovalent H2PO(4)(-). The SCL34 family comprises both electrogenic and electroneutral members that are expressed in various epithelia and other polarized cells. Through regulated activity in apical membranes of the gut and kidney, they maintain body P(i) homeostasis, and in salivary and mammary glands, liver, and testes they play a role in modulating the P(i) content of luminal fluids. The two SLC20 family members PiT-1 and PiT-2 are electrogenic and ubiquitously expressed and may serve a housekeeping role for cell P(i) homeostasis; however, also more specific roles are emerging for these transporters in, for example, bone mineralization. In this review, we focus on recent advances in the characterization of the transport kinetics, structure-function relationships, and physiological implications of having two distinct Na+/P(i) cotransporter families.

Deciphering PiT transport kinetics and substrate specificity using electrophysiology and flux measurements.

Members of the SLC20 family or type III Na(+) -coupled P(i) cotransporters (PiT-1, PiT-2) are ubiquitously expressed in mammalian tissue and are thought to perform a housekeeping function for intracellular P(i) homeostasis. Previous studies have shown that PiT-1 and PiT-2 mediate electrogenic P(i) cotransport when expressed in Xenopus oocytes, but only limited kinetic characterizations were made. To address this shortcoming, we performed a detailed analysis of SLC20 transport function. Three SLC20 clones (Xenopus PiT-1, human PiT-1, and human PiT-2) were expressed in Xenopus oocytes. Each clone gave robust Na(+)-dependent (32)P(i) uptake, but only Xenopus PiT-1 showed sufficient activity for complete kinetic characterization by using two-electrode voltage clamp and radionuclide uptake. Transport activity was also documented with Li(+) substituted for Na(+). The dependence of the P(i)-induced current on P(i) concentration was Michaelian, and the dependence on Na(+) concentration indicated weak cooperativity. The dependence on external pH was unique: the apparent P(i) affinity constant showed a minimum in the pH range 6.2-6.8 of approximately 0.05 mM and increased to approximately 0.2 mM at pH 5.0 and pH 8.0. Xenopus PiT-1 stoichiometry was determined by dual (22)Na-(32)P(i) uptake and suggested a 2:1 Na(+):P(i) stoichiometry. A correlation of (32)P(i) uptake and net charge movement indicated one charge translocation per P(i). Changes in oocyte surface pH were consistent with transport of monovalent P(i). On the basis of the kinetics of substrate interdependence, we propose an ordered binding scheme of Na(+):H(2)PO(4)(-):Na(+). Significantly, in contrast to type II Na(+)-P(i) cotransporters, the transport inhibitor phosphonoformic acid did not inhibit PiT-1 or PiT-2 activity.

Temperature dependence of steady-state and presteady-state kinetics of a type IIb Na+/P i cotransporter.

The temperature dependence of the transport kinetics of flounder Na(+)-coupled inorganic phosphate (P(i)) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. (32)P(i) uptake was strongly temperature-dependent and decreased by approximately 80% at a temperature change from 25 degrees C to 5 degrees C. The corresponding activation energy (E (a)) was approximately 14 kcal mol(-1) for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM P(i) and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the P(i)- and PFA-induced changes in holding current decreased with temperature. E (a) at -100 mV for the cotransport and leak modes was approximately 16 kcal mol(-1) and approximately 11 kcal mol(-1), respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E (a) for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM P(i) over a range of external Na(+). The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na(+)-debinding rate compared with the other voltage-dependent rate constants.

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(+)/P(i) 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 P(i)-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.

The human NBCe1-A mutant R881C, associated with proximal renal tubular acidosis, retains function but is mistargeted in polarized renal epithelia.

The human electrogenic renal Na-HCO(3) cotransporter (NBCe1-A; SLC4A4) is localized to the basolateral membrane of proximal tubule cells. Mutations in the SLC4A4 gene cause an autosomal recessive proximal renal tubular acidosis (pRTA), a disease characterized by impaired ability of the proximal tubule to reabsorb HCO(3)(-) from the glomerular filtrate. Other symptoms can include mental retardation and ocular abnormalities. Recently, a novel homozygous missense mutant (R881C) of NBCe1-A was reported from a patient with a severe pRTA phenotype. The mutant protein was described as having a lower than normal activity when expressed in Xenopus oocytes, despite having normal Na(+) affinity. However, without trafficking data, it is impossible to determine the molecular basis for the phenotype. In the present study, we expressed wild-type NBCe1-A (WT) and mutant NBCe1-A (R881C), tagged at the COOH terminus with enhanced green fluorescent protein (EGFP). This approach permitted semiquantification of surface expression in individual Xenopus oocytes before assay by two-electrode voltage clamp or measurements of intracellular pH. These data show that the mutation reduces the surface expression rather than the activity of the individual protein molecules. Confocal microscopy on polarized mammalian epithelial kidney cells [Madin-Darby canine kidney (MDCK)I] expressing nontagged WT or R881C demonstrates that WT is expressed at the basolateral membrane of these cells, whereas R881C is retained in the endoplasmic reticulum. In summary, the pathophysiology of pRTA caused by the R881C mutation is likely due to a deficit of NBCe1-A at the proximal tubule basolateral membrane, rather than a defect in the transport activity of individual molecules.

Steady-state kinetic characterization of the mouse B(0)AT1 sodium-dependent neutral amino acid transporter.

The members of the neurotransmitter transporter family SLC6A exhibit a high degree of structural homology; however differences arise in many aspects of their transport mechanisms. In this study we report that mouse B(0)AT1 (mouse Slc6a19) mediates the electrogenic transport of a broad range of neutral amino acids but not of the chemically similar substrates transported by other SLC6A family members. Cotransport of L: -Leu and Na(+) generates a saturable, reversible, inward current with Michaelis-Menten kinetics (Hill coefficient approximately 1) yielding a K(0.5) for L: -Leu of 1.16 mM and for Na(+) of 16 mM at a holding potential of -50 mV. Changing the membrane voltage influences both substrate binding and substrate translocation. Li(+) can substitute partially for Na(+) in the generation of L: -Leu-evoked inward currents, whereas both Cl(-) and H(+) concentrations influence its magnitude. The simultaneous measurement of charge translocation and L: -Leu uptake in the same cell indicates that B(0)AT1 transports one Na(+) per neutral amino acid. This appears to be accomplished by an ordered, simultaneous mechanism, with the amino acid binding prior to the Na(+), followed by the simultaneous translocation of both co-substrates across the plasma membrane. From this kinetic analysis, we conclude that the relatively constant [Na(+)] along the renal proximal tubule both drives the uptake of neutral amino acids via B(0)AT1 thermodynamically and ensures that, upon binding, these are translocated efficiently into the cell.

Renouncing electroneutrality is not free of charge: switching on electrogenicity in a Na+-coupled phosphate cotransporter.

Renal type IIa Na+-coupled inorganic phosphate (Pi) cotransporters (NaPi-IIa) mediate divalent Pi transport in an electrogenic manner, whereas the renal type IIc isoform (NaPi-IIc) is electroneutral, yet it shows high sequence identity with NaPi-IIa. Dual uptake (32Pi/22Na) assays confirmed that NaPi-IIc displayed Na+-coupled Pi cotransport with a 2:1 (Na+:Pi) stoichiometry compared with 3:1 established for NaPi-IIa. This finding suggested that the electrogenicity of NaPi-IIa arises from the interaction of an additional Na+ ion compared with NaPi-IIc. To identify the molecular elements responsible for the functional difference between isoforms, we used chimera and amino acid replacement approaches. Transport activity of chimeras constructed with NaPi-IIa and NaPi-IIc indicated that residues within the first six transmembrane domains were essential for the electrogenicity of NaPi-IIa. Sequence comparison between electrogenic and electroneutral isoforms revealed differences in the charge and polarity of residues clustered in three areas, one of which included part of the predicted third transmembrane domain. Here, substitution of three residues with their NaPi-IIa equivalents in NaPi-IIc (S189A, S191A, and G195D) resulted in a transporter that displayed a 1:1 charge/Pi coupling, a 3:1 Na+:Pi stoichiometry, and transient currents that resembled pre-steady-state relaxations. The mutant's weaker voltage dependency and 10-fold lower apparent Pi affinity compared with NaPi-IIa indicated that other residues important for the NaPi-IIa kinetic fingerprint exist. Our findings demonstrate that, through a minimal number of side chain substitutions, we can effect a switch from electroneutral to electrogenic cotransporter function, concomitant with the appearance of a cosubstrate interaction site.

Na+-dependent HCO3- uptake into the rat choroid plexus epithelium is partially DIDS sensitive.

Several studies suggest the involvement of Na+ and HCO3- transport in the formation of cerebrospinal fluid. Two Na+-dependent HCO3- transporters were recently localized to the epithelial cells of the rat choroid plexus (NBCn1 and NCBE), and the mRNA for a third protein was also detected (NBCe2) (Praetorius J, Nejsum LN, and Nielsen S. Am J Physiol Cell Physiol 286: C601-C610, 2004). Our goal was to immunolocalize the NBCe2 to the choroid plexus by immunohistochemistry and immunogold electronmicroscopy and to functionally characterize the bicarbonate transport in the isolated rat choroid plexus by measurements of intracellular pH (pHi) using a dual-excitation wavelength pH-sensitive dye (BCECF). Both antisera derived from COOH-terminal and NH2-terminal NBCe2 peptides localized NBCe2 to the brush-border membrane domain of choroid plexus epithelial cells. Steady-state pHi in choroidal cells increased from 7.03 +/- 0.02 to 7.38 +/- 0.02 (n=41) after addition of CO2/HCO3- into the bath solution. This increase was Na+ dependent and inhibited by the Cl- and HCO3- transport inhibitor DIDS (200 muM). This suggests the presence of Na+-dependent, partially DIDS-sensitive HCO3- uptake. The pHi recovery after acid loading revealed an initial Na+ and HCO3- -dependent net base flux of 0.828 +/- 0.116 mM/s (n = 8). The initial flux in the presence of CO2/HCO3- was unaffected by DIDS. Our data support the existence of both DIDS-sensitive and -insensitive Na+- and HCO3- -dependent base loader uptake into the rat choroid plexus epithelial cells. This is consistent with the localization of the three base transporters NBCn1, Na+-driven Cl- bicarbonate exchanger, and NBCe2 in this tissue.

Substrate interactions in the human type IIa sodium-phosphate cotransporter (NaPi-IIa).

We have characterized the kinetics of substrate transport in the renal type IIa human sodium-phosphate cotransporter (NaPi-IIa). The transporter was expressed in Xenopus laevis oocytes, and steady-state and pre-steady-state currents and substrate uptakes were characterized by voltage-clamp and isotope flux. First, by measuring simultaneous uptake of a substrate (32Pi, 22Na) and charge in voltage-clamped oocytes, we established that the human NaPi-IIa isoform operates with a Na:Pi:charge stoichiometry of 3:1:1 and that the preferred transported Pi species is HPO4(2-). We then probed the complex interrelationship of substrates, pH, and voltage in the NaPi-IIa transport cycle by analyzing both steady-state and pre-steady-state currents. Steady-state current measurements show that the apparent HPO4(2-) affinity is voltage dependent and that this voltage dependency is abrogated by lowering the pH or the Na+ concentration. In contrast, the voltage dependency of the apparent Na+ affinity increased when pH was lowered. Pre-steady-state current analysis shows that Na+ ions bind first and influence the preferred orientation of the transporter in the absence of Pi. Pre-steady-state charge movement was partially suppressed by complete removal of Na+ from the bath, by reducing extracellular pH (both in the presence and absence of Na+), or by adding Pi (in the presence of 100 mM Na). None of these conditions suppressed charge movement completely. The results allowed us to modify previous models for the transport cycle of NaPi-II transporters by including voltage dependency of HPO4(2-) binding and proton modulation of the first Na+ binding step.

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

We have functionally characterized Na+-driven bicarbonate transporter (NBC)4, originally cloned from human heart by Pushkin et al. (Pushkin A, Abuladze N, Newman D, Lee I, Xu G, and Kurtz I. Biochem Biophys Acta 1493: 215-218, 2000). Of the four NBC4 variants currently present in GenBank, our own cloning efforts yielded only variant c. We expressed NBC4c (GenBank accession no. AF293337) in Xenopus laevis oocytes and assayed membrane potential (Vm) and pH regulatory function with microelectrodes. Exposing an NBC4c-expressing oocyte to a solution containing 5% CO2 and 33 mM HCO elicited a large hyperpolarization, indicating that the transporter is electrogenic. The initial CO2-induced decrease in intracellular pH (pH(i)) was followed by a slow recovery that was reversed by removing external Na+. Two-electrode voltage clamp of NBC4c-expressing oocytes revealed large HCO- and Na+-dependent currents. When we voltage clamped V(m) far from NBC4c's estimated reversal potential (E(rev)), the pH(i) recovery rate increased substantially. Both the currents and pH(i) recovery were blocked by 200 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). We estimated the transporter's HCO:Na+ stoichiometry by measuring E(rev) at different extracellular Na+ concentration ([Na+]o) values. A plot of E(rev) against log[Na+]o was linear, with a slope of 54.8 mV/log[Na+]o. This observation, as well as the absolute E(rev) values, are consistent with a 2:1 stoichiometry. In conclusion, the behavior of NBC4c, which we propose to call NBCe2-c, is similar to that of NBCe1, the first electrogenic NBC.

Cloning of a Na+-driven Cl/HCO3 exchanger from squid giant fiber lobe.

We extracted RNA from the giant fiber lobe (GFL) of the squid Loligo pealei and performed PCR with degenerate primers that were based on highly conserved regions of Na+-coupled HCO3- transporters. This approach yielded a novel, 290-bp sequence related to the bicarbonate transporter superfamily. Using an L. opalescens library, we extended the initial fragment in the 3' and 5' directions by a combination of library screening and PCR and obtained the full-length clone (1,198 amino acids) by PCR from L. pealei GFL. The amino acid sequence is 46% identical to mammalian electrogenic and electroneutral Na-HCO3 cotransporters and 33% identical to the anion exchanger AE1. Northern blot analysis showed strong signals in L. pealei GFL, optic lobe, and heart and weaker signals in gill and stellate ganglion. To assess function, we injected in vitro-transcribed cRNA into Xenopus oocytes and subsequently used microelectrodes to monitor intracellular pH (pHi) and membrane voltage (Vm). Superfusing these oocytes with 5% CO2-33 mM HCO3- caused a CO2-induced fall in pHi, followed by a slow recovery. The absence of a rapid HCO3- -induced hyperpolarization indicates that the pHi recovery mechanism is electroneutral. Ion substitutions showed that Na+ and Cl- are required on opposite sides of the membrane. Transport was blocked by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The characteristics of our novel clone fit those of a Na+-driven Cl/HCO3 exchanger (NDCBE).

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

We have cloned a novel aquaporin (AQP) from Xenopus 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 approximately 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)) increased P(f) and to a lesser extent urea uptake but not glycerol uptake. Remarkably, low HgCl(2) concentrations (0.3-10 microm) reduced P(f) and urea uptake, whereas high concentrations (300-1000 microm) reversed the inhibition. We propose that AQPxlo is a new AQP paralogue unknown in mammals.