Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport
|
|
- Nancy George
- 5 years ago
- Views:
Transcription
1 Am J Physiol Gastrointest Liver Physiol 293: G271 G278, First published April 5, 2007; doi: /ajpgi Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport Chitrawina Mahagita, 1,3 Steven M. Grassl, 2 Pawinee Piyachaturawat, 1 and Nazzareno Ballatori 3 1 Department of Physiology, Faculty of Science, Mahidol University, Bangkok, Thailand; 2 Department of Pharmacology, State University of New York Upstate Medical University, Syracuse; and 3 Department of Environmental Medicine, University of Rochester School of Medicine, Rochester, New York Submitted 12 February 2007; accepted in final form 3 April 2007 Mahagita C, Grassl SM, Piyachaturawat P, Ballatori N. Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport. Am J Physiol Gastrointest Liver Physiol 293: G271 G278, First published April 5, 2007; doi: /ajpgi Organic anion transporting polypeptides (OATP/SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 (Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 (SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C (SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C crna was unaffected by depolarization of the membrane potential or by changes in ph, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/ OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity. membrane transport; anion exchange; driving force; glutathione; organic solute transporters THE CLEARANCE of endogenous substances and xenobiotics from blood is one of the vital functions of the liver. This elaborate system requires specific uptake, biotransformation, and excretion mechanisms. Hepatic uptake is mediated in large part by three types of transporters: the sodium-taurocholate cotransporter (SLC10) (9), organic anion or cation transporters (SLC22A) (6, 15), and organic anion transporting polypeptides (OATP/SLCO) (12, 20). Members of the OATP/SLCO family of solute carriers function as transporters for a large variety of amphipathic organic compounds. These proteins mediate Na - and ATP-independent transport of bile salts, steroids, thyroid hormones, anionic peptides, drugs, and xenobiotics (7, 8). Among the OATP/ SLCO family members, OATP1B1/OATP-C (SLCO1B1) and OATP1B3/OATP-8 (SLCO1B3) are expressed selectively in the liver (1, 11, 16) and have also been called liver-specific organic anion transporters (LST-1 and LST-2, respectively). The current nomenclature for the OATP/SLCO family was introduced in 2004 by Hagenbuch and Meier (7) to prevent confusion and to permit unequivocal, species-independent identification. SLCO1B1 and SLCO1B3 genes, encoding OATP1B1/ OATP-C and OATP1B3/OATP-8 proteins, were identified and cloned in (1, 11, 16, 17) and are thought to function as the major hepatic uptake systems for endogenous compounds, drugs, and albumin-bound organic anions. More than 20 single-nucleotide polymorphisms (SNPs) of the SLCO1B1 gene have been identified in African-American, European-American, and Japanese people (18), and 3 nonsynonymous SNPs in human blood samples have been reported for SLCO1B3, consisting of 334T G, encoding OTP1B3/OATP- 8-S112A; 699G A, encoding OTP1B3/OATP-8-M233I; and 1564G T, encoding OTP1B3/OATP-8-G522C (18, 21). The 334T G and 699G A gene mutations are detected frequently in European and American Caucasians ( 70% allelic frequency for each), whereas the 1564G T mutant is seen in 2% of individuals in these populations (21, 30). The OATP1B3/OATP-8 reference sequence and the S112A and M233I sequences localize correctly to the basolateral membrane of hepatocytes and exhibit similar transport properties (21). In contrast, OTP1B3/OATP-8-G522C is retained inside cells and is thus unable to mediate cellular transport activity (21). In recent years, numerous studies (4, 10, 13, 24, 26, 30, 32) have focused on the substrates transported by human OATP1B1/OATP-C and OATP1B3/OATP-8, their roles in liver disease, and their regulation of drug disposition. On the other hand, the energy coupling transport mechanisms of human liver OATPs remain poorly understood. OATP/Oatpmediated transport is independent of sodium, chloride, and potassium gradients, membrane potential, and ATP levels and is generally believed to occur by electroneutral exchange, in which the cellular uptake of organic anions is coupled to the efflux of anions such as HCO 3, GSH, and/or glutathione-sconjugates (12, 14, 19, 22, 23, 27 29, 33). However, a coupled exchange mechanism has been demonstrated for only one member of this large protein family, namely, rat Oatp1a1 (22, 28). Studies with other members of this family have been Address for reprint requests and other correspondence: N. Ballatori, Dept. of Environmental Medicine, Box EHSC, Univ. of Rochester School of Medicine, 575 Elmwood Ave., Rochester, NY ( Ned_Ballatori@urmc. rochester.edu). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact /07 $8.00 Copyright 2007 the American Physiological Society G271
2 G272 unable to identify the driving force and have also not implicated a role for GSH or HCO 3 in the transport cycle. In contrast with the electroneutral transport observed in these previous studies, a recent study (3) with OATP1B3/OATP-8 suggests that this protein functions as a GSH-bile acid cotransporter and thus would presumably result in the net movement of charge across the plasma membrane, although this was not directly examined. Based on a comparative analysis of OATPs from multiple species, Meier-Abt et al. (25) proposed a common transport mechanism for all OATPs/Oatps, whereby substrates are translocated through a central, positively charged pore in a rockerswitch type of mechanism. However, this analysis did not establish whether transport is coupled (i.e., is driven by exchange or cotransport with another molecule) or simply occurs by facilitated diffusion through this putative central pore. The present study aimed to investigate the transport mechanism mediated by OATP1B1/OATP-C and OATP1B3/OATP-8 and to examine whether GSH is required for their energy coupling mechanism. Because OATP1B3/OATP-8 has some common polymorphisms, three of these were also examined (namely, S112A, M233I, and both S112A and M233I) to compare their transport functions. The latter amino acid sequence is the most prevalent among Europeans and Americans (56% allelic frequency) (21), and most of the present experiments were carried out with this sequence (i.e., OATP1B3/OATP-8- S112A&M233I). MATERIALS AND METHODS Materials. [ 3 H]estrone sulfate (46 Ci/mmol), [ 3 H]taurocholate (1.19 Ci/mmol), and [ 3 H]GSH (41.5 Ci/mmol) were purchased from NEN Life Science Products (Boston, MA). Other chemical and reagents were obtained from Sigma-Aldrich Chemical (St Louis, MO) or from J. T. Baker (Philipsburg, NJ). Mature Xenopus laevis were purchased from Nasco (Fort Atkinson, WI). Animals were maintained under constant light. SLCO1B1 and SLCO1B3-S112A&M233I cdnas were kindly provided by Drs. Bruno Stieger and Bruno Hagenbuch (Zurich, Switzerland, and Kansas City, MO, respectively). Capped crna was transcribed in vitro with T7 RNA polymerase (Ambion, Austin, TX), and crna was precipitated with lithium chloride and resuspended in RNase-free water for oocyte injection. Generation of OATP crna was verified by gel electrophoresis. Site-directed mutagenesis of SLCO1B3. Mutations were introduced into the cdna of SLCO1B3-S112A&M233I, which contains both 334T G and 699G A in psport, using the QuickChange II Site- Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Primers were purchased from Operon Biotechnologies (Huntsville, AL). For mutation of SLCO1B3 334G T, the forward primer was 5 -GGG AAC TGG AAG TAT TTT GAC ATC TTT ACC ACA TTT CTT CAT GGG-3 and the reverse primer was 5 -CCC ATG AAG AAA TGT GGT AAA GAT GTC AAA ATA CTT CCA GTT CCC-3. For the SLCO1B3 699A G mutation, the forward primer was 5 -GGG ATC TCT GTT TGC TAA AAT GTA CGT GGA TAT TGG ATA TGT AG-3 and the reverse primer was 5 -CTA CAT ATC CAA TAT CCA CGT ACA TTT TAG CAA ACA GAG ATC CC-3. DNA sequencing confirmed the intended mutations (underlined). cdna sequence analysis. cdnas were sequenced at the DNA Sequencing Facility of the Mount Desert Island Biological Laboratory (Salisbury Cove, ME). cdna sequences were aligned against the reference sequences (GenBank/EMBL Accession Nos. NM_ and NM_ for SLCO1B1 and SLCO1B3 genes, respectively). Xenopus laevis oocyte preparation and injection. Isolation of Xenopus laevis oocytes was performed as described by Goldin (5) and previously employed in our laboratory (2, 22). Frogs were anesthetized by immersion for min in ice-cold 0.3% tricane. Oocytes were removed from the ovary and washed with Ca 2 -free OR-2 solution (82.5 mm NaCl, 2 mm KCl, 1 mm MgCl 2,and5mM HEPES-Tris; ph 7.5) and incubated at room temperature with gentle shaking for 90 min in OR-2 solution containing 2 mg/ml collagenase (type IA, Sigma). Oocytes were transferred to fresh collagenase solution after the first 45 min. Collagenase was removed by a wash with OR-2 solution at room temperature. Defolliculated stage V and VI oocytes were selected and incubated at 18 C in modified Barth s solution (ph ) [88 mm NaCl, 1 mm KCl, 2.4 mm NaHCO 3, 0.82 mm MgSO 4, 0.33 mm Ca(NO 3) 2, 0.41 mm CaCl 2, and 20 mm HEPES-Tris (ph 7.5)] or U-medium (100 mm choline chloride, 2 mm KCl, 1 mm CaCl 2, 1 mm MgCl 2, 10 mm HEPES; ph was adjusted to 7.0 with Tris) supplemented with gentamycin (0.5 mg/ml). U-medium was used in some experiments, reproducing the conditions used by Briz et al. (3). After 2 h of incubation, oocytes were injected with 50 nl of OATP1B1/OATP-C crna, OATP1B3/OATP-8 crna (5 ng/ oocyte), or sterile water. Injected oocytes were cultured at 18 C with a daily change of modified Barth s media or U-medium containing gentamycin. Oocytes with a homogeneous brown animal half and distinct equator line were selected for transport experiments after 3 days. Cis-inhibition of [ 3 H]estrone sulfate and [ 3 H]taurocholate uptake into oocytes. Control or crna-injected oocytes were incubated at 25 C in 100 l of modified Barth s solution or U-medium in the presence of [ 3 H]estrone sulfate or [ 3 H]taurocholate with or without other substrates. The ph of GSH solutions was adjusted to 7.4 using dilute NaOH. For some experiments, the ph of the modified Barth s solution was adjusted to 6.5 using dilute HCl and to 8.5 using dilute NaOH. Uptake was stopped by rapid dilution with 2.5 ml of ice-cold modified Barth s solution or U-medium, and oocytes were washed three times with these same solutions. Two oocytes were placed in a polypropylene scintillation vial and dissolved in 200 l of 10% SDS by vortexing. Five milliliters of Opti-Fluor (Packard Instruments, Downers Grove, IL) scintillation cocktail were added. Samples were left for at least 3 h before being counted in a Beckman 6500 scintillation counter. Trans-stimulation of [ 3 H]taurocholate and [ 3 H]estrone sulfate uptake into oocytes. Control and crna-injected oocytes were reinjected with 50 nl of each of the following solutions: 550 M estrone sulfate, 550 M taurocholate, 220 mm GSH, 275 mm NaHCO 3,55 mm Na 2SO 4,or550 M 17 -estradiol-d-17 -glucuronide (E 217 G). Oocytes were washed three times at room temperature and then incubated for min in ice-cold modified Barth s solution or U-medium. [ 3 H]estrone sulfate or [ 3 H]taurocholate uptake was measured at 25 C for 30 min or 1 h, respectively. Efflux of [ 3 H]GSH and [ 3 H]taurocholate from oocytes. Fifty nanoliters of either 20 mm [ 3 H]GSH, 300 M [ 3 H]taurocholate, or 300 M [ 3 H]taurocholate with 20 mm unlabled GSH were injected into control and crna-injected oocytes, which were washed three times at room temperature and then allowed to recover for min in ice-cold U-medium solution. The ph of the injected solutions was adjusted to 7.4 using dilute NaOH. Efflux was measured in individual oocytes for 1hat25 C in 200 l of U-medium. Acivicin (0.5 mm) was used in experiments that measured [ 3 H]GSH efflux (2, 22, 23). The medium was removed to terminate efflux and counted separately from the oocyte. Trans-stimulation of [ 3 H]estrone sulfate efflux from oocytes. Control and crna-expressing oocytes were preloaded by a 1-h incubation at 25 C with 50 nm [ 3 H]estrone. Oocytes were washed, and efflux was measured at 25 C for 30 min in the presence and absence of either 0.1 mm estrone sulfate, 0.1 mm taurocholate, 0.1 M leukotriene C 4, or 10 mm GSH in 200 l of modified Barth s solution. Amounts of [ 3 H]estrone sulfate in the oocytes and the efflux buffer were measured.
3 G273 Membrane potential measurements. Single oocytes were placed in a small (0.2 ml) Plexiglas chamber and superfused with modified Barth s solution or a high-k modified Barth s solution in which the NaCl was replaced with KCl (88 mm). Membrane potential determinations were made as previously described using a custom-made voltage-clamp amplifier (31). Both reference electrodes and intracellular recording electrodes were filled with 3 M KCl, and oocytes were impaled at the vegetal pole. All measurements were made at room temperature (22 24 C). Statistical analysis. Statistical significance was determined using an unpaired t-test for two means where P 0.05 was taken as the limit denoting significant differences. Values are means SE. RESULTS OATP1B3/OATP-8 and three of its polymorphisms do not mediate GSH-bile acid cotransport. To replicate the finding that OATP1B3/OATP-8 can mediate cotransport of bile acids and GSH (3), the effects of GSH were examined under multiple experimental conditions using four different OATP1B3/ OATP-8 sequences: the reference sequence (Refseq), S112A (SLCO1B3-334T G), M223I (SLCO1B3-699G A), and a construct containing both S112A and M233I (S112A&M233I). As noted above, the sequence containing both S112A and M233I is the most common sequence found in European and American Caucasians. When expressed in Xenopus laevis oocytes, each of these four constructs was able to mediate taurocholate transport activity and to roughly similar levels (Fig. 1). To examine the inhibitor sensitivity of the four constructs, cis-inhibition of taurocholate uptake was measured (Fig. 2). Surprisingly, GSH and GSSG did not cis-stimulate taurocholate uptake by OATP1B3/OATP-8, as expected based on the results of Briz and coworkers (3). As expected, E 2 17 G, taurocholate, and estrone sulfate cis-inhibited the uptake of taurocholate by all four sequences, whereas HCO 3 had no significant effect (Fig. 2). Fig. 2. Cis-inhibition of TC uptake by OATP1B3/OATP-8 and its polymorphisms. A D: cis-inhibition of 10 M [ 3 H]TC uptake by OATP1B3/OATP- 8-refseq (A), S112A (B), and M233I (C), and S112A&M233I (D). Uptake of 10 M [ 3 H]TC by control or OATP1B3/OATP-8-expressing oocytes was measured at 25 C for 1 h in U-medium with or without the substrates shown. E 217 G, 17 -estradiol-d-17 -glcuronide; ES, estrone sulfate. Values are means SE. *P 0.05 compared with control. Fig. 1. Taurocholate (TC) uptake by organic anion transporter (OATP)1B3/ OATP-8 and its polymorphic forms [S112A, M233I, and both (S112A&M233I)]. Control or OATP1B3/OATP-8-expressing oocytes were incubated at 25 C for 1 h in U-medium containing 10 M [ 3 H]TC. Values are means SE and are shown as the uptake ratio relative to control. To further examine whether OATP1B3/OATP-8 functions as a GSH-bile acid cotransporter, trans-stimulation of taurocholate uptake was investigated in oocytes expressing OATP1B3/OATP-8 and two of its polymorphic forms. In agreement with the cis-inhibition results (Fig. 2), E 2 17 G,
4 G274 taurocholate, and estrone sulfate trans-stimulated taurocholate uptake with these constructs, albeit at slightly different levels (Fig. 3). However, taurocholate uptake was not affected by GSH or HCO 3 (Fig. 3), suggesting that GSH is neither cotransported nor countertransported with taurocholate. In the experiments described above, all solutions containing GSH were supplemented with either NaOH or KOH to adjust the ph to the physiological range (i.e., to raise the ph from 3.7 to 7.4). Interestingly, when the ph was not adjusted, taurocholate uptake was cis-stimulated (Fig. 4). Similar results were noted for GSSG (Fig. 4). However, because these low ph values are well beyond the physiological range, and are possibly toxic to the oocytes, the observed transport stimulation may be of no significance with regard to transport mechanism. Fig. 4. Effect of adjusted and nonadjusted ph of GSH and GSSG solutions on TC uptake by OATP1B3/OATP-8-S112A&M233I. Cis-inhibition of 10 M [ 3 H]TC uptake by OATP1B3/OATP-8 was measured in U-medium for 1hat 25 C. Values are means SE; n 3 5 experiments in different oocyte preparations. *P 0.05 compared with control. Fig. 3. Trans-stimulation of TC uptake by OATP1B3/OATP-8 and its polymorphic forms. A C: ocytes injected with water (control) or crna of OATP1B3/OATP-8-refseq (A), S112A (B), or M233I (C) were microinjected with the substrates shown. [ 3 H]TC uptake (10 M) was measured for 1hin U-medium. Values are means SE; n 3 6 experiments in different oocyte preparations. *P 0.05 compared with control. OATP1B3/OATP-8-S112A&M233I-mediated uptake of estrone sulfate is not cis-inhibited or trans-stimulated by GSH. Cis-inhibition and trans-stimulation of another substrate, estrone sulfate, was examined to further test the hypothesis that GSH is not required for OATP1B3/OATP-8-S112A&M233I function. As expected, [ 3 H]estrone sulfate uptake by OATP1B3/ OATP-8-S112A&M233I was cis-inhibited by taurocholate, estrone sulfate, dehydroepiandrosterone sulfate (DHEAS), E 2 17 G, sulfobromophthalein sodium (BSP), and probenecid but not by GSH, HCO 3, SO 4 2, glutamate, glutarate, lactate, and citrate (Fig. 5A). [ 3 H]estrone sulfate uptake was trans-stimulated by intracellular estrone sulfate and taurocholate but not by GSH, HCO 3, and SO 4 2 (Fig. 5B). These results are in agreement with the findings presented in Figs. 1 3 and also suggest that glutamate, glutarate, lactate, and citrate are unlikely to be substrates of this transporter. OATP1B3/OATP8-S112A&M233I does not mediate [ 3 H]GSH export, and intracellular GSH does not stimulate [ 3 H]taurocholate efflux. To investigate whether GSH is a direct substrate for OATP1B3/OATP8-S112A&M233I, [ 3 H]GSH efflux and GSH cis-stimulation of [ 3 H]taurocholate efflux was measured. As shown in Fig. 6A, GSH efflux was not enhanced in OATP1B3/OATP-8-expressing oocytes ( 2% release/h), whereas Briz et al. (3) demonstrated an extensive efflux of GSH in both control and OATP1B3/OATP-8-expressing oocytes ( 30% and 60% release/h, respectively). Oatp1a1/Oatp1 was used as a positive control for GSH release, and this transporter did increase GSH efflux (Fig. 6A), as previously reported (22). As shown in Fig. 6B, high intracellular GSH did not promote [ 3 H]taurocholate efflux in OATP1B3/OATP8-S112A&M233Iinjected oocytes compared with control, supporting the present conclusion that OATP1B3/OATP-8-S112A&M233I does not function as a GSH-bile acid cotransporter.
5 G275 or 8.5 (Fig. 8). However, estrone sulfate uptake was unaffected either at ph 6.5 or 8.5, indicating that H and OH are probably not involved. The effect of membrane potential on the transport mechanism of OATP1B1/OATP-C and OATP1B3/ OATP-8-S112A&M233I was investigated by isoosmotic substitution of NaCl for KCl in modified Barth s solution, resulting in a high K and a depolarization of the membrane potential. Oocyte resting membrane potentials were approximately 40 mv (Fig. 9A). As expected, a marked depolarization was observed upon K substitution, and this depolarization was reversed upon return to buffer with Na (Fig. 9A). If transport is mediated by a uniport mechanism or by a cotransport with another anion, it would be electrogenic and thus should be sensitive to membrane potential alteration. However, membrane potential depolarization induced by increased K had no effect on estrone sulfate uptake by OATP1B1/OATP-C or OATP1B3/OATP-8 (Fig. 9, B and C). Therefore, the transport on both OATPs is independent of membrane potential and Fig. 5. Cis-inhibition and trans-stimulation of ES uptake by OATP1B3/ OATP-8-S112A&M233I. A: control and crna-expressing oocytes were incubated at 25 C in modified Barth s solution containing 10 M [ 3 H]ES with or without the indicated substrates, and uptake was measured for 30 min at 25 C. B: oocytes were microinjected with the indicated substrates. The uptake of 10 M [ 3 H]ES was measured for 30 min at 25 C. DHEAS, dehydroepiandrosterone sulfate; BSP, sulfobromophthalein sodium. Values are means of 6 8 oocytes, represented as a percentage of control; uptake measured in the absence of substrate. Values are means SE; n 5 8 experiments in different oocyte preparations. *P 0.05 compared with control. OATP1B1/OATP-C transport of taurocholate and estrone sulfate is also not cis-inhibited or trans-stimulated by GSH. Similar to OATP1B3/OATP-8, taurocholate and estrone sulfate uptake by OATP1B1/OATP-C was cis-inhibited by taurocholate, DHEAS, E 2 17 G, BSP, and probenecid but not by GSH, glutamate, glutarate, lactate, and citrate (Fig. 7, A and B). HCO 3 and SO 4 2 slightly inhibited taurocholate uptake (Fig. 7A) but had no effect on estrone sulfate uptake (Fig. 7B). [ 3 H]estrone sulfate efflux was trans-stimulated by unlabeled estrone sulfate and taurocholate but not by GSH (Fig. 7C). Therefore, GSH is also not required for OATP1B1/OATP-C transport activity. ph and membrane potential insensitivity of OATP1B1/ OATP-C and OATP1B3/OATP-8-S112A&M233I. To examine if the energy coupling mechanism involves the ph gradient, estrone sulfate uptake was measured in media of ph 6.5, 7.5, Fig. 6. OATP1B3/OATP-8-S112A&M233I-mediated efflux of GSH and TC. Control and OATP1B3/OATP-8-S112A&M233I-injected oocytes were microinjected with 20 mm [ 3 H]GSH (A) or with 300 M[ 3 H]TC without or with 20 mm GSH (adjusted to neutral ph using dilute NaOH) (B). Individual oocytes were incubated in U-medium at 25 C for 1 h. The medium contained 0.5 mm acivicin for [ 3 H]GSH measurements. Values are means SE; n 3 5 experiments in different oocyte preparations. *P 0.05.
6 G276 proteins remains largely unknown. Although OATPs are generally regarded as electroneutral anion exchangers, direct evidence for this contention has only been provided for one member of this large family of proteins, rat Oatp1a1. However, even for this one protein, there is uncertainty as to whether it functions as a GSH exchanger or as a HCO 3 exchanger (22, 28). The present data provide support for the hypothesis that human OATP1B1 and OATP1B3 function as bidirectional facilitated diffusion transporters rather than exchangers or cotransporters. That is, transport on OATP1B1 and OATP1B3 appears to be energized by the electrochemical substrate gradient and is not directly coupled to the simultaneous movement of another substrate across the membrane. The evidence for this is as follows: 1) none of a variety of plausible cotrasported or countertransported ions tested had any significant effect on the rate of transport; and 2) a modest trans-stimulation of transport was observed only when known organic solute substrates were placed on opposite sides of the plasma membrane. This modest trans-stimulation is consistent with accelerated exchange diffusion (i.e., more rapid movement of the loaded carrier) rather than coupled transport; and 3) OATP-mediated export from the oocytes was observed despite the fact that the cell culture medium consisted of a simple salt solution and thus was presumably devoid of countertransportable organic ions. Nevertheless, these data cannot conclusively rule out the possibility that an as-yet-unidentified ion may have been present and may have energized the transport event. Thus, additional studies are needed to resolve this critical question. The present results also demonstrate that GSH has no effect on substrate uptake or efflux on OATP1B1/OATP-C and OATP1B3/OATP-8, indicating that GSH is not required for transport activity. These findings contrast with those of Briz et al. (3), who reported that OATP1B3/OATP-8 mediates the cotransport of bile acids with either GSH or GSSG. In an attempt to resolve this discrepancy, the present study characterized the transport properties of both the reference sequence for OATP1B3/OATP-8 and three of its common polymorphisms, since SNPs may alter their transport acivity (22). However, the four OATP1B3/OATP-8 sequences examined in this study exhibited comparable transport properties, and none were dependent on GSH for their activity. In addition, exper- Fig. 7. Cis-inhibition of ES and TC uptake and trans-stimulation of ES efflux by OATP1B1/OATP-C. Control and crna-expressing oocytes were incubated at 25 C for 30 min in modified Barth s solution containing 10 M [ 3 H]TC (A) or 50 nm [ 3 H]ES (B) with or without the indicated substrates. For the trans-stimulation experiment, oocytes were remicroinjected with the substrates shown (C). LTC 4, leukotriene C 4. Values are means SE; n 5 8 experiments in different oocyte preparations. *P 0.05 compared with control. the Na gradient and appears to be electroneutral. Because estrone sulfate is a monovalent anion, the voltage insensitivity of its uptake suggests that it associated with either the outward movement of a monovalent anion or the uptake of a monovalent cation; however, the identity of these ions remains unknown. DISCUSSION Despite many years of work by several laboratories, the energetic coupling mechanism for the large family of OATP Fig. 8. Effect of ph on ES uptake by OATP1B1/OATP-C and OATP1B3/ OATP-8-S112A&M233I. Oocytes were incubated at 25 C for 30 min in modified Barth s solution containing 50 nm and 10 M[ 3 H]ES for OATP1B1/ OATP-C and OATP1B3/OATP-8, respectively. Uptake in control oocytes was also unaffected by these ph changes (data not shown). Values are means SE; n 5 experiments in different oocyte preparations.
7 Fig. 9. Membrane potential insensitivity of OATP1B1/OATP-C and OATP1B3/OATP-8-S112A&M233I-mediated transport. A: membrane potential was measured in oocytes cultured in media in which KCl (K) was substituted for 88 mm NaCl in modified Barth s solution (MB). Measurements were made in sets of 6 oocytes with bath changes from MB to high K and from high K to MB. Control and crna-injected oocytes were incubated at 25 C in MB containing 50 nm and 10 M [ 3 H]ES for OATP1B1/OATP-C (B) and OATP1B3/OATP-8 (C), respectively. Values are means SE; n 3 5 experiments in different oocyte preparations. *P 0.01 compared with the MB control of each group. iments were repeated using different culture media, substrates, and ph. None of these maneuvers elicited sensitivity to GSH except when the ph of the GSH and GSSG incubation solutions was not adjusted. The ph of nonadjusted 20 mm GSH and GSSG in U-medium are 3.7 and 3.5, respectively, which are below the working range of the HEPES-Tris buffer included in this medium. At these low ph values, taurocholate uptake was significantly increased to 160% of control, but this is likely an artifact rather than evidence for H -taurocholate cotransport or OH -taurocholate exchange. Briz and cowork- ers (3) also reported that OATP1B3/OATP-8 mediates the efflux of GSH itself; however, their reported rates of GSH release appear to be nonphysiological. Control oocytes were found to release intracellular [ 3 H]GSH at a surprisingly high rate ( 30%/h), and this was further enhanced in oocytes expressing OATP1B3/OATP-8 ( 60%/h). In contrast, our present (Fig. 6) and previous studies (2, 22) indicated that oocytes release [ 3 H]GSH at rate of 2 3%/h, which is more consistent with cellular GSH turnover rates (2). As shown in Fig. 6A, GSH release was not enhanced in OATP1B3/ OATP-8-expressing oocytes, and it was not affected by loading the oocytes with taurocholate (Fig. 6B). It is important to note that, in contrast with GSH, oocytes have an active endogenous taurocholate export mechanism (31) and that the baseline rate of taurocholate export is relatively high (31) (Fig. 6B). This high background rate of taurocholate release by oocytes probably masks any small release that may be mediated by OATP1B3/OATP-8 under these conditions. Except for known substrates, none of the anions tested altered transport activity on either OATP1B1/OATP-C or OATP1BC/OATP-8. Although HCO 3 and SO 2 4 showed significant cis-inhition of taurocholate uptake by OATP1B1/ OATP-C, the magnitude was small compared with that produced by taurocholate, estrone sulfate, BSP, or probenecid (Fig. 7A). Estrone sulfate uptake by OATP1B1/OATP-C was also not cis-inhibited by HCO 3 and SO 2 4 (Fig. 7B). Transport of taurocholate and estrone sulfate occurred in both the inward and outward direction, indicating that transport is bidirectional. Furthermore, we found that estrone sulfate uptake was not significantly altered when the ph was changed from 7.5 to 6.5 or 8.5 (Fig. 8). The uptake of estrone sulfate by OATP1B1/OATP-C and OATP1B3/OATP-8 was also not significantly change after substitution of NaCl for KCl in modified Barth s solution (Fig. 9, B and C). This Na -independent transport is in agreement with previous studies (1, 7, 17) indicating that OATP proteins do not require Na for their transport function. Because membrane potential was markedly depolarized when Na was replaced with K (Fig. 9A), these data also argue against an electrogenic GSH-bile acid cotransport mode of transport of OATP1B3/OATP-8. Given that both GSH and taurocholate are anions, cells would lose (or gain) two negative charges with each transport cycle, and thus this process should be sensitive to membrane potential. Although the present and previous results indicate that transport is electroneutral, the nature of the neutralizing ion is unknown. G277 ACKNOWLEDGMENTS We thank Albert Koh and Whitney Christian for expert technical assistance and Dr. Ted Begenisich (Department of Pharmacology and Physiology, University of Rochester) for assistance in making electrophysiology measurements. GRANTS This work was supported in part by National Institute of Health Grants DK-48823, DK , ES-01247, and ES Additional financial support for C. Mahagita and P. Piyachaturawat was provided by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant PHD/0266/2545).
8 G278 REFERENCES 1. Abe T, Kakyo M, Tokui T, Nakagomi R, Nishio T, Nakai D, Nomura H, Unno M, Suzuki M, Naitoh T, Matsuno S, Yawo H. Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1. J Biol Chem 274: , Ballatori N, Wang W, Li L, Truong AT. An endogenous ATP-sensitive glutathione S-conjugate efflux mechanism in Xenopus laevis oocytes. Am J Physiol Regul Integr Comp Physiol 270: R1156 R1162, Briz O, Romero MR, Martinez-Becerra P, Macias RI, Perez MJ, Jimenez F, San Martin FG, Marin JJ. OATP8/1B3-mediated cotransport of bile acids and glutathione: an export pathway for organic anions from hepatocytes? J Biol Chem 281: , Cvetkovic M, Leake B, Fromm MF, Wilkinson GR, Kim RB. OATP and P-glycoprotein transporters mediate the cellular uptake and excretion of fexofenadine. Drug Metab Dispos 27: , Goldin AL. Maintenance of Xenopus laevis and oocyte injection. Methods Enzymol 207: , Grundemann D, Gorboulev V, Gambaryan S, Veyhl M, Koepsell H. Drug excretion mediated by a new prototype of polyspecific transporter. Nature 372: , Hagenbuch B, Meier PJ. Organic anion transporting polypeptides of the OATP/ SLC21 family: phylogenetic classification as OATP/SLCO superfamily, new nomenclature and molecular/functional properties. Pflugers Arch 447: , Hagenbuch B, Meier PJ. The superfamily of organic anion transporting polypeptides. Biochim Biophys Acta 1609: 1 18, Hagenbuch B, Stieger B, Foguet M, Lubbert H, Meier PJ. Functional expression cloning and characterization of the hepatocyte Na /bile acid cotransport system. Proc Natl Acad Sci USA 88: , Ho RH, Tirona RG, Leake BF, Glaeser H, Lee W, Lemke CJ, Wang Y, Kim RB. Drug and bile acid transporters in rosuvastatin hepatic uptake: function, expression, and pharmacogenetics. Gastroenterology 130: , Hsiang B, Zhu Y, Wang Z, Wu Y, Sasseville V, Yang WP, Kirchgessner TG. A novel human hepatic organic anion transporting polypeptide (OATP2). Identification of a liver-specific human organic anion transporting polypeptide and identification of rat and human hydroxymethylglutaryl-coa reductase inhibitor transporters. J Biol Chem 274: , Jacquemin E, Hagenbuch B, Stieger B, Wolkoff AW, Meier PJ. Expression cloning of a rat liver Na -independent organic anion transporter. Proc Natl Acad Sci USA 91: , Kamath AV, Yao M, Zhang Y, Chong S. Effect of fruit juices on the oral bioavailability of fexofenadine in rats. J Pharm Sci 94: , Kanai N, Lu R, Satriano JA, Bao Y, Wolkoff AW, Schuster VL. Identification and characterization of a prostaglandin transporter. Science 268: , Koepsell H, Endou H. The SLC22 drug transporter family. Pflugers Arch 447: , Konig J, Cui Y, Nies AT, Keppler D. Localization and genomic organization of a new hepatocellular organic anion transporting polypeptide. J Biol Chem 275: , Konig J, Cui Y, Nies AT, Keppler D. A novel human organic anion transporting polypeptide localized to the basolateral hepatocyte membrane. Am J Physiol Gastrointest Liver Physiol 278: G156 G164, Konig J, Seithel A, Gradhand U, Fromm MF. Pharmacogenomics of human OATP transporters. Naunyn Schmiedebergs Arch Pharmacol 372: , Kullak-Ublick GA, Hagenbuch B, Stieger B, Schteingart CD, Hofmann AF, Wolkoff AW, Meier PJ. Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver. Gastroenterology 109: , Kullak-Ublick GA, Hagenbuch B, Stieger B, Wolkoff AW, Meier PJ. Functional characterization of the basolateral rat liver organic anion transporting polypeptide. Hepatology 20: , Letschert K, Keppler D, Konig J. Mutations in the SLCO1B3 gene affecting the substrate specificity of the hepatocellular uptake transporter OATP1B3 (OATP8). Pharmacogenetics 14: , Li L, Lee TK, Meier PJ, Ballatori N. Identification of glutathione as a driving force and leukotriene C4 as a substrate for oatp1, the hepatic sinusoidal organic solute transporter. J Biol Chem 273: , Li L, Meier PJ, Ballatori N. Oatp2 mediates bidirectional organic solute transport: a role for intracellular glutathione. Mol Pharmacol 58: , Maeda K, Ieiri I, Yasuda K, Fujino A, Fujiwara H, Otsubo K, Hirano M, Watanabe T, Kitamura Y, Kusuhara H, Sugiyama Y. Effects of organic anion transporting polypeptide 1B1 haplotype on pharmacokinetics of pravastatin, valsartan, and temocapril. Clin Pharmacol Ther 79: , Meier-Abt F, Mokrab Y, Mizuguchi K. Organic anion transporting polypeptides of the OATP/SLCO superfamily: identification of new members in nonmammalian species, comparative modeling and a potential transport mode. J Membr Biol 208: , Nakai D, Nakagomi R, Furuta Y, Tokui T, Abe T, Ikeda T, Nishimura K. Human liver-specific organic anion transporter, LST-1, mediates uptake of pravastatin by human hepatocytes. J Pharmacol Exp Ther 297: , Noe B, Hagenbuch B, Stieger B, Meier PJ. Isolation of a multispecific organic anion and cardiac glycoside transporter from rat brain. Proc Natl Acad Sci USA 94: , Satlin LM, Amin V, Wolkoff AW. Organic anion transporting polypeptide mediates organic anion/hco 3 exchange. J Biol Chem 272: , Shi X, Bai S, Ford AC, Burk RD, Jacquemin E, Hagenbuch B, Meier PJ, Wolkoff AW. Stable inducible expression of a functional rat liver organic anion transport protein in HeLa cells. J Biol Chem 270: , Smith NF, Marsh S, Scott-Horton TJ, Hamada A, Mielke S, Mross K, Figg WD, Verweij J, McLeod HL, Sparreboom A. Variants in the SLCO1B3 gene: interethnic distribution and association with paclitaxel pharmacokinetics. Clin Pharmacol Ther 81: 76 82, Stampe P, Begenisich T. Unidirectional K fluxes through recombinant Shaker potassium channels expressed in single Xenopus oocytes. J Gen Physiol 107: , Vavricka SR, Van Montfoort J, Ha HR, Meier PJ, Fattinger K. Interactions of rifamycin SV and rifampicin with organic anion uptake systems of human liver. Hepatology 36: , Walters HC, Craddock AL, Fusegawa H, Willingham MC, Dawson PA. Expression, transport properties, and chromosomal location of organic anion transporter subtype 3. Am J Physiol Gastrointest Liver Physiol 279: G1188 G1200, 2000.
SUPPORTING INFORMATION FOR. SEquence-Enabled Reassembly of β-lactamase (SEER-LAC): a Sensitive Method for the Detection of Double-Stranded DNA
SUPPORTING INFORMATION FOR SEquence-Enabled Reassembly of β-lactamase (SEER-LAC): a Sensitive Method for the Detection of Double-Stranded DNA Aik T. Ooi, Cliff I. Stains, Indraneel Ghosh *, David J. Segal
More informationSUPPLEMENTARY DATA - 1 -
- 1 - SUPPLEMENTARY DATA Construction of B. subtilis rnpb complementation plasmids For complementation, the B. subtilis rnpb wild-type gene (rnpbwt) under control of its native rnpb promoter and terminator
More informationPractical Bioinformatics
5/2/2017 Dictionaries d i c t i o n a r y = { A : T, T : A, G : C, C : G } d i c t i o n a r y [ G ] d i c t i o n a r y [ N ] = N d i c t i o n a r y. h a s k e y ( C ) Dictionaries g e n e t i c C o
More informationComparison of Type I and Type II Organic Cation Transport by Organic Cation Transporters and Organic Anion- Transporting Polypeptides
0022-3565/01/2981-110 115$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 298, No. 1 Copyright 2001 by The American Society for Pharmacology and Experimental Therapeutics 3765/911050
More informationBuilding a Multifunctional Aptamer-Based DNA Nanoassembly for Targeted Cancer Therapy
Supporting Information Building a Multifunctional Aptamer-Based DNA Nanoassembly for Targeted Cancer Therapy Cuichen Wu,, Da Han,, Tao Chen,, Lu Peng, Guizhi Zhu,, Mingxu You,, Liping Qiu,, Kwame Sefah,
More informationNumber-controlled spatial arrangement of gold nanoparticles with
Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 2016 Number-controlled spatial arrangement of gold nanoparticles with DNA dendrimers Ping Chen,*
More informationAdvanced topics in bioinformatics
Feinberg Graduate School of the Weizmann Institute of Science Advanced topics in bioinformatics Shmuel Pietrokovski & Eitan Rubin Spring 2003 Course WWW site: http://bioinformatics.weizmann.ac.il/courses/atib
More informationFunctional and structural relevance of conserved positively charged. lysine residues in organic anion transporting polypeptide 1B3
Molecular Pharmacology This article has not Fast been Forward. copyedited and Published formatted. The on final June version 3, 2011 may differ as doi:10.1124/mol.111.071282 from this version. MOL #71282
More informationABC Transporter Vesicle Assay: Comparison of Cold (LC-MS/MS) and Hot (Radiolabeled) Methods. Na Li, PhD. BD Biosciences Discovery Labware May 29, 2012
ABC Transporter Vesicle Assay: Comparison of Cold (LC-MS/MS) and Hot (Radiolabeled) Methods Na Li, PhD BD Biosciences Discovery Labware May 29, 212 Today s Topics Overview of the important role of ABC
More informationSupporting Information
Supporting Information T. Pellegrino 1,2,3,#, R. A. Sperling 1,#, A. P. Alivisatos 2, W. J. Parak 1,2,* 1 Center for Nanoscience, Ludwig Maximilians Universität München, München, Germany 2 Department of
More informationNa Li, PhD. Application of a Fluorescent Substrate / Inside-Out Transporter Vesicle Assay for Identifying Inhibitors of MRP Transport
Application of a Fluorescent Substrate / Inside-Out Transporter Vesicle Assay for Identifying Inhibitors of MRP Transport Na Li, PhD BD Biosciences October 20, 2010 BD Gentest Transporter Seminar Series
More informationSupplemental Table 1. Primers used for cloning and PCR amplification in this study
Supplemental Table 1. Primers used for cloning and PCR amplification in this study Target Gene Primer sequence NATA1 (At2g393) forward GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT CAT GGC GCC TCC AAC CGC AGC
More informationSupporting Information for. Initial Biochemical and Functional Evaluation of Murine Calprotectin Reveals Ca(II)-
Supporting Information for Initial Biochemical and Functional Evaluation of Murine Calprotectin Reveals Ca(II)- Dependence and Its Ability to Chelate Multiple Nutrient Transition Metal Ions Rose C. Hadley,
More informationRenal handling of substances. Dr.Charushila Rukadikar Assistance Professor Physiology
Renal handling of substances Dr.Charushila Rukadikar Assistance Professor Physiology GENERAL PRINCIPLES OF RENAL TUBULAR TRANSPORT Transport mechanisms across cell membrane 1) Passive transport i. Diffusion
More informationSupplemental data. Pommerrenig et al. (2011). Plant Cell /tpc
Supplemental Figure 1. Prediction of phloem-specific MTK1 expression in Arabidopsis shoots and roots. The images and the corresponding numbers showing absolute (A) or relative expression levels (B) of
More informationTransport of glucose across epithelial cells: a. Gluc/Na cotransport; b. Gluc transporter Alberts
Figure 7 a. Secondary transporters make up the largest subfamily of transport proteins. TAGI 2000. Nature 408, 796 1. Na+- or H+-coupled cotransporters - Secondary active transport 2/7-02 Energy released
More informationChapter 3 Part 1! 10 th ed.: pp ! 11 th ed.: pp !! Cellular Transport Mechanisms! The Cell Cycle!
Chapter 3 Part 1! 10 th ed.: pp. 87 105! 11 th ed.: pp. 90 107!! Cellular Transport Mechanisms! The Cell Cycle! Transport Processes: Passive and Active (1 of 2)! 1. Passive transport! Does not use ATP!
More informationChapter 3 Part 1! 10 th ed.: pp ! 11 th ed.: pp !! Cellular Transport Mechanisms! The Cell Cycle!
Chapter 3 Part 1! 10 th ed.: pp. 87 105! 11 th ed.: pp. 90 107!! Cellular Transport Mechanisms! The Cell Cycle! Transport Processes: Passive and Active (1 of 2)! 1. Passive transport! Does not use ATP!
More informationBiol2174 Cell Physiology in Health & Disease
Biol2174 Cell Physiology in Health & Disease Lecture 4: Membrane Transport Proteins Kiaran Kirk Research School of Biology Learning objectives To understand: The need for membrane transport proteins in
More informationCh. 3: Cells & Their Environment
Ch. 3: Cells & Their Environment OBJECTIVES: 1. Understand cell membrane permeability 2. To recognize different types of cellular transport (passive vs active) 3. To understand membrane potential and action
More informationHigh throughput near infrared screening discovers DNA-templated silver clusters with peak fluorescence beyond 950 nm
Electronic Supplementary Material (ESI) for Nanoscale. This journal is The Royal Society of Chemistry 2018 High throughput near infrared screening discovers DNA-templated silver clusters with peak fluorescence
More informationMembrane Potentials, Action Potentials, and Synaptic Transmission. Membrane Potential
Cl Cl - - + K + K+ K + K Cl - 2/2/15 Membrane Potentials, Action Potentials, and Synaptic Transmission Core Curriculum II Spring 2015 Membrane Potential Example 1: K +, Cl - equally permeant no charge
More informationClay Carter. Department of Biology. QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture.
QuickTime and a TIFF (Uncompressed) decompressor are needed to see this picture. Clay Carter Department of Biology QuickTime and a TIFF (LZW) decompressor are needed to see this picture. Ornamental tobacco
More informationActive Efflux across the Blood-Brain Barrier: Role of the Solute Carrier Family
NeuroRx : The Journal of the American Society for Experimental NeuroTherapeutics Active Efflux across the Blood-Brain Barrier: Role of the Solute Carrier Family Hiroyuki Kusuhara and Yuichi Sugiyama Graduate
More informationName: TF: Section Time: LS1a ICE 5. Practice ICE Version B
Name: TF: Section Time: LS1a ICE 5 Practice ICE Version B 1. (8 points) In addition to ion channels, certain small molecules can modulate membrane potential. a. (4 points) DNP ( 2,4-dinitrophenol ), as
More informationSEQUENCE ALIGNMENT BACKGROUND: BIOINFORMATICS. Prokaryotes and Eukaryotes. DNA and RNA
SEQUENCE ALIGNMENT BACKGROUND: BIOINFORMATICS 1 Prokaryotes and Eukaryotes 2 DNA and RNA 3 4 Double helix structure Codons Codons are triplets of bases from the RNA sequence. Each triplet defines an amino-acid.
More informationCrick s early Hypothesis Revisited
Crick s early Hypothesis Revisited Or The Existence of a Universal Coding Frame Ryan Rossi, Jean-Louis Lassez and Axel Bernal UPenn Center for Bioinformatics BIOINFORMATICS The application of computer
More informationSSR ( ) Vol. 48 No ( Microsatellite marker) ( Simple sequence repeat,ssr),
48 3 () Vol. 48 No. 3 2009 5 Journal of Xiamen University (Nat ural Science) May 2009 SSR,,,, 3 (, 361005) : SSR. 21 516,410. 60 %96. 7 %. (),(Between2groups linkage method),.,, 11 (),. 12,. (, ), : 0.
More informationChapter 1 subtitles Ion gradients
CELLULAR NEUROPHYSIOLOGY CONSTANCE HAMMOND Chapter 1 subtitles Ion gradients Introduction In this first chapter, I'll explain the basic knowledge required to understand the electrical signals generated
More informationNature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1
Supplementary Figure 1 Zn 2+ -binding sites in USP18. (a) The two molecules of USP18 present in the asymmetric unit are shown. Chain A is shown in blue, chain B in green. Bound Zn 2+ ions are shown as
More informationInvolvement of Multiple Transporters in the Efflux of 3- Hydroxy-3-methylglutaryl-CoA Reductase Inhibitors across the Blood-Brain Barrier
0022-3565/04/3113-1147 1153$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 311, No. 3 Copyright 2004 by The American Society for Pharmacology and Experimental Therapeutics 71621/1179070
More informationThallium and Rubidium Permeability of Human and Rat Erythrocyte Membrane
Gen. Physiol. Biophys. (1990), 9, 39 44 39 Thallium and Rubidium Permeability of Human and Rat Erythrocyte Membrane I. A. SKULSKII, V. MANNINEN* and V. V. GLASUNOV Institute of Evolutionary Physiology
More informationSupplementary Information
Electronic Supplementary Material (ESI) for RSC Advances. This journal is The Royal Society of Chemistry 2014 Directed self-assembly of genomic sequences into monomeric and polymeric branched DNA structures
More information10/16/17 ACIDS AND BASES, DEFINED WATER IS AMPHOTERIC OUTLINE. 9.1 Properties of Acids and Bases. 9.2 ph. 9.3 Buffers
ACIDS AND BASES, DEFINED A hydrogen atom contains a proton and an electron, thus a hydrogen ion (H + ) is a proton: Acids: Proton (H + ) transfer between molecules is the basis of acid/base chemistry Ø
More informationCharacterization of Pathogenic Genes through Condensed Matrix Method, Case Study through Bacterial Zeta Toxin
International Journal of Genetic Engineering and Biotechnology. ISSN 0974-3073 Volume 2, Number 1 (2011), pp. 109-114 International Research Publication House http://www.irphouse.com Characterization of
More informationNSCI Basic Properties of Life and The Biochemistry of Life on Earth
NSCI 314 LIFE IN THE COSMOS 4 Basic Properties of Life and The Biochemistry of Life on Earth Dr. Karen Kolehmainen Department of Physics CSUSB http://physics.csusb.edu/~karen/ WHAT IS LIFE? HARD TO DEFINE,
More informationBiology 112 Practice Midterm Questions
Biology 112 Practice Midterm Questions 1. Identify which statement is true or false I. Bacterial cell walls prevent osmotic lysis II. All bacterial cell walls contain an LPS layer III. In a Gram stain,
More informationNuclear Medicine Department, Academic Medical Center, Amsterdam, The Netherlands;
[ 3 H]-Spiperone Competition Binding to Dopamine D2, D3 and D4 Receptors Jan-Peter van Wieringen 1 and Martin C. Michel 2* 1 Nuclear Medicine Department, Academic Medical Center, Amsterdam, The Netherlands;
More informationChapter 10. Thermodynamics of Transport. Thermodynamics of Transport, con t. BCH 4053 Summer 2001 Chapter 10 Lecture Notes. Slide 1.
BCH 4053 Summer 2001 Chapter 10 Lecture Notes 1 Chapter 10 Membrane Transport 2 3 Thermodynamics of Transport Free Energy change is given by difference in electrochemical potential and the quantity transported
More informationMEMBRANE STRUCTURE. Lecture 9. Biology Department Concordia University. Dr. S. Azam BIOL 266/
MEMBRANE STRUCTURE Lecture 9 BIOL 266/4 2014-15 Dr. S. Azam Biology Department Concordia University RED BLOOD CELL MEMBRANE PROTEINS The Dynamic Nature of the Plasma Membrane SEM of human erythrocytes
More informationFUNCTIONAL CHARACTERIZATION OF HUMAN MONOCARBOXYLATE TRANSPORTER 6 (SLC16A5)
0090-9556/05/3312-1845 1851$20.00 DRUG METABOLISM AND DISPOSITION Vol. 33, No. 12 Copyright 2005 by The American Society for Pharmacology and Experimental Therapeutics 5264/3066373 DMD 33:1845 1851, 2005
More informationIII. Voltage Independence of Basolateral Membrane Na - Efflux
Na' and K+ Transport at Basolateral Membranes of Epithelial Cells III. Voltage Independence of Basolateral Membrane Na - Efflux THOMAS C. COX and SANDY I. HELMAN From the Department of Physiology and Biophysics,
More informationcotransport expressed in Xenopus oocytes
JBC Papers in Press. Published on April 27, 2004 as Manuscript M402401200 Voltage-dependent H+ buffering Voltage dependence of H+ buffering mediated by sodium-bicarbonate cotransport expressed in Xenopus
More informationElectronic supplementary material
Applied Microbiology and Biotechnology Electronic supplementary material A family of AA9 lytic polysaccharide monooxygenases in Aspergillus nidulans is differentially regulated by multiple substrates and
More informationSupplementary Information for
Supplementary Information for Evolutionary conservation of codon optimality reveals hidden signatures of co-translational folding Sebastian Pechmann & Judith Frydman Department of Biology and BioX, Stanford
More informationCELL BIOLOGY - CLUTCH CH. 9 - TRANSPORT ACROSS MEMBRANES.
!! www.clutchprep.com K + K + K + K + CELL BIOLOGY - CLUTCH CONCEPT: PRINCIPLES OF TRANSMEMBRANE TRANSPORT Membranes and Gradients Cells must be able to communicate across their membrane barriers to materials
More informationIdentification of a Novel Voltage-driven Organic Anion Transporter Present at Apical Membrane of Renal Proximal Tubule*
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 278, No. 30, Issue of July 25, pp. 27930 27938, 2003 2003 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Identification
More informationProduction of Recombinant Annexin V from plasmid pet12a-papi
Tait Research Laboratory Page 1 of 5 Principle Production of Recombinant Annexin V from plasmid pet12a-papi Annexin V is expressed cytoplasmically in BL21(DE3) E. coli (Novagen) with the pet vector system
More informationMembranes 2: Transportation
Membranes 2: Transportation Steven E. Massey, Ph.D. Associate Professor Bioinformatics Department of Biology University of Puerto Rico Río Piedras Office & Lab: NCN#343B Tel: 787-764-0000 ext. 7798 E-mail:
More informationModule Membrane Biogenesis and Transport Lecture 15 Ion Channels Dale Sanders
Module 0220502 Membrane Biogenesis and Transport Lecture 15 Ion Channels Dale Sanders 9 March 2009 Aims: By the end of the lecture you should understand The principles behind the patch clamp technique;
More informationLecture 2. Excitability and ionic transport
Lecture 2 Excitability and ionic transport Selective membrane permeability: The lipid barrier of the cell membrane and cell membrane transport proteins Chemical compositions of extracellular and intracellular
More informationThe Role of G-Protein Coupled Estrogen Receptor (GPER) in Early Neurite Development. Kyle Pemberton
The Role of G-Protein Coupled Estrogen Receptor (GPER) in Early Neurite Development Kyle Pemberton Acknowledgement Dr. Xu Lab Members Brittany Mersman Nicki Patel Pallavi Mhaskar Jason Cocjin Committee
More informationRegulatory Sequence Analysis. Sequence models (Bernoulli and Markov models)
Regulatory Sequence Analysis Sequence models (Bernoulli and Markov models) 1 Why do we need random models? Any pattern discovery relies on an underlying model to estimate the random expectation. This model
More informationAD-" IONIC BASIS OF POTENTIAL REGULATION(U) BAYLOR COLLO / U U ijejmedicine L HOUSTON TX DEPT OF PHYSIOLOGY AND MOLECULAR 7 MEEE"..
AD-"19 459 IONIC BASIS OF POTENTIAL REGULATION(U) BAYLOR COLLO / U U ijejmedicine L HOUSTON TX DEPT OF PHYSIOLOGY AND MOLECULAR 7 MEEE"..,E NCLA SIFIE BIOPHYSIC S D C CHANG 6 i N 1988 Neg@14-85-K-6424
More informationMidterm Review Guide. Unit 1 : Biochemistry: 1. Give the ph values for an acid and a base. 2. What do buffers do? 3. Define monomer and polymer.
Midterm Review Guide Name: Unit 1 : Biochemistry: 1. Give the ph values for an acid and a base. 2. What do buffers do? 3. Define monomer and polymer. 4. Fill in the Organic Compounds chart : Elements Monomer
More informationTM1 TM2 TM3 TM4 TM5 TM6 TM bp
a 467 bp 1 482 2 93 3 321 4 7 281 6 21 7 66 8 176 19 12 13 212 113 16 8 b ATG TCA GGA CAT GTA ATG GAG GAA TGT GTA GTT CAC GGT ACG TTA GCG GCA GTA TTG CGT TTA ATG GGC GTA GTG M S G H V M E E C V V H G T
More informationMembrane Protein Channels
Membrane Protein Channels Potassium ions queuing up in the potassium channel Pumps: 1000 s -1 Channels: 1000000 s -1 Pumps & Channels The lipid bilayer of biological membranes is intrinsically impermeable
More informationMEMBRANE POTENTIALS AND ACTION POTENTIALS:
University of Jordan Faculty of Medicine Department of Physiology & Biochemistry Medical students, 2017/2018 +++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ Review: Membrane physiology
More informationb) What is the gradient at room temperature? Du = J/molK * 298 K * ln (1/1000) = kj/mol
Chem350 Practice Problems Membranes 1. a) What is the chemical potential generated by the movement of glucose by passive diffusion established by a 1000 fold concentration gradient at physiological temperature?
More informationMembrane transport 1. Summary
Membrane transport 1. Summary A. Simple diffusion 1) Diffusion by electrochemical gradient no energy required 2) No channel or carrier (or transporter protein) is needed B. Passive transport (= Facilitated
More informationSupplementary information. Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization.
Supplementary information Porphyrin-Assisted Docking of a Thermophage Portal Protein into Lipid Bilayers: Nanopore Engineering and Characterization. Benjamin Cressiot #, Sandra J. Greive #, Wei Si ^#,
More informationBile salt transport profiling of hepatic transporters. Peter Krajcsi
Bile salt transport profiling of hepatic transporters Peter Krajcsi The human bile Carey and Duane: The liver, 3rd edition Bile acids / salts Cholesterol Hepatocytes Primary bile acids: Chenodeoxycholic
More informationSupplemental Figure 1.
A wt spoiiiaδ spoiiiahδ bofaδ B C D E spoiiiaδ, bofaδ Supplemental Figure 1. GFP-SpoIVFA is more mislocalized in the absence of both BofA and SpoIIIAH. Sporulation was induced by resuspension in wild-type
More informationChapter Cells and the Flow of Energy A. Forms of Energy 1. Energy is capacity to do work; cells continually use energy to develop, grow,
Chapter 6 6.1 Cells and the Flow of Energy A. Forms of Energy 1. Energy is capacity to do work; cells continually use energy to develop, grow, repair, reproduce, etc. 2. Kinetic energy is energy of motion;
More informationTHALLIUM AND CESIUM IN MUSCLE CELLS COMPETE FOR THE ADSORPTION SITES NORMALLY OCCUPlED BY K+
THALLIUM AND CESIUM IN MUSCLE CELLS COMPETE FOR THE ADSORPTION SITES NORMALLY OCCUPlED BY K+ GILBERT N. LING Department of Molecular Biology. Pennsylvania Hospital. Philadelphia, Pennsylvania 19107 Reprit~red
More informationElectrical Properties of the Membrane
BIOE 2520 Electrical Properties of the Membrane Reading: Chapter 11 of Alberts et al. Stephen Smith, Ph.D. 433 Biotech Center shs46@pitt.edu Permeability of Lipid membrane Lipid bilayer is virtually impermeable
More informationChapter 2 Cellular Homeostasis and Membrane Potential
Chapter 2 Cellular Homeostasis and Membrane Potential 2.1 Membrane Structure and Composition The human cell can be considered to consist of a bag of fluid with a wall that separates the internal, or intracellular,
More informationevoglow - express N kit distributed by Cat.#: FP product information broad host range vectors - gram negative bacteria
evoglow - express N kit broad host range vectors - gram negative bacteria product information distributed by Cat.#: FP-21020 Content: Product Overview... 3 evoglow express N -kit... 3 The evoglow -Fluorescent
More informationHole s Human Anatomy and Physiology Tenth Edition. Chapter 2
PowerPoint Lecture Outlines to accompany Hole s Human Anatomy and Physiology Tenth Edition Shier w Butler w Lewis Chapter 2 Copyright The McGraw-Hill Companies, Inc. Permission required for reproduction
More informationSupporting Information
Supporting Information Mullins et al. 10.1073/pnas.0906781106 SI Text Detection of Calcium Binding by 45 Ca 2 Overlay. The 45 CaCl 2 (1 mci, 37 MBq) was obtained from NEN. The general method of 45 Ca 2
More informationTable S1. Primers and PCR conditions used in this paper Primers Sequence (5 3 ) Thermal conditions Reference Rhizobacteria 27F 1492R
Table S1. Primers and PCR conditions used in this paper Primers Sequence (5 3 ) Thermal conditions Reference Rhizobacteria 27F 1492R AAC MGG ATT AGA TAC CCK G GGY TAC CTT GTT ACG ACT T Detection of Candidatus
More informationOxisResearch A Division of OXIS Health Products, Inc.
OxisResearch A Division of OXIS Health Products, Inc. BIOXYTECH GSH-400 Colorimetric Assay for Glutathione For Research Use Only. Not For Use In Diagnostic Procedures. Catalog Number 21011 INTRODUCTION
More informationCellular Electrophysiology. Cardiac Electrophysiology
Part 1: Resting and Action Potentials Cardiac Electrophysiology Theory Simulation Experiment Scale The membrane: structure, channels and gates The cell: resting potential, whole cell currents, cardiac
More informationAntibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria
Antibiotic efflux pumps in eucaryotic cells: consequences for activity against intracellular bacteria Françoise Van Bambeke on behalf of J.M. Michot, C. Seral, M. Heremans, M.P. Mingeot-Leclercq, P.M.
More informationOXFORD BIOMEDICAL RESEARCH
Colorimetric Assay for Glutathione Product No. GT 10 For Research Use Only INTRODUCTION Glutathione (gamma-glutamylcysteinylglycine or GSH) is a naturally occuring tripeptide whose nucleophilic and reducing
More informationRNA Synthesis and Processing
RNA Synthesis and Processing Introduction Regulation of gene expression allows cells to adapt to environmental changes and is responsible for the distinct activities of the differentiated cell types that
More informationChapter 2 Concepts of Chemistry
Anatomy Physiology and Disease for the Health Professions 3rd Edition Booth Test Bank Full Download: http://testbanklive.com/download/anatomy-physiology-and-disease-for-the-health-professions-3rd-edition-booth-te
More informationEFFECT OF ph AND AMMONIUM IONS ON THE PERMEABILITY
EFFECT OF ph AND AMMONIUM IONS ON THE PERMEABILITY OF BACILLUS PASTEURII W. R. WILEY AND J. L. STOKES Department of Bacteriology and Public Health, Washington State University, Pullman, Washington ABSTRACT
More informationMain idea of this lecture:
Ac#ve Transport Main idea of this lecture: How do molecules, big and small, get in OR out of a cell? 2 Main ways: Passive Transport (Does not require energy) Lecture 1 Ac=ve Transport (Requires energy)
More informationCurriculum Links. AQA GCE Biology. AS level
Curriculum Links AQA GCE Biology Unit 2 BIOL2 The variety of living organisms 3.2.1 Living organisms vary and this variation is influenced by genetic and environmental factors Causes of variation 3.2.2
More informationلجنة الطب البشري رؤية تنير دروب تميزكم
1) Hyperpolarization phase of the action potential: a. is due to the opening of voltage-gated Cl channels. b. is due to prolonged opening of voltage-gated K + channels. c. is due to closure of the Na +
More informationCELL SIGNALLING and MEMBRANE TRANSPORT. Mark Louie D. Lopez Department of Biology College of Science Polytechnic University of the Philippines
CELL SIGNALLING and MEMBRANE TRANSPORT Mark Louie D. Lopez Department of Biology College of Science Polytechnic University of the Philippines GENERIC SIGNALLING PATHWAY CELL RESPONSE TO SIGNALS CELL RESPONSE
More informationOverview of ion channel proteins. What do ion channels do? Three important points:
Overview of ion channel proteins Protein Structure Membrane proteins & channels Specific channels Several hundred distinct types Organization Evolution We need to consider 1. Structure 2. Functions 3.
More informationThe solvent is the dissolving agent -- i.e., the most abundant component of the solution
SOLUTIONS Definitions A solution is a system in which one or more substances are homogeneously mixed or dissolved in another substance homogeneous mixture -- uniform appearance -- similar properties throughout
More informationMULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. 1)
MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. 1) 1) Which of the following statements about the atom A) It has 12 neutrons in its nucleus. B) It
More informationTrioMol Isolation Reagent
TrioMol Isolation Reagent Technical Manual No. 0242 Version 06142007 I Description... 1 II Key Features... 1 III Storage..... 1 IV General Protocol Using Triomol Isolation Reagent 1 V Troubleshooting.
More informationInstantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a ph-assisted and Surfactant-Free Route
Supporting Information Instantaneous and Quantitative Functionalization of Gold Nanoparticles with Thiolated DNA Using a ph-assisted and Surfactant-Free Route Xu Zhang,, Mark R. Servos and Juewen Liu *
More informationTrioMol Isolation Reagent
TrioMol Isolation Reagent Technical Manual No. 0242 Version 06142007 I Description... 1 II Key Features... 1 III Storage..... 1 IV General Protocol Using Triomol Isolation Reagent 1 V Troubleshooting.
More informationElectronic Supplementary Information
Electronic Supplementary Information Polymer-coated spherical mesoporous silica for ph-controlled delivery of insulin Sae Rom Choi a,, Dong-jin Jang b,, Sanghyun Kim a, Sunhyung An c, Jinwoo Lee c, Euichaul
More informationMULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. C is FALSE?
Exam Name MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. 1) Which of the following statements about the atom 12 6 C is FALSE? 1) A) It has 12 neutrons
More informationHuman Coagulation Factor XII Total Antigen ELISA Kit
Human Coagulation Factor XII Total Antigen ELISA Kit Catalog No: IHFXIIKT-TOT Lot No: SAMPLE INTENDED USE This human coagulation Factor XII antigen assay is intended for the quantitative determination
More informationMULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. Figure 2.1
Exam Name MULTIPLE CHOICE. Choose the one alternative that best completes the statement or answers the question. Figure 2.1 1) Which compound in Figure 2.1 is an ester? 1) A) a b c d e Answer: D 2) A scientist
More informationDOUBLE DISPLACEMENT REACTIONS. Double your pleasure, double your fun
DOUBLE DISPLACEMENT REACTIONS Double your pleasure, double your fun Industrial processes produce unwanted by-products. Dissolved toxic metal ions-copper, mercury, and cadmium-are common leftovers in the
More informationNeurons and the membrane potential. N500 John Beggs 23 Aug, 2016
Neurons and the membrane potential N500 John Beggs 23 Aug, 2016 My background, briefly Neurons Structural elements of a typical neuron Figure 1.2 Some nerve cell morphologies found in the human
More informationBiophysics I (BPHS 3090)
Biophysics I (BPHS 3090) Instructors: Prof. Christopher Bergevin (cberge@yorku.ca) Website: http://www.yorku.ca/cberge/3090w2015.html York University Winter 2015 Lecture 16 Reference/Acknowledgement: -
More informationIon Chromatography. Anion Exchange. Chromatography Ion Exchange Theory. Dr. Shulamit Levin
Ion Exchange Chromatography Chromatographic Process BA Mobile phase Stationary Phase A Shula Levin Bioforum B Distribution: K = C s/c m B shulal@zahav.net.il http://shulalc.co.il/ A Elution through the
More informationBRIEF COMMUNICATION 3,4-DIAMINOPYRIDINE A POTENT NEW POTASSIUM CHANNEL BLOCKER
BRIEF COMMUNICATION 3,4-DIAMINOPYRIDINE A POTENT NEW POTASSIUM CHANNEL BLOCKER GLENN E. KIRSCH AND ToSHIo NARAHASHI, Department ofpharmacology, Northwestem University Medical School, Chicago, Illinois
More informationACTIVE TRANSPORT AND GLUCOSE TRANSPORT. (Chapter 14 and 15, pp and pp )
ACTIVE TRANSPORT AND GLUCOSE TRANSPORT (Chapter 14 and 15, pp 140-143 and pp 146-151) Overview Active transport is the movement of molecules across a cell membrane in the direction against their concentration
More informationGlutathione S-Transferase (GST) Assay Kit
Manual Glutathione S-Transferase (GST) Assay Kit For the determination of GST activity in biological samples Valid from 31.01.2013 K 2631 100 8 1. INTENDED USE The Glutathione S-Transferase (GST) Assay
More informationMembrane Physiology. Dr. Hiwa Shafiq Oct-18 1
Membrane Physiology Dr. Hiwa Shafiq 22-10-2018 29-Oct-18 1 Chemical compositions of extracellular and intracellular fluids. 29-Oct-18 2 Transport through the cell membrane occurs by one of two basic processes:
More information