CryoEM structures of anion exchanger 1 capture multiple states of inward- and outward-facing conformations.

Anion exchanger 1 (AE1, band 3) is a major membrane protein of red blood cells and plays a key role in acid-base homeostasis, urine acidification, red blood cell shape regulation, and removal of carbon dioxide during respiration. Though structures of the transmembrane domain (TMD) of three SLC4 transporters, including AE1, have been resolved previously in their outward-facing (OF) state, no mammalian SLC4 structure has been reported in the inward-facing (IF) conformation. Here we present the cryoEM structures of full-length bovine AE1 with its TMD captured in both IF and OF conformations. Remarkably, both IF-IF homodimers and IF-OF heterodimers were detected. The IF structures feature downward movement in the core domain with significant unexpected elongation of TM11. Molecular modeling and structure guided mutagenesis confirmed the functional significance of residues involved in TM11 elongation. Our data provide direct evidence for an elevator-like mechanism of ion transport by an SLC4 family member.

Cryo-EM structure of the sodium-driven chloride/bicarbonate exchanger NDCBE.

SLC4 transporters play significant roles in pH regulation and cellular sodium transport. The previously solved structures of the outward facing (OF) conformation for AE1 (SLC4A1) and NBCe1 (SLC4A4) transporters revealed an identical overall fold despite their different transport modes (chloride/bicarbonate exchange versus sodium-carbonate cotransport). However, the exact mechanism determining the different transport modes in the SLC4 family remains unknown. In this work, we report the cryo-EM 3.4 Å structure of the OF conformation of NDCBE (SLC4A8), which shares transport properties with both AE1 and NBCe1 by mediating the electroneutral exchange of sodium-carbonate with chloride. This structure features a fully resolved extracellular loop 3 and well-defined densities corresponding to sodium and carbonate ions in the tentative substrate binding pocket. Further, we combine computational modeling with functional studies to unravel the molecular determinants involved in NDCBE and SLC4 transport.

Identification of multiple substrate binding sites in SLC4 transporters in the outward-facing conformation: Insights into the transport mechanism.

Solute carrier family 4 (SLC4) transporters mediate the transmembrane transport of HCO3-, CO32-, and Cl- necessary for pH regulation, transepithelial H+/base transport, and ion homeostasis. Substrate transport with varying stoichiometry and specificity is achieved through an exchange mechanism and/or through coupling of the uptake of anionic substrates to typically co-transported Na+. Recently solved outward-facing structures of two SLC4 members (human anion exchanger 1 [hAE1] and human electrogenic sodium bicarbonate cotransporter 1 [hNBCe1]) with different transport modes (Cl-/HCO3- exchange versus Na+-CO32- symport) revealed highly conserved three-dimensional organization of their transmembrane domains. However, the exact location of the ion binding sites and their protein-ion coordination motifs are still unclear. In the present work, we combined site identification by ligand competitive saturation mapping and extensive molecular dynamics sampling with functional mutagenesis studies which led to the identification of two substrate binding sites (entry and central) in the outward-facing states of hAE1 and hNBCe1. Mutation of residues in the identified binding sites led to impaired transport in both proteins. We also showed that R730 in hAE1 is crucial for anion binding in both entry and central sites, whereas in hNBCe1, a Na+ acts as an anchor for CO32- binding to the central site. Additionally, protonation of the central acidic residues (E681 in hAE1 and D754 in hNBCe1) alters the ion dynamics in the permeation cavity and may contribute to the transport mode differences in SLC4 proteins. These results provide a basis for understanding the functional differences between hAE1 and hNBCe1 and may facilitate potential drug development for diseases such as proximal and distal renal tubular acidosis.

SLC4A11 function: evidence for H+(OH-) and NH3-H+ transport.

Whether SLC4A11 transports ammonia and its potential mode of ammonia transport (NH4+, NH3, or NH3-2H+ transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 mediates significant conductive H+(OH-) transport is also controversial. The present study was performed to determine the mechanism of human SLC4A11 ammonia transport and whether the transporter mediates conductive H+(OH-) transport in the absence of ammonia. We quantitated H+ flux by monitoring changes in intracellular pH (pHi) and measured whole cell currents in patch-clamp studies of HEK293 cells expressing the transporter in the absence and presence of NH4Cl. Our results demonstrate that SLC4A11 mediated conductive H+(OH-) transport that was stimulated by raising the extracellular pH (pHe). Ammonia-induced HEK293 whole cell currents were also stimulated by an increase in pHe. In studies using increasing NH4Cl concentrations with equal NH4+ extracellular and intracellular concentrations, the shift in the reversal potential (Erev) due to the addition of ammonia was compatible with NH3-H+ transport competing with H+(OH-) rather than NH3-nH+ (n ≥ 2) transport. The increase in equivalent H+(OH-) flux observed in the presence of a transcellular H+ gradient was also compatible with SLC4A11-mediated NH3-H+ flux. The NH3 versus Erev data fit a theoretical model suggesting that NH3-H+ and H+(OH-) competitively interact with the transporter. Studies of mutant SLC4A11 constructs in the putative SLC4A11 ion coordination site showed that both H+(OH-) transport and ammonia-induced whole cell currents were blocked suggesting that the H+(OH-) and NH3-H+ transport processes share common features involving the SLC4A11 transport mechanism.

CryoEM structure of the human SLC4A4 sodium-coupled acid-base transporter NBCe1.

Na+-coupled acid-base transporters play essential roles in human biology. Their dysfunction has been linked to cancer, heart, and brain disease. High-resolution structures of mammalian Na+-coupled acid-base transporters are not available. The sodium-bicarbonate cotransporter NBCe1 functions in multiple organs and its mutations cause blindness, abnormal growth and blood chemistry, migraines, and impaired cognitive function. Here, we have determined the structure of the membrane domain dimer of human NBCe1 at 3.9 Å resolution by cryo electron microscopy. Our atomic model and functional mutagenesis revealed the ion accessibility pathway and the ion coordination site, the latter containing residues involved in human disease-causing mutations. We identified a small number of residues within the ion coordination site whose modification transformed NBCe1 into an anion exchanger. Our data suggest that symporters and exchangers utilize comparable transport machinery and that subtle differences in their substrate-binding regions have very significant effects on their transport mode.

Multifunctional ion transport properties of human SLC4A11: comparison of the SLC4A11-B and SLC4A11-C variants.

Congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (CHED with progressive sensorineural deafness), and potentially a subset of individuals with late-onset Fuchs' endothelial corneal dystrophy are caused by mutations in the SLC4A11 gene that results in corneal endothelial cell abnormalities. Originally classified as a borate transporter, the function of SLC4A11 as a transport protein remains poorly understood. Elucidating the transport function(s) of SLC4A11 is needed to better understand how its loss results in the aforementioned posterior corneal dystrophic disease processes. Quantitative PCR experiments demonstrated that, of the three known human NH2-terminal variants, SLC4A11-C is the major transcript expressed in human corneal endothelium. We studied the expression pattern of the three variants in mammalian HEK-293 cells and demonstrated that the SLC4A11-B and SLC4A11-C variants are plasma membrane proteins, whereas SLC4A11-A is localized intracellularly. SLC4A11-B and SLC4A11-C were shown to be multifunctional ion transporters capable of transporting H+ equivalents in both a Na+-independent and Na+-coupled mode. In both transport modes, SLC4A11-C H+ flux was significantly greater than SLC4A11-B. In the presence of ammonia, SLC4A11-B and SLC4A11-C generated inward currents that were comparable in magnitude. Chimera SLC4A11-C-NH2-terminus-SLC4A11-B experiments demonstrated that the SLC4A11-C NH2-terminus functions as an autoactivating domain, enhancing Na+-independent and Na+-coupled H+ flux without significantly affecting the electrogenic NH3-H(n)+ cotransport mode. All three modes of transport were significantly impaired in the presence of the CHED causing p.R109H (SLC4A11-C numbering) mutation. These complex ion transport properties need to be addressed in the context of corneal endothelial disease processes caused by mutations in SLC4A11.

Interplay between disulfide bonding and N-glycosylation defines SLC4 Na+-coupled transporter extracellular topography.

The extracellular loop 3 (EL-3) of SLC4 Na(+)-coupled transporters contains 4 highly conserved cysteines and multiple N-glycosylation consensus sites. In the electrogenic Na(+)-HCO3(-) cotransporter NBCe1-A, EL-3 is the largest extracellular loop and is predicted to consist of 82 amino acids. To determine the structural-functional importance of the conserved cysteines and the N-glycosylation sites in NBCe1-A EL-3, we analyzed the potential interplay between EL-3 disulfide bonding and N-glycosylation and their roles in EL-3 topological folding. Our results demonstrate that the 4 highly conserved cysteines form two intramolecular disulfide bonds, Cys(583)-Cys(585) and Cys(617)-Cys(642), respectively, that constrain EL-3 in a folded conformation. The formation of the second disulfide bond is spontaneous and unaffected by the N-glycosylation state of EL-3 or the first disulfide bond, whereas formation of the first disulfide bond relies on the presence of the second disulfide bond and is affected by N-glycosylation. Importantly, EL-3 from each monomer is adjacently located at the NBCe1-A dimeric interface. When the two disulfide bonds are missing, EL-3 adopts an extended conformation highly accessible to protease digestion. This unique adjacent parallel location of two symmetrically folded EL-3 loops from each monomer resembles a domain-like structure that is potentially important for NBCe1-A function in vivo. Moreover, the formation of this unique structure is critically dependent on the finely tuned interplay between disulfide bonding and N-glycosylation in the membrane processed NBCe1-A dimer.

Human SLC4A11-C functions as a DIDS-stimulatable H⁺(OH⁻) permeation pathway: partial correction of R109H mutant transport.

The SLC4A11 gene mutations cause a variety of genetic corneal diseases, including congenital hereditary endothelial dystrophy 2 (CHED2), Harboyan syndrome, some cases of Fuchs' endothelial dystrophy (FECD), and possibly familial keratoconus. Three NH2-terminal variants of the human SLC4A11 gene, named SLC4A11-A, -B, and -C are known. The SLC4A11-B variant has been the focus of previous studies. Both the expression of the SLC4A11-C variant in the cornea and its functional properties have not been characterized, and therefore its potential pathophysiological role in corneal diseases remains to be explored. In the present study, we demonstrate that SLC4A11-C is the predominant SLC4A11 variant expressed in human corneal endothelial mRNA and that the transporter functions as an electrogenic H(+)(OH(-)) permeation pathway. Disulfonic stilbenes, including 4,4'-diisothiocyano-2,2'-stilbenedisulfonate (DIDS), 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonate (H2DIDS), and 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonate (SITS), which are known to bind covalently, increased SLC4A11-C-mediated H(+)(OH(-)) flux by 150-200% without having a significant effect in mock-transfected cells. Noncovalently interacting 4,4'-diaminostilbene-2,2'-disulfonate (DADS) was without effect. We tested the efficacy of DIDS on the functionally impaired R109H mutant (SLC4A11-C numbering) that causes CHED2. DIDS (1 mM) increased H(+)(OH(-)) flux through the mutant transporter by ∼40-90%. These studies provide a basis for future testing of more specific chemically modified dilsulfonic stilbenes as potential therapeutic agents to improve the functional impairment of specific SLC4A11 mutant transporters.

Single particle electron microscopy analysis of the bovine anion exchanger 1 reveals a flexible linker connecting the cytoplasmic and membrane domains.

Anion exchanger 1 (AE1) is the major erythrocyte membrane protein that mediates chloride/bicarbonate exchange across the erythrocyte membrane facilitating CO2 transport by the blood, and anchors the plasma membrane to the spectrin-based cytoskeleton. This multi-protein cytoskeletal complex plays an important role in erythrocyte elasticity and membrane stability. An in-frame AE1 deletion of nine amino acids in the cytoplasmic domain in a proximity to the membrane domain results in a marked increase in membrane rigidity and ovalocytic red cells in the disease Southeast Asian Ovalocytosis (SAO). We hypothesized that AE1 has a flexible region connecting the cytoplasmic and membrane domains, which is partially deleted in SAO, thus causing the loss of erythrocyte elasticity. To explore this hypothesis, we developed a new non-denaturing method of AE1 purification from bovine erythrocyte membranes. A three-dimensional (3D) structure of bovine AE1 at 2.4 nm resolution was obtained by negative staining electron microscopy, orthogonal tilt reconstruction and single particle analysis. The cytoplasmic and membrane domains are connected by two parallel linkers. Image classification demonstrated substantial flexibility in the linker region. We propose a mechanism whereby flexibility of the linker region plays a critical role in regulating red cell elasticity.

Topology of NBCe1 protein transmembrane segment 1 and structural effect of proximal renal tubular acidosis (pRTA) S427L mutation.

In the kidney proximal tubule, NBCe1-A plays a critical role in absorbing HCO3(-) from cell to blood. NBCe1-A transmembrane segment 1 (TM1) is involved in forming part of the ion permeation pathway, and a missense mutation S427L in TM1 impairs ion transport, causing proximal renal tubular acidosis. In the present study, we examined the topology of NBCe1-A-TM1 in detail and its structural perturbation induced by S427L. We analyzed the N-terminal cytoplasmic region (Cys-389-Gln-424) of NBCe1-A-TM1 using the substituted cysteine scanning accessibility method combined with extensive chemical stripping, in situ chemical probing, and functional transport assays. NBCe1-A-TM1 was previously modeled on the anion exchanger 1 TM1 (AE1-TM1); however, our data demonstrated that the topology of AE1-TM1 differs significantly from NBCe1-A-TM1. Our findings revealed that NBCe1-A-TM1 is unusually long, consisting of 31 membrane-embedded amino acids (Phe-412 to Thr-442). The linker region (Arg-394-Pro-411) between the N terminus of TM1 and the cytoplasmic domain is minimally exposed to aqueous and is potentially folded in a helical structure that intimately interacts with the NBCe1-A cytoplasmic domain. In contrast, AE1-TM1 contains 25 amino acids connected to an aqueous-exposed cytoplasmic region. Based on our new NBCe1-A-TM1 model, Ser-427 resides in the middle of TM1. Leucine substitution at Ser-427 blocks the normal aqueous access to Thr-442, Ala-435, and Lys-404, implying a significant alteration of NBCe1-TM1 orientation. Our study provides novel structural insights into the pathogenic mechanism of S427L in mediating proximal renal tubular acidosis.

Aminoacylase 3 binds to and cleaves the N-terminus of the hepatitis C virus core protein.

Aminoacylase 3 (AA3) mediates deacetylation of N-acetyl aromatic amino acids and mercapturic acids. Deacetylation of mercapturic acids of exo- and endobiotics are likely involved in their toxicity. AA3 is predominantly expressed in kidney, and to a lesser extent in liver, brain, and blood. AA3 has been recently reported to interact with the hepatitis C virus core protein (HCVCP) in the yeast two-hybrid system. Here we demonstrate that AA3 directly binds to HCVCP (K(d) ~10 µM) that may by implicated in HCV pathogenesis. AA3 also revealed a weak endopeptidase activity towards the N-terminus of HCVCP.

Inhibition of aminoacylase 3 protects rat brain cortex neuronal cells from the toxicity of 4-hydroxy-2-nonenal mercapturate and 4-hydroxy-2-nonenal.

4-Hydroxy-2-nonenal (4HNE) and acrolein (ACR) are highly reactive neurotoxic products of lipid peroxidation that are implicated in the pathogenesis and progression of Alzheimer's and Parkinson's diseases. Conjugation with glutathione (GSH) initiates the 4HNE and ACR detoxification pathway, which generates the mercapturates of 4HNE and ACR that can be excreted. Prior work has shown that the efficiency of the GSH-dependent renal detoxification of haloalkene derived mercapturates is significantly decreased upon their deacetylation because of rapid transformation of the deacetylated products into toxic compounds mediated by ß-lyase. The enzymes of the GSH-conjugation pathway and ß-lyases are expressed in the brain, and we hypothesized that a similar toxicity mechanism may be initiated in the brain by the deacetylation of 4HNE- and ACR-mercapturate. The present study was performed to identify an enzyme(s) involved in 4HNE- and ACR-mercapturate deacetylation, characterize the brain expression of this enzyme and determine whether its inhibition decreases 4HNE and 4HNE-mercapturate neurotoxicity. We demonstrated that of two candidate deacetylases, aminoacylases 1 (AA1) and 3 (AA3), only AA3 efficiently deacetylates both 4HNE- and ACR-mercapturate. AA3 was further localized to neurons and blood vessels. Using a small molecule screen we generated high-affinity AA3 inhibitors. Two of them completely protected rat brain cortex neurons expressing AA3 from the toxicity of 4HNE-mercapturate. 4HNE-cysteine (4HNE-Cys) was also neurotoxic and its toxicity was mostly prevented by a ß-lyase inhibitor, aminooxyacetate. The results suggest that the AA3 mediated deacetylation of 4HNE-mercapturate may be involved in the neurotoxicity of 4HNE.

A new technique for multiple re-use of planar patch clamp chips.

The patch clamp technique is widely used for recording the activity of ion channels in single cells and lipid bilayers. Most platforms utilize borosilicate glass configured as a pipette, however more recently planar patch clamp chips have been developed that require less technical expertise. Planar patch clamp chips in systems like the Nanion Port-a-Patch are useful in that they allow more rapid throughput in drug screening studies. This technique also has the ability to perform rapid solution changes from the intracellular side. A current drawback with the planar patch clamp chips is the need to utilize a separate chip for each experiment. This increases the cost of each experiment and is due to the fact that the ∼1µm aperture used for cell attachment is thought to retain cellular debris thereby preventing subsequent cell attachment and formation of GΩ seals. In the present study we have for the first time solved the technical problem of developing a simple protocol for re-use of Nanion planar patch clamp chips. The re-use methodology is demonstrated in whole cell patch clamp studies of HEK-293 cells expressing the electrogenic sodium bicarbonate cotransporter NBCe1-A in protocols involving external and internal solution changes, and CHO-K1 cells with incorporated gramicidin channels.

Determination of membrane protein transporter oligomerization in native tissue using spatial fluorescence intensity fluctuation analysis.

Membrane transporter proteins exist in a complex dynamic equilibrium between various oligomeric states that include monomers, dimers, dimer of dimers and higher order oligomers. Given their sub-optical microscopic resolution size, the oligomerization state of membrane transporters is difficult to quantify without requiring tissue disruption and indirect biochemical methods. Here we present the application of a fluorescence measurement technique which combines fluorescence image moment analysis and spatial intensity distribution analysis (SpIDA) to determine the oligomerization state of membrane proteins in situ. As a model system we analyzed the oligomeric state(s) of the electrogenic sodium bicarbonate cotransporter NBCe1-A in cultured cells and in rat kidney. The approaches that we describe offer for the first time the ability to investigate the oligomeric state of membrane transporter proteins in their native state.

The sodium-driven chloride/bicarbonate exchanger in presynaptic terminals.

The sodium-driven chloride/bicarbonate exchanger (NDCBE), a member of the SLC4 family of bicarbonate transporters, was recently found to modulate excitatory neurotransmission in hippocampus. By using light and electron microscopic immunohistochemistry, we demonstrate here that NDCBE is expressed throughout the adult rat brain, and selectively concentrates in presynaptic terminals, where it is closely associated with synaptic vesicles. NDCBE is in most glutamatergic axon terminals, and is also present in the terminals of parvalbumin-positive γ-aminobutyric acid (GABA)ergic cells. These findings suggest that NDCBE can regulate glutamatergic transmission throughout the brain, and point to a role for NDCBE as a possible regulator of GABAergic neurotransmission.

Structures of aminoacylase 3 in complex with acetylated substrates.

Trichloroethylene (TCE) is one of the most widespread environmental contaminants, which is metabolized to N-acetyl-S-1,2-dichlorovinyl-L-cysteine (NA-DCVC) before being excreted in the urine. Alternatively, NA-DCVC can be deacetylated by aminoacylase 3 (AA3), an enzyme that is highly expressed in the kidney, liver, and brain. NA-DCVC deacetylation initiates the transformation into toxic products that ultimately causes acute renal failure. AA3 inhibition is therefore a target of interest to prevent TCE induced nephrotoxicity. Here we report the crystal structure of recombinant mouse AA3 (mAA3) in the presence of its acetate byproduct and two substrates: N(α)-acetyl-L-tyrosine and NA-DCVC. These structures, in conjunction with biochemical data, indicated that AA3 mediates substrate specificity through van der Waals interactions providing a dynamic interaction interface, which facilitates a diverse range of substrates.

Structural and functional characterization of the C-terminal transmembrane region of NBCe1-A.

NBCe1-A and AE1 both belong to the SLC4 HCO(3)(-) transporter family. The two transporters share 40% sequence homology in the C-terminal transmembrane region. In this study, we performed extensive substituted cysteine-scanning mutagenesis analysis of the C-terminal region of NBCe1-A covering amino acids Ala(800)-Lys(967). Location of the introduced cysteines was determined by whole cell labeling with a membrane-permeant biotin maleimide and a membrane-impermeant 2-((5(6)-tetramethylrhodamine)carboxylamino) ethyl methanethiosulfonate (MTS-TAMRA) cysteine-reactive reagent. The results show that the extracellular surface of the NBCe1-A C-terminal transmembrane region is minimally exposed to aqueous media with Met(858) accessible to both biotin maleimide and TAMRA and Thr(926)-Ala(929) only to TAMRA labeling. The intracellular surface contains a highly exposed (Met(813)-Gly(828)) region and a cryptic (Met(887)-Arg(904)) connecting loop. The lipid/aqueous interface of the last transmembrane segment is at Asp(960). Our data clearly determined that the C terminus of NBCe1-A contains 5 transmembrane segments with greater average size compared with AE1. Functional assays revealed only two residues in the region of Pro(868)-Leu(967) (a functionally important region in AE1) that are highly sensitive to cysteine substitution. Our findings suggest that the C-terminal transmembrane region of NBCe1-A is tightly folded with unique structural and functional features that differ from AE1.

Topological location and structural importance of the NBCe1-A residues mutated in proximal renal tubular acidosis.

NBCe1-A electrogenically cotransports Na(+) and HCO(3)(-) across the basolateral membrane of renal proximal tubule cells. Eight missense mutations and 3 nonsense mutations in NBCe1-A cause severe proximal renal tubular acidosis (pRTA). In this study, the topologic properties and structural importance of the 8 endogenous residues mutated in pRTA and the in situ topology of NBCe1-A were examined by the substituted cysteine accessibility method. Of the 55 analyzed individually introduced cysteines, 8 were labeled with both membrane permeant (biotin maleimide (BM)) and impermeant (2-((5(6)-tetramethylrhodamine)carboxylamino)ethyl methanethiosulfonate (MTS-TAMRA)) sulfhydryl reagents, 4 with only BM, and 3 with only MTS-TAMRA. The location of the labeled and unlabeled introduced cysteines clearly indicates that the transmembrane region of NBCe1-A contains 14 transmembrane segments (TMs). In this in situ based NBCe1-A topology, residues mutated in pRTA (pRTA residues) are assigned as: Ser(427), TM1; Thr(485) and Gly(486), TM3; Arg(510) and Leu(522), TM4; Ala(799), TM10; and Arg(881), TM12. Substitution of pRTA residues with cysteines impaired the membrane trafficking of R510C and R881C, the remaining membrane-processed constructs had various impaired transport function. Surprisingly, none of the membrane-processed constructs was accessible to labeling with BM and MTS-TAMRA, nor were they functionally sensitive to the inhibition by (2-aminoethyl)methanethiosulfonate. Functional analysis of Thr(485) with different amino acid substitutions indicated it resides in a unique region important for NBCe1-A function. Our findings demonstrate that the pRTA residues in NBCe1-A are buried in the protein complex/lipid bilayer where they perform important structural roles.

Transport of N-acetyl-S-(1,2-dichlorovinyl)-L-cysteine, a metabolite of trichloroethylene, by mouse multidrug resistance associated protein 2 (Mrp2).

N-acetyl-S-(1,2-dichlorovinyl)-l-cysteine (Ac-DCVC) and S-(1,2-dichlorovinyl)-l-cysteine (DCVC) are the glutathione conjugation pathway metabolites of a common industrial contaminant and potent nephrotoxicant trichloroethylene (TCE). Ac-DCVC and DCVC are accumulated in the renal proximal tubule where they may be secreted into the urine by an unknown apical transporter(s). In this study, we explored the hypothesis that the apical transport of Ac-DCVC and/or DCVC may be mediated by the multidrug resistance associated protein 2 (Mrp2, ABCC2), which is known to mediate proximal tubular apical ATP-dependent transport of glutathione and numerous xenobiotics and endogenous substances conjugated with glutathione. Transport experiments using membrane vesicles prepared from mouse proximal tubule derived cells expressing mouse Mrp2 utilizing ATPase assay and direct measurements of Ac-DCVC/DCVC using liquid chromatography/tandem mass-spectrometry (LC/MS/MS) demonstrated that mouse Mrp2 mediates ATP-dependent transport of Ac-DCVC. Expression of mouse Mrp2 antisense mRNA significantly inhibited the vectorial basolateral to apical transport of Ac-DCVC but not DCVC in mouse proximal tubule derived cells endogenously expressing mouse Mrp2. The results suggest that Mrp2 may be involved in the renal secretion of Ac-DCVC.