scholarly journals Mutational analysis of histidine residues in human organic anion transporter 4 (hOAT4)

2004 ◽  
Vol 384 (1) ◽  
pp. 87-92 ◽  
Author(s):  
Fanfan ZHOU ◽  
Zui PAN ◽  
Jianjie MA ◽  
Guofeng YOU
Keyword(s):  
Mutational Analysis ◽  
Organic Anion ◽  
The Body ◽  
Organic Anion Transporter ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Membrane Expression ◽  
Histidine Residues ◽  
Anion Transporter

Human organic anion transporter 4 (hOAT4) belongs to a family of organic anion transporters which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, antitumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. hOAT4-mediated transport of the organic anion oestrone sulphate in COS-7 cells was inhibited by the histidine-modifying reagent DEPC (diethyl pyrocarbonate). Therefore the role of histidine residues in the function of hOAT4 was examined by site-directed mutagenesis. All five histidine residues of hOAT4 were converted into alanine, singly or in combination. Single replacement of His-47, or simultaneous replacement of His-47/52/83 or His-47/52/83/305/469 (H-less) led to a 50–80% decrease in transport activity. The decreased transport activity of these mutants was correlated with a decreased amount of cell-surface expression, although the total cell expression of these mutants was similar to that of wild-type hOAT4. These results suggest that mutation at positions 47, 47/52/83 and 47/52/83/305/469 impaired membrane expression rather than function. We also showed that, although most of the histidine mutants of hOAT4 were sensitive to inhibition by DEPC, H469A (His-469→Ala) was completely insensitive to inhibition by this reagent. Therefore modification of His-469 is responsible for the inhibition of hOAT4 by DEPC.

scholarly journals Mutational analysis of histidine residues in the rabbit Na+/dicarboxylate co-transporter NaDC-1

1998 ◽  
Vol 331 (1) ◽  
pp. 257-264 ◽  
Author(s):  
Ana M. PAJOR ◽  
Ning SUN ◽  
Heidi G. VALMONTE
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Membrane Expression ◽  
Histidine Residues ◽  
Cell Surface Biotinylation

Succinate transport by the rabbit Na+/dicarboxylate co-transporter, NaDC-1, expressed in Xenopusoocytes was inhibited by the histidyl-selective reagent diethyl pyrocarbonate (DEPC). Therefore the role of histidine residues in the function of NaDC-1 was examined by site-directed mutagenesis. All 11 histidine residues in NaDC-1 were converted to alanine, but only mutant H106A exhibited a decrease in succinate transport. Additional mutations of NaDC-1 at position 106 showed that aspartic acid and asparagine, but not arginine, can substitute for histidine. Examination of succinate and citrate kinetics of H106A revealed a decrease in Vmax with no change in Km. Cell surface biotinylation experiments showed that the transport activity of all four mutants at position 106 was correlated with the amount of cell surface expression, suggesting a role of His-106 in membrane expression rather than function. Two of the histidine mutants, H153A and H569A, exhibited insensitivity to inhibition by DEPC, indicating that these residues are involved in binding DEPC. Neither of these residues is required for transport activity; thus DEPC probably inhibits NaDC-1 function by hindrance of the mobility of the carrier. We conclude that histidine residues are not critical for transport function in NaDC-1, although His-106 might be involved in determining protein expression or stability in the membrane.

scholarly journals Cysteine residues in the organic anion transporter mOAT1

2004 ◽  
Vol 380 (1) ◽  
pp. 283-287 ◽  
Author(s):  
Kunihiko TANAKA ◽  
Fanfan ZHOU ◽  
Kogo KUZE ◽  
Guofeng YOU
Keyword(s):  
Organic Anion ◽  
Cysteine Residue ◽  
The Body ◽  
Organic Anion Transporter ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Transport Activity ◽  
Anion Transporters ◽  
Anion Transporter

Mouse organic anion transporter 1 (mOAT1) belongs to a family of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-HIV therapeutics, anti-tumour drugs, antibiotics, anti-hypertensives and anti-inflammatories. mOAT1-mediated transport of organic anion PAH (p-aminohippurate) in HeLa cells was inhibited by the cysteine-modifying reagent PCMBS (p-chloromercuribenzenesulphonate). Therefore the role of cysteine residues in the function of mOAT1 was examined by site-directed mutagenesis. All 13 cysteine residues in mOAT1 were replaced by alanine, singly or in combination. Single replacement of these residues had no significant effect on mOAT1-mediated PAH transport, indicating that no individual cysteine residue is necessary for function. Multiple replacements at a C-terminal region (C335/379/427/434A; Cys335/379/427/434→Ala) resulted in a substantial decrease in transport activity. A simultaneous replacement of all 13 cysteine residues (C-less) led to a complete loss of transport function. The decreased or lack of transport activity of the mutants C335/379/427/434A and C-less was due to the impaired trafficking of the mutant transporters to the cell surface. These results suggest that although cysteine residues are not required for function in mOAT1, their presence appears to be important for the targeting of the transporter to the plasma membrane. We also showed that, although all cysteine mutants of mOAT1 were sensitive to the inhibition by PCMBS, C49A was less sensitive than the wild-type mOAT1, suggesting that the modification of Cys49 may play a role in the inhibition of mOAT1 by PCMBS.

scholarly journals Nedd4-2 but not Nedd4-1 is critical for protein kinase C-regulated ubiquitination, expression, and transport activity of human organic anion transporter 1

2016 ◽  
Vol 310 (9) ◽  
pp. F821-F831 ◽  
Author(s):  
Da Xu ◽  
Haoxun Wang ◽  
Qiang Zhang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Organic Anion ◽  
The Body ◽  
Organic Anion Transporter ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter ◽  
In Cells

Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.

scholarly journals Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4–2

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhou Yu ◽  
Chenchang Liu ◽  
Jinghui Zhang ◽  
Zhengxuan Liang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Organic Anion ◽  
Surface Expression ◽  
Organic Anion Transporter ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter

Abstract Background Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4–2 (Nedd4–2). Nedd4–2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4–2 is a direct substrate for PKC-induced phosphorylation is unknown. Results In this study, we investigated the role of Nedd4–2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4–2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4–2 partially blocked the effects of PKC on Nedd4–2 phosphorylation and on OAT1 transport activity. Conclusions Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4–2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4–2 in combination play an important role in this regulatory process.

scholarly journals Regulation of human organic anion transporter 1 by ANG II: involvement of protein kinase Cα

2009 ◽  
Vol 296 (2) ◽  
pp. E378-E383 ◽  
Author(s):  
Shanshan Li ◽  
Peng Duan ◽  
Guofeng You
Keyword(s):  
Protein Kinase ◽  
Cell Surface ◽  
Organic Anion ◽  
Specific Inhibitor ◽  
The Body ◽  
Organic Anion Transporter ◽  
Cell Surface Expression ◽  
Maximal Velocity ◽  
Ang Ii ◽  
Anion Transporter

Human organic anion transporter 1 (hOAT1) belongs to a family of organic anion transporters that play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, antihypertensives, and anti-inflammatories. hOAT1 is abundantly expressed in the kidney. In the current study, we examined the regulation of hOAT1 by ANG II in kidney COS-7 cells. ANG II induced a concentration- and time-dependent inhibition of hOAT1 transport activity. Such inhibition mainly resulted from a decreased cell surface expression without a change in total cell expression of the transporter, kinetically revealed as a decreased maximal velocity without significant change in Michaelis constant. ANG II-induced inhibition of hOAT1 activity could be prevented by treating hOAT1-expressing cells with staurosporine, a general protein kinase C (PKC) inhibitor. To obtain further information on which PKC isoform mediates ANG II regulation of hOAT1 activity, cellular distribution of various PKC isoforms was examined in cells treated with or without ANG II. We showed that ANG II treatment resulted in a significant translocation of PKCα from cytosol to membrane, and such translocation was blocked by treating hOAT1-expressing cells with Gö-6976, a PKCα-specific inhibitor. We further showed that ANG II-induced inhibition of hOAT1 activity and retrieval of hOAT1 from the cell surface could also be prevented by treating hOAT1-expressing cells with Gö-6976. We concluded that ANG II inhibited hOAT1 activity through activation of PKCα, which led to the redistribution of the transporter from the cell surface to the intracellular compartments.

scholarly journals Cationic Amino Acids Involved in Dicarboxylate Binding of the Flounder Renal Organic Anion Transporter

2001 ◽  
Vol 12 (10) ◽  
pp. 2012-2018 ◽  
Author(s):  
NATASCHA A. WOLFF ◽  
BETTINA GRÜNWALD ◽  
BJÖRN FRIEDRICH ◽  
FLORIAN LANG ◽  
STEFAN GODEHARDT ◽  
...  
Keyword(s):  
Amino Acids ◽  
Organic Anion ◽  
Surface Expression ◽  
Organic Anion Transporter ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Anion Transporter ◽  
Cationic Amino Acids

Abstract. Three conserved cationic amino acids in predicted transmembrane domains 1, 8, and 11, respectively, of the flounder renal organic anion transporter, fROAT, were changed by site-directed mutagenesis and the resulting mutants functionally characterized inXenopus laevisoocytes. Uptake ofp-aminohippurate (PAH) in oocytes that expressed mutant H34I, K394A, or R478D was markedly reduced compared with oocytes that expressed wild-type fROAT, but was still several-fold higher than that in water-injected control oocytes. Immunocytochemically, no decrease in cell surface expression of the mutants could be detected. Only mutant R478D appeared to have a lower PAH affinity than the wild type. Similar to wild-type—dependent PAH transport, uptake induced by mutant H34I was sensitive to glutarate (GA) cis-inhibition. In contrast, mutants K394A and R478D could not be significantly affected by up to 10 mM GA, although the cRNA-dependent PAH uptake could still be almost completely suppressed by probenecid. Moreover, again in contrast to the wild type, neither PAH influx nor PAH efflux mediated by these two mutants could be trans-stimulated by GA, nor did they induce GA transport. These data suggest that amino acids K394 and R478 in fROAT are required for dicarboxylate binding and PAH/dicarboxylate exchange.

Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

2007 ◽  
Vol 293 (1) ◽  
pp. G271-G278 ◽  
Author(s):  
Chitrawina Mahagita ◽  
Steven M. Grassl ◽  
Pawinee Piyachaturawat ◽  
Nazzareno Ballatori
Keyword(s):  
Bile Acid ◽  
Transport Mechanism ◽  
Organic Anion ◽  
Large Family ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Facilitated Diffusion ◽  
Transport Activity ◽  
Estrone Sulfate ◽  
Anion Transporter

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.

scholarly journals Evaluation of the endothelin receptor antagonists ambrisentan, darusentan, bosentan, and sitaxsentan as substrates and inhibitors of hepatobiliary transporters in sandwich-cultured human hepatocytesThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

2010 ◽  
Vol 88 (6) ◽  
pp. 682-691 ◽  
Author(s):  
J. Craig Hartman ◽  
Kenneth Brouwer ◽  
Arun Mandagere ◽  
Lawrence Melvin ◽  
Richard Gorczynski
Keyword(s):  
Organic Anion ◽  
Hepatic Uptake ◽  
Organic Anion Transporter ◽  
Endothelin Receptor Antagonists ◽  
Endothelin Receptor ◽  
Transport Activity ◽  
Receptor Antagonists ◽  
P Glycoprotein ◽  
Anion Transporter ◽  
Hepatic Transporters

To evaluate potential mechanisms of clinical hepatotoxicity, 4 endothelin receptor antagonists (ERAs) were examined for substrate activity and inhibition of hepatic uptake and efflux transporters in sandwich-cultured human hepatocytes. The 4 transporters studied were sodium-dependent taurocholate cotransporter (NTCP), organic anion transporter (OATP), bile salt export pump (BSEP), and multidrug resistance-associated protein 2 (MRP2). ERA transporter inhibition was examined using the substrates taurocholate (for NTCP and BSEP), [3H]estradiol-17β-d-glucuronide (for OATP), and [2-d-penicillamine, 5-d-penicillamine]enkephalin (for MRP2). ERA substrate activity was evaluated using probe inhibitors ritonavir (OATP and BSEP), bromosulfalein (OATP), erythromycin (P-glycoprotein), probenecid (MRP2 and OATP), and cyclosporin (NTCP). ERAs were tested at 2, 20, and 100 µmol·L–1 for inhibition and at 2 µmol·L–1 as substrates. OATP, NTCP, or BSEP transport activity was not reduced by ambrisentan or darusentan. Bosentan and sitaxsentan attenuated NTCP transport at higher concentrations. Only sitaxsentan decreased OATP transport (52%), and only bosentan reduced BSEP transport (78%). MRP2 transport activity was unaltered. OATP inhibitors decreased influx of all ERAs. Darusentan influx was least affected (84%–100% of control), whereas bosentan was most affected (32%–58% of control). NTCP did not contribute to influx of ERAs. Only bosentan and darusentan were shown as substrates for both BSEP and P-glycoprotein efflux. All ERAs tested were substrates for at least one hepatic transporter. Bosentan and sitaxsentan, but not ambrisentan and darusentan, inhibited human hepatic transporters, which provides a potential mechanism for the increased hepatotoxicity observed for these agents in the clinical setting.

The human organic anion transporter 3 (OAT3; SLC22A8): genetic variation and functional genomics

2006 ◽  
Vol 290 (4) ◽  
pp. F905-F912 ◽  
Author(s):  
Andrew R. Erdman ◽  
Lara M. Mangravite ◽  
Thomas J. Urban ◽  
Leah L. Lagpacan ◽  
Richard A. Castro ◽  
...  
Keyword(s):  
Genetic Variation ◽  
Asian Americans ◽  
Mexican Americans ◽  
Organic Anion ◽  
Critical Role ◽  
Organic Anion Transporter ◽  
Site Directed Mutagenesis ◽  
Sample Population ◽  
Coding Region ◽  
Anion Transporter

The human organic anion transporter, OAT3 ( SLC22A8), plays a critical role in renal drug elimination, by mediating the entry of a wide variety of organic anions, including a number of commonly used pharmaceuticals, into the renal proximal tubular cells. To understand the nature and extent of genetic variation in OAT3, and to determine whether such variation affects its function, we identified OAT3 variants in a large, ethnically diverse sample population and studied their transport activities in cellular assays. We identified a total of 10 distinct coding-region variants, which altered the encoded amino acid sequence, in DNA samples from 270 individuals (80 African-Americans, 80 European-Americans, 60 Asian-Americans, and 50 Mexican-Americans). The overall prevalence of these OAT3 variants was relatively low among the screened population, with only three variants having allele frequencies of >1% in a particular ethnic group. Clones of each variant were created by site-directed mutagenesis, expressed in HEK-293 cells, and tested for function using the model substrates, estrone sulfate (ES) and cimetidine (CIM). The results revealed a high degree of functional heterogeneity among OAT3 variants, with three variants (p. Arg149Ser, p. Gln239Stop, and p. Ile260Arg) that resulted in complete loss of function, and several others with significantly reduced function. One of the more common variants (p. Ile305Phe), found in 3.5% of Asian-Americans, appeared to have altered substrate specificity. This variant exhibited a reduced ability to transport ES, but a preserved ability to transport CIM. These data suggest that genetic variation in OAT3 may contribute to variation in the disposition of drugs.

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