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Am J Physiol Renal Physiol 293: F360-F370, 2007. First published April 18, 2007; doi:10.1152/ajprenal.00102.2007
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Molecular identification and functional characterization of rabbit MATE1 and MATE2-K

Xiaohong Zhang,1 Nathan J. Cherrington,2 and Stephen H. Wright1

Departments of 1Physiology and 2Pharmacology and Toxicology, University of Arizona, Tucson, Arizona

Submitted 27 February 2007 ; accepted in final form 8 April 2007


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
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An electroneutral organic cation (OC)/proton exchanger in the apical membrane of proximal tubules mediates the final step of renal OC excretion. Two members of the multidrug and toxin extrusion family, MATE1 and MATE2-K, were recently identified in human and rodent kidney and proposed to be the molecular basis of renal OC/H+ exchange. To take advantage of the comparative value of the large database on the kinetic and selectivity characteristics of OC/H+ exchange that exists for rabbit kidney, we cloned rbMATE1 and rbMATE2-K. The rabbit homologs have 75% (MATE1) and 74% (MATE2-K) amino acid identity to their human counterparts (and 51% identity with each other). rbMATE1 and rbMATE2-K exhibited H+ gradient-dependent uptake and efflux of tetraethylammonium (TEA) when expressed in Chinese hamster ovary cells. Both transporters displayed similar affinities for selected compounds [IC50 values within 2-fold for TEA, 1-methyl-4-phenylpyridinium, and quinidine] and very different affinities for others (IC50 values differing by 8- to 80-fold for choline and cimetidine, respectively). These results indicate that rbMATE1 and rbMATE2-K are multispecific OC/H+ exchangers with similar, but distinct, functional characteristics. Overall, the selectivity of MATE1 and MATE2-K correlated closely with that observed in rabbit renal brush-border membrane vesicles.

organic cation; transport; tetraethylammonium; proximal tubule; kidney

RENAL EXCRETION OF organic cations (OCs) plays a critical role in clearing the body of a diverse array of cationic metabolites of both exogenous and endogenous origin. The principal site of renal OC secretion is the proximal tubule (33), and the current model of the cellular basis of this process is little changed from the one proposed 25 years ago by Holohan and Ross (11), i.e., transepithelial OC transport reflects the activity of a basolateral entry step that is dominated by electrogenic facilitated diffusion, followed by an apical exit step in the tubular filtrate that is dominated by OC/H+ exchange (12). Insight into the molecular characteristics of the basolateral entry step followed the cloning of an electrogenic OC transporter from rat kidney [OCT1 (7)], and, in short order, by the cloning of two related isoforms [OCT2 (29) and OCT3 (16)], all of which are members of the larger SLC22A family of solute carriers (9). The molecular identity of apical OC/H+ exchange has, however, remained elusive.

The "physiological fingerprint" of apical OC transport, i.e., the carrier-mediated exchange of OC for H+, arose from studies using isolated renal brush-border membranes vesicles (BBMV) from human, rabbit, rat, dog, chicken, and snake kidneys (33, 44). The process supports the electroneutral, and obligatory, exchange of one OC+ for one H+ [46, 47], and permits OCs to leave renal proximal tubule (RPT) cells and develop a luminal concentration as large as or larger than (depending on the size of the transluminal H+ gradient) that in the cytoplasm, resulting in net transepithelial secretion. OC/H+ exchange is the active step in renal OC secretion because it depends on the displacement of H+ away from electrochemical equilibrium, a state maintained through the activity in the luminal membrane of the Na+/H+ exchanger and, to a lesser extent, a V-type H-ATPase (44). The first molecular candidate for an OC transporter capable of supporting OC/H+ exchange was a novel member of the SLC22A family of transporters, OCTN1, because of the following two key characteristics: 1) it is expressed in the apical membrane of RPT (37), and 2) it supports a transport of the prototypic OC, tetraethylammonium (TEA), that is trans-stimulated by an oppositely oriented H+ gradient (53). However, OCTN1 has recently been shown to support the Na+-dependent transport of the fungal antioxidant ergothioneine (6) with much greater catalytic efficiency than for OCs like TEA, and this fact, combined with the comparatively low level of expression of OCTN1 in the kidney (27) and a kinetic, selectivity, and tissue distribution profile that differs markedly from that of physiologically determined OC/H+ exchange (44), make it unlikely that OCTN1 is the predominant OC transporter in the apical membrane of RPT cells.

The recent isolation from human and rodent kidney of members of the multidrug and toxin extrusion (MATE) family of transporters (31) (from the multidrug/oligosaccharidyl-lipid/polysaccharide, or MOP, superfamily; see Ref. 13) introduced two new candidates for the molecular identity of the apical OC/H+ exchanger of RPT cells, i.e., MATE1 and MATE2. The MATEs support a transport of TEA, 1-methyl-4-phenylpyridinium (MPP), and other cations typically associated with renal OC/H+ exchange that is trans-stimulated by oppositely oriented H+ concentration ([H+]) gradients (21, 31). In addition, the tissue and cellular distribution of MATE1 and MATE2 is consistent with the functional distribution of OC/H+ exchange that is, in turn, associated with renal OC secretion. In other words, MATE1 is predominantly expressed in the kidney and liver and is effectively restricted to the apical membrane of RPT cells and the canalicular membrane of liver cells (31); and MATE2 (or its splice variant, MATE2-K) is predominantly expressed in the apical membrane of RPT cells (21), and these profiles of activity and distribution are consistent with the contention that MATE1 and/or MATE2/2-K serve the role of OC/H+ exchange in the process of renal (and hepatic) OC secretion.

Despite the fact that MATE1 and MATE2-K display properties similar to those of "renal OC/H+ exchange," the extent to which either of these proteins contributes to renal OC transport is unclear. Recalling that OCTN1 was initially proposed as contributing significantly to this process (53), it is evident that a more detailed assessment of the functional properties of MATE1 and MATE2/2-K is required if their contribution to the renal handling of OCs is to be understood. In the present paper, we examine the properties of the rabbit orthologs of these two transporters, thereby permitting comparison of their functional properties with that detailed in the database we (19, 46–49, 51) and others (22–25) have generated on the properties of OC/H+ exchange as expressed in rabbit renal BBMV. We found that rabbit (rb) MATE1 and rbMATE2-K both support the [H+]-sensitive transport of TEA, MPP, and cimetidine. Furthermore, whereas the two transporters discriminate markedly between selected substrates, they have an overlapping selectivity for most OCs tested. Finally, comparison of the selectivity of rbMATE1 and rbMATE2-K with that of OC/H+ exchange in rabbit renal BBMV suggests that the latter could reflect that combined activity of these processes.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
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Chemicals. [14C]TEA (55 Ci/mmol) and [3H]MPP (85 Ci/mmol) were purchased from American Radiochemicals; [3H]cimetidine (12.7 Ci/mmol) was purchased from Amersham Biosciences. Other chemicals were typically of the highest grade available and, unless otherwise indicated, were purchased from Sigma Chemical.

Cloning of rbMATE1 and rbMATE2-K. To clone rbMATE1, degenerate sense and antisense oligonucleotide primers were designed from consensus sequences of the human, rat, and mouse orthologs of MATE1, resulting in 5'-GTCAGAACYCCTCAGATAAG-3'(sense) and 5'-GATGCCRCCRATGTCAC-3'(antisense). For first-strand synthesis, 0.5 µg of rabbit kidney poly(A)+ RNA was reverse transcribed using Moloney murine leukemia virus RT H– at 37°C for 20 min. After incubation at 70°C for 15 min, RNase H was added, and the reactions were kept again at 37°C for 20 min. The RT reaction (2 µl) was used directly for amplification. The PCR solution was assembled and heated at 94°C for 3 min before Pfu DNA polymerase was added. Subsequently, PCR was performed using the following profile: 94°C for 1 min, 54°C for 1 min, and 72°C for 2 min for 35 cycles. The last cycle was terminated after an elongation time of 7 min. A 420-bp RT-PCR product was gel purified and sequenced. To obtain the remaining 5'- and 3'-portions of the rabbit kidney MATE1 sequence, the PCR-based FirstChoice RLM-RACE Kit (Ambion) was used. Briefly, two gene-specific primers, [5'-GGAAGCACACCTGCATCTTG-3' (sense) and 5'-GAGATTCCTGATGAACGTGG-3' (antisense)], were designed from the partial rbMATE1 sequence. The 5'- and 3'-RACE reactions were primed with an internal gene-specific primer and an adapter primer. The PCR reactions were performed according to the manufacturer's protocols. The RACE products were gel purified and subcloned into the mammalian expression vector pcDNA5/FRT/V5-His (Invitrogen). The two overlapped RACE products were digested by BamHI/EcoRI and then ligated to form a full-length cDNA for rbMATE1. The same strategy was applied for cloning rbMATE2-K: the degenerate primers 5'-CTGCTGCTCTTCAGRCAGGAC-3'(sense) and 5'-ACRATGGCTCCAACCTTCTG-3'(antisense) were designed based upon published human, mouse, and rat sequences; gene-specific primers 5'-TCGTCTGGCCACAAGTCCTT-3' (sense; for 3'-RACE) and 5'-TGCACAAACTGGGAGATGGTG-3' (antisense; for 5'-RACE) were used to obtain the remaining sequences; and the resulting products were ligated to form the full-length cDNA of rbMATE2-K. Both rbMATE1 and rbMATE2-K sequences were confirmed in the sense and antisense strands by an Applied Biosystems model 373A sequencing unit at the University of Arizona sequencing facility.

Branched DNA signal amplification assay. MATE1 and MATE2-K mRNA was measured using the branched DNA (bDNA) assay (Quantigene bDNA signal amplification kit) with modifications (8). Rabbit MATE gene sequences of interest were derived from the present study. Multiple oligonucleotide probes (containing capture, label, and blocker probes) specific to a single mRNA transcript (i.e., MATE1 or MATE2-K) were designed using Probe Designer software version 1.0, with a melting temperature of ~63°C, enabling hybridization conditions to be held constant (i.e., 53°C) during each hybridization step and for each probe set. Every probe developed in Probe Designer was submitted to the National Center for Biotechnological Information (Bethesda, MD) for nucleotide comparison by the basic logarithmic alignment search tool (BLASTn) to ensure minimal cross-reactivity with other known rabbit sequences and expressed sequence tags. The nucleotide sequence and function of these probes are given in Table 1. Total RNA (1 µg/µl; 10 µl/well) was added to each well of a 96-well plate containing 50 µl capture hybridization buffer and 50 µl of the desired probe set diluted in lysis buffer per the manufacturer's protocol. For each gene, total RNA was allowed to hybridize to the probe set overnight at 53°C. Subsequent hybridization steps were carried out according to the manufacturer's protocol, and luminescence was measured with a Quantiplex 320 bDNA luminometer interfaced with Quantiplex Data Management software version 5.02 for analysis of luminescence from the 96-well plates. The luminescence for each well is reported as relative light units per 10 µg of total RNA.


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  		Table 1. Oligonucleotide probes generated for analysis of MATE expression by branched DNA signal amplification

 
Cell culture and transfection. Chinese hamster ovary (CHO) cells were grown at 37°C in a humidified atmosphere (5% CO2) in plastic culture flasks. The medium was Kaighn's modified Ham's F-12 medium supplemented with 10% FCS. Cultures were split every 3 days. Cells were transfected via electroporation (ECM 630; BTX, San Diego, CA) with a plasmid containing the cDNA sequence for the desired construct. Briefly, cells were transfected with 10 µg of DNA at 260 volts (time constant of ~25 ms) and seeded into 12-well plates at 320,000 cells/well. Uptake was typically measured 48 h after transfection, which was sufficient for the cells to reach confluence.

Measurement of transport. CHO cells were incubated at room temperature (~25°C) in Waymouth buffer (in mM: 135 NaCl, 13 HEPES-NaOH, pH 7.4, 28 D-glucose, 5 KCl, 1.2 MgCl2, 2.5 CaCl2, and 0.8 MgSO4) to which labeled substrate and appropriate test agents were added. Uptake was stopped by rinsing the cells three times with 2 ml of ice-cold Waymouth buffer. The cells were then solubilized in 400 µl of 0.5 N NaOH with 1% (vol/vol) SDS, and the extract was subsequently neutralized with 200 µl of 1 N HCl. Accumulated radioactivity was determined by liquid scintillation spectrometry. Rates of uptake are expressed as millimoles per square centimeter of nominal cell surface of the confluent monolayer.

Immunocytochemistry. CHO cells were electroporated with plasmid DNA containing a COOH-terminal V5 epitope-tagged transporter and seeded on cover slips. Immunocytochemistry was generally performed on a confluent monolayer 24 h after plating. Cells were fixed in ice-cold 100% methanol for 20 min (permeabilized cells) and washed with PBS (in mM: 137 NaCl, 2.7 KCl, 8.0 Na2HPO4, and 1.5 KH2PO4, pH 8). For nonpermeabilized cells, cells were fixed in 2% paraformaldehyde (in PBS) for 2 min and then washed with 0.3 M glycine. All washes were done in triplicate. The permeabilized and nonpermeabilized cells were then incubated for 1 h with anti-V5 antibody (Invitrogen) diluted 1:500 in PBS. The cells were then washed with PBS and incubated for 1 h in the dark with FITC-conjugated goat anti-mouse antibody (Molecular Probes) diluted 1:1,000 in PBS. To visualize the nuclei, cells were treated with propidium iodide for 10 min.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Molecular properties and secondary structure of rabbit MATE1 and MATE2-K. A PCR-based cloning approach was used to clone the rabbit MATE1 and MATE2-K, as described previously (54). Briefly, PCR amplification using oligonucleotide primers designed from consensus sequences of human, mouse, and rat MATE1 generated a 420-bp RT-PCR product of rbMATE1. PCR-based 5'- and 3'-RACE systems were then used to obtain the remaining 5'- and 3'-portions of the rbMATE1 sequence. The full-length cDNA for rbMATE1 obtained using this approach was 2,478 bp and contained a 20-bp 5'-untranslated region, a 1,707-bp open reading frame, and a 471-bp 3'-untranslated region (GenBank accession no. EF120627). Our effort to clone the rabbit ortholog of MATE2 resulted in a 546-bp RT-PCR product that led to the cloning of a full-length cDNA that was 1,963 bp and contained a 16-bp 5'-untranslated region, a 1,806-bp open reading frame, and a 126-bp 3'-untranslated region (GenBank accession no. EF121852). As discussed below, this sequence proved to be the ortholog of human MATE2-K, rather than MATE2. The rabbit isoforms have 75% (MATE1) and 74% (MATE2-K) amino acid identity to their human counterparts (and 51% identity with each other).

The original report of the cloning of human MATE1 suggested that its secondary structure contained 12 transmembrane helices (TMHs) with internal NH2- and COOH-termini (31). However, our assessment of the putative secondary structure of the rabbit and human (and rat) orthologs of MATE1 (Fig. 1) predicted 13 TMHs for both proteins (HMMTOP; see Ref 40 and http://www.enzim.hu/hmmtop/html/document.html), with an intracellular NH2 terminus and an extracellular COOH terminus. Interestingly, HMMTOP predicts 12 TMHs for mouse MATE1 (Fig. 1), consistent with the previous report for the mouse MATE1 (10). Comparison of the multiple alignment of human, rabbit, rat, and mouse MATE1 amino acid sequences reveals close homology except at the COOH terminus where differences include the absence of the final 29 amino acid residues found in human, rabbit, and rat MATE1. The carboxy end of human, rabbit, and rat MATE1 appears to include a comparatively short 13th TMH and an extracellular COOH terminus, whereas mMATE1 lacks this last TMH and, consequently, ends with a long cytoplasmic COOH terminus (Fig. 1).


Figure 1
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  		Fig. 1. Sequence alignment (ClustalW; http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html) for the rabbit, human, rat, and mouse orthologs of multidrug and toxin extrusion family (MATE) 1. Highlighted in yellow are the predicted transmembrane helices (TMHs) as determined using HMMTOP (http://www.enzim.hu/hmmtop/html/document.html). The depicted topology (TOPO2; http://www.sacs.ucsf.edu/TOPO/) highlights the 13th predicted TMH common to rabbit, human, and rat MATE1 that is absent in the mouse.

 
To test the prediction of an external COOH terminus for rbMATE1 vs. an internal COOH terminus for mMATE1, we added the V5-epitope to the COOH terminal end of both transporters and then tested the accessibility of the COOH terminus to anti-v5 antibody in permeabilized and nonpermeabilized CHO cells (Fig. 2). Both constructs supported TEA transport (data not shown), indicating that the presence of the V5 tag neither prevented trafficking of the protein to the membrane nor compromised its function. Mouse MATE1 was clearly expressed in the plasma membrane but was only evident in permeabilized CHO cells, whereas rabbit MATE1 was detected in the plasma membrane of both permeabilized and nonpermeabilized CHO cells (Fig. 2). These data support the hypothesis that the COOH terminus of rbMATE1 (and, presumably, that of the structurally homologous human and rat MATE1 orthologs) is located on the extracellular side of the plasma membrane, whereas the COOH terminus of mMATE1 is within the cytoplasm. These data also support the contention that rabbit (human and rat) MATE1 has 13 TMH compared with 12 for mMATE1.


Figure 2
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  		Fig. 2. Confocal microscopy of nonpermeabilized (D–F) and permeabilized (A–C) Chinese hamster ovary (CHO) cells expressing rabbit (rb) MATE1/V5 or mouse (m) MATE1/V5 protein. Cells were transiently transfected with constructs containing the V5 epitope on the COOH terminus of MATE1s. Nuclei were stained using propidium iodide (red), and MATE1 proteins were stained using the antibody against the COOH-terminal V5 tag (green).

 
The sequence alignments of the several MATE2/MATE2-K orthologs also revealed an interesting profile. The distinguishing difference between human MATE2 and its splice variant, MATE-K, as described by Inui and colleagues (21), is the absence in MATE2-K of a 36-amino acid sequence in the intracellular loop between putative TMHs four and five (Fig. 3). The rabbit sequence we isolated excludes the same 36-amino acid sequence (as does mouse MATE2; Fig. 3). Importantly, as noted below, the function of the cloned rabbit transporter corresponds closely with that of hMATE2-K, whereas the function of hMATE2 remains unclear (21). Consequently, to be consistent with current MATE nomenclature, we refer to this rabbit MATE2 homologue as "rbMATE2-K."


Figure 3
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  		Fig. 3. Sequence alignment (ClustalW; http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html) for the rabbit, human, and mouse orthologs of MATE2/2-K. Highlighted in yellow are the predicted TMHs as determined using HMMTOP [http://www.enzim.hu/hmmtop/html/document.html; note: the 13th TMH, although recognized in human (h) MATE2 by HMMTOP, was not recognized in hMATE2-K; our shading of TMH 13 in hMATE2-K therefore reflects the consensus view derived from several other TMH algorithms, including TMHMM, SPLIT 4.0, and ConPred II]. The depicted topology (TOPO2; http://www.sacs.ucsf.edu/TOPO) highlights (arrow) the site of insertion of the 36-amino acid sequence that distinguishes hMATE2 from hMATE2-K (and the rabbit and mouse orthologs as well).

 
MATE mRNA levels in rabbit kidney. Figure 4 compares the mRNA levels determined for MATE1 and MATE2-K in total RNA isolated from renal cortex collected from male and female rabbits. Two observations are worth emphasizing. First, mRNA for both transporters was expressed in rabbit renal cortical tissue. Second, there was no significant difference between the levels of expression of the mRNAs for either MATE1 or MATE2-K in male vs. female rabbit kidney. In addition, cortical expression of MATE2-K mRNA appeared to be approximately sevenfold higher than that of MATE1. Comparison of the signals obtained with the three probe sets used in the bDNA signal amplification method must be made cautiously because of possible differences in amplification efficiency. However, the presence of multiple hybridization sequences in the probe sets for each transporter reduces problems arising from variable hybridization efficiency (8) and supports the contention that the differences in signal levels between MATE1 and MATE2-K evident in Fig. 4 may reflect real differences in the relative expression of the two mRNA species.


Figure 4
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  		Fig. 4. Relative expression of mRNA for MATE1 and MATE2-K in rabbit renal cortical tissue. Cortical total RNA was isolated from 60- to 70-day-old male and female rabbits and analyzed by the branched DNA (bDNA) signal amplification assay for MATE mRNA content. Each bar represents the mean (expressed as relative light units; ±SE) determined from tissues isolated from 6 animals.

 
Functional characteristics of rbMATE1 and rbMATE2-K. We first determined the transport of a set of structurally diverse compounds (TEA, MPP, and cimetidine) in CHO cells transiently expressing rbMATE1 or rbMATE2-K (Fig. 5). At pH 8.5, all three compounds were transported by rbMATE1, exhibiting 10- to 12-fold greater uptake in rbMATE1-transfected cells compared with that in mock cells after 10 min of uptake. Rabbit MATE2-K exhibited similar levels of TEA and MPP transport but relatively weaker transport for cimetidine (only twofold higher than the control). These results suggest that rbMATE1 and rbMATE2-K are both multispecific transporters that display overlapping yet distinct selectivities for hydrophilic OCs.


Figure 5
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  		Fig. 5. Transport of tetraethylammonium (TEA; A), 1-methyl-4-phenylpyridinium (MPP; B), and cimetidine (CIM; C) mediated by rbMATE1 or rbMATE2-K. Uptakes (10 min) of 20 µM [14C]TEA or ~15 nM [3H]MPP or ~70 nM [3H]CIM were measured at pH 8.5 in triplicate wells of cells transiently transfected with either rbMATE1 or rbMATE2, with uptake expressed relative to uptake measured in wild-type CHO cells.

 
Next, we compared the kinetics of TEA and cimetidine interaction with rabbit MATE1 and MATE2-K. CHO cells transfected with one of these two transporters were exposed to [14C]TEA and increasing concentrations of unlabeled TEA (0–2.5 mM) or cimetidine (0–100 µM). Unlabeled TEA blocked the uptake of labeled TEA by both transporters with comparable profiles (Fig. 6A) that were adequately described by the Michaelis-Menten equation for the competitive interaction of labeled and unlabeled TEA (20):

Formula 1(1)
where J is the rate of [14C]TEA transport from a concentration of labeled substrate equal to [T*]; Jmax is the maximum rate of mediated TEA transport; Kt is the TEA concentration that resulted in half-maximal transport (Michaelis constant); [T] is the concentration of unlabeled TEA in the transport reaction, and C is a constant that represents the component of total [14C]TEA uptake that was not saturated (over the range of substrate concentrations tested) and presumably reflects the combined influence of diffusive flux, nonspecific binding, and/or incomplete rinsing of the cell layer. In three separate experiments, the Kt for TEA uptake was 160 ± 15 (SE) µM for rbMATE1 and 77 ± 12 µM for rbMATE2-K. In contrast to the similar apparent affinity of MATE1 and MATE2-K for TEA, these transporters had markedly different apparent affinities for cimetidine (Fig. 6B), as determined from the kinetics of competitive inhibition [5]:

Formula 2(2)
where Japp is defined as (Ki/Kt)Jmax, [I] is the concentration of the test agent (cimetidine, in this case), and Kapp is an apparent competitive inhibitory constant (Ki) for the test agent that is defined as Ki(1 + [T*]/Kt). When [T*] is <<Kt, Kapp {approx} Ki. The application of this equation carries with it the tacit assumption that the inhibitory interactions observed are competitive in nature and reflect binding of substrate and inhibitor at a common binding site or a set of mutually exclusive binding sites. Although this is likely the case for TEA and cimetidine, both of which are shown here to be substrates for MATE1 and MATE2-K (Fig. 5), we have not rigorously proven this to be the case for all compounds used in this study. Consequently, we will henceforth refer to the kinetic constants calculated through application of Eq. 2 as "IC50" values. In three separate experiments, the IC50 for cimetidine's interaction with rbMATE2-K was 80-fold higher than that for rbMATE1 (17.6 ± 2.5 and 0.22 ± 0.05 µM, respectively).


Figure 6
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  		Fig. 6. Inhibition of MATE1 or MATE2-K transport by TEA (A) or CIM (B). CHO cells were transfected with rbMATE1 or rbMATE2 and then 10-min (rbMATE1) or 5-min (rbMATE2) uptakes of [14C]TEA were measured at pH 8.5 in the presence of increasing concentrations of unlabeled TEA or CIM. The kinetics of total substrate accumulation were adequately described by a relationship comprised of a single mediated uptake process and a parallel first-order process. Each point is the mean ± SE of uptakes measured in 3 wells in a single representative experiment.

 
pH dependence of rbMATE1 and rbMATE2-K transport. Human, mouse, and rat MATE1 (31, 38, 39) and human MATE2-K (21) have been suggested to function as OC/proton exchangers. To determine the pH sensitivity of rbMATE1 and rbMATE2-K, we first examined the effect of an inwardly directed [H+] gradient on the efflux of [14C]TEA from CHO cells that expressed one of these two transporters. When cells expressing either MATE1 or MATE2-K were preloaded with [14C]TEA at pH 8.5, exposure to a buffer at pH 6.0 markedly stimulated efflux (compared with efflux in a control buffer of pH 8.5; Fig. 7), consistent with TEA/H+ exchange. We then examined the effect of extracellular [H+] on uptake of TEA. Figure 8 shows that increasing concentrations of extracellular H+ produced a hyperbolic inhibition of TEA transport in CHO cells expressing rbMATE1 or rbMATE2-K. In three separate experiments, the IC50 values for inhibition by H+ of TEA transport were 17.9 ± 3.1 and 8.9 ± 0.9 nM for rbMATE1 and rbMATE2-K, respectively.


Figure 7
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  		Fig. 7. pH-dependent efflux of TEA by rbMATE1 (A) or rbMATE2 (B). CHO cells expressing rbMATE1 or rbMATE2 were incubated with Waymouth buffer (pH 8.5) containing 20 µM [14C]TEA for 10 min and then the cells were transferred to buffer of the indicated pH (time 0) and incubated for another 10 min, after which the remaining radioactivity was assayed. Each point represents the mean uptake ± SE measured in 3 separate experiments (*P < 0.05, Student's t-test).

 

Figure 8
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  		Fig. 8. Effect of extracellular H+ concentration ([H+]) on transport of TEA by rbMATE1 (A) and rbMATE2 (B). Uptakes (10 min) of 20 µM [14C]TEA were measured in CHO cells transfected with vector only (mock; {circ}) or rbMATE1 or rbMATE2 cDNA (bullet) at the indicated [H+]. Data points are means ± SE of uptakes measured in 3 wells from single representative transfections.

 
Substrate specificity of rbMATE1 and rbMATE2-K. The cloning of the rabbit orthologs of MATE1 and MATE2-K offered a unique opportunity to compare the selectivity characteristics of these individual proteins with those of the OC/H+ exchange process as expressed in native renal membranes. In previous studies, we examined in some detail the molecular determinants that influence substrate binding to (49, 51) and transport by (48) the OC/H+ exchanger of BBMV isolated from rabbit kidney. From the data reported in these previous studies, we selected nine compounds of widely different structures that display IC50 interactions with the OC/H+ exchanger that vary by >1,000-fold (Table 2) and tested them as inhibitors of TEA transport in CHO cells that expressed rbMATE1 or rbMATE2-K (Table 2). Figure 9A presents the IC50 values for this set of OCs as inhibitors of OC/H+ exchange in rabbit renal BBMV compared with their IC50 values as inhibitors of TEA transport mediated by rbMATE1 or rbMATE2-K. Two observations offer themselves. First, there was a clear correlation between the affinity of the test compounds for MATE1 and MATE2-K and their affinity for OC/H+ exchange, as expressed in renal BBMV (r values of 0.84 and 0.94 for MATE1 and MATE2-K, respectively). Second, whereas most of the test compounds displayed a similar affinity for MATE1 and MATE2-K, selected compounds discriminated markedly between the two transporters (Fig. 9A, inset, and 9B). Cimetidine showed the most marked difference in apparent affinity for the two transporters, with an IC50 80 times higher for rbMATE2-K than for rbMATE1 (17.6 vs. 0.22 µM; Table 2). Choline, on the other hand, displayed a substantially higher affinity for rbMATE2-K (IC50 = 657 µM) than for rbMATE1 (IC50 = 5,324 µM; Table 2). It should be noted that rbMATE1 and rbMATE2-K both displayed a higher apparent affinity for N'-methylnicotinamide (NMN) than did the OC/H+ exchange activity of renal BBMV noted in our earlier work. The basis of this apparent discrepancy is not clear, but it underscores the caution that should be used when making quantitative comparisons between kinetic values determined in native membrane preparations with those obtained for cloned transporters expressed in different heterologous expression systems (44).


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  		Table 2. IC50 values for inhibition of [14C]TEA transport (20 µM) mediated by rbMATE1, rbMATE2-K (as expressed in CHO cells), or by OC/H+ exchange in BBMV isolated from rabbit renal cortex

 

Figure 9
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  		Fig. 9. A: relationship between the IC50 values measured for 10 test agents as inhibitors of TEA/H+ exchange in brush-border membrane vesicles (BBMV) isolated from rabbit renal cortex (abscissa; data taken from Refs. 46, 47, 49, 51) and the IC50 values measured for the test agents as inhibitors of [14C]TEA transport in CHO cells that expresses either rbMATE1 (bullet) or rbMATE2-K ({circ}; ordinate; data from Table 2). The solid line is the theoretical line of identity for IC50 values for renal TEA/H+ exchange in BBMV and corresponding values for MATE1 and MATE2-K. A, inset: relationship between IC50 values for rbMATE1 and rbMATE2-K. Solid line is the "line of identity" for the IC50 values of the transporters. B: ratio of rbMATE2-K and rbMATE1 IC50 values for the 10 test agents.

 

   DISCUSSION
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 ABSTRACT
 METHODS
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It is generally accepted that apical OC secretion, with its hallmark multispecificity (41, 49, 51), involves the mediated exchange of H+ for OCs (44). The apical membrane of RPT contains several transporters that accept OCs as substrates, including OCTN1, OCTN2, and plasma membrane monoamine transporter (4, 17, 44), as well as several processes that have been physiologically characterized but for which no molecular candidates have been identified (e.g., electrogenic choline transport and guanidine/H+ exchange; see Refs. 23 and 50). However, these are all either too restrictive in their selectivity or operate in a mode other than the obligatory exchange of OC for H+. Nevertheless, the apical exit step in renal OC secretion could involve contributions from several distinct processes, similar to basolateral OC entry with its involvement, in at least some species, of multiple OCTs (14, 15, 45). The recently described multidrug and toxin extrusion transporters MATE1 (31) and MATE2-K (21) are the strongest candidates yet for the molecular identity of the exit step in renal OC secretion, and the results presented here support the hypothesis that MATE1 and MATE2-K may both contribute significantly to the OC/H+ exchange observed in native renal membranes.

OC/H+ exchange activity has been identified in isolated luminal membrane vesicles from every mammalian species examined, including dog, rat, rabbit, and human (e.g., Refs. 12, 32, 36, 46), and in all nonmammalian species examined, including birds (42, 43) and snakes (3). Following its description in 1981 by Holohan and Ross (12), substantial interest in this process reflected the conclusion that apical OC/H+ exchange is both the rate-limiting and active step in renal OC secretion (25, 34, 47). Efforts to clone a molecular candidate for this process met with little success until the recent report of the first eukaryotic members of the MOP superfamily of transporters (31), namely the multidrug and toxicant extruders MATE1 and MATE2. These proteins were isolated from human and mouse kidney (31) and were localized to the apical membrane of RPT and the canalicular membrane of hepatocytes, sites known to express multispecific OC/H+ exchange activity (e.g., Refs. 26 and 46). Of particular importance is the fact that MATE1 supports a pH-sensitive transport of TEA that is stimulated by oppositely oriented [H+] gradients (31). MATE2-K, a splice variant of human MATE2 expressed predominantly in the apical membrane of RPT cells, also supports multispecific, H+ gradient-stimulated OC transport (21). Importantly, Tsuda and colleagues (39) showed that membrane vesicles isolated from HEK293 cells that express rMATE1 support concentrative TEA transport in the presence of an oppositely oriented [H+] gradient, thereby providing unambiguous evidence that MATEs operate as coupled OC/H+ exchangers.

Despite the above-noted characteristics of MATE1 and MATE2-K, it is not known whether either contributes significantly to total OC flux across the apical membrane of RPT; one or more heretofore undescribed transporters could prove to play a more important role. In the absence of appropriate genetic models to test the influence of these transporters on OC secretion, we sought to correlate the quantitative characteristics of heterologously expressed MATE-mediated transport to the known profile of OC/H+ exchange in native renal membranes as a means of testing the hypothesis that MATE1 and/or MATE2-K contribute to apical OC transport. Unfortunately, information on human multispecific renal OC/H+ exchange is limited to the demonstration that BBMV from human kidney support [H+] gradient-dependent transport of TEA and NMN (2, 32). Consequently, we elected to clone the rabbit orthologs of MATE1 and MATE2-K and then compare the kinetic and selectivity characteristics of these processes with those observed in studies of multispecific OC/H+ exchange in rabbit renal BBMV.

Examination of the sequences of cloned rabbit MATE1 and MATE2-K, which showed marked structural and functional homology with their human orthologs (Figs. 1 and 3), provided interesting insights concerning structure/function relationships for MATE transporters. In the initial description of human MATE1 (31), the authors suggested that it contains 12 putative TMH with cytoplasmic NH2 and COOH termini, structural characteristics common to prokaryotic and plant members of the MATE family (30). However, our assessment of predicted TMHs for the human, rabbit, rat, and mouse MATE1 amino acid sequences [using the web-based algorithms TMHMM (18, 35), HMMTOP (40), and ConPred II (1, 52)] consistently resulted in 13 putative TMHs for the human, rabbit, and rat orthologs of MATE1 compared with 12 for mouse MATE1 (Fig. 1). The difference is the presence of an extended, hydrophobic sequence at the COOH terminus in the former three species that is predicted to form a terminal TMH. In addition, all three known orthologs of MATE2/2-K (i.e., human, rabbit, and mouse) also appear to end with a COOH-terminal TMH. This 13th predicted TMH results in placing the COOH terminus of MATE proteins at the extracellular face of the membrane. Immunocytochemical localization of transfected rbMATE1 and mouse MATE1 in the plasma membrane of CHO cells (Fig. 2) confirmed that rabbit (and, presumably, human) MATE1 has an extracellular COOH terminus, whereas the COOH terminus is intracellular in mouse MATE1. An intriguing conclusion supported by these observations is that the presence of a COOH-terminal 13th THM does not profoundly influence the functional characteristics of a protein that, in other family members, appears to display a highly conserved helical organization comprised of 12 TMHs.

The interaction of H+ and TEA at the OC/H+ exchanger of rabbit renal BBMV has been examined in some detail (e.g., Refs. 19, 46, 47, 51), and is a starting point for the comparison of renal OC/H+ exchange with the activity of MATEs. An inwardly directed [H+] gradient stimulated TEA efflux from CHO cells that expressed rabbit MATE1 or MATE2-K (Fig. 7), a characteristic seen with other MATE1 and MATE2-K orthologs (10, 21, 28, 31, 38, 39) and one consistent with the fact that trans-H+ increases the turnover of the OC/H+ exchange process in rabbit BBMV (47). We also observed that, as seen with both the human (21, 31) and rodent (10, 28) orthologs, preloading cells with H+ (using the ammonia-pulse method) stimulated TEA uptake in cells expressing rabbit MATE1 and MATE2-K (Zhang, unpublished observations). H+ is a competitive inhibitor of OC transport via OC/H+ exchange (47), and in rabbit BBMV the Ki for H+ inhibition of TEA/H+ exchange is 30 nM (47). Consistent with this observation, TEA transport mediated by rabbit MATE1 and MATE2-K showed a marked, and similar, sensitivity to ambient pH, with IC50 values for H+ inhibition of TEA transport of 18 and 9 nM, respectively (Fig. 8). The apparent affinities for TEA of rbMATE1 and rbMATE2-K also proved to be similar (at 160 and 77 µM, respectively), and corresponded rather closely to the range of values for the Kt of TEA transport (100–200 µM) observed in our studies on TEA transport in rabbit renal BBMV (19, 46, 47, 51). The qualitative and quantitative similarity between the interactions of TEA and H+ with both MATE transporters and with OC/H+ exchange activity of native renal membranes supports the contention that MATEs play a role in apical OC transport.

Comparison of the selectivity characteristics of cloned proteins with those expressed in native renal tubules has provided a powerful means to assess the fractional contribution of basolateral transport proteins to transport activity in the kidney (45, 54, 55). For example, the comparatively high affinity of OCT2 for cimetidine, compared with OCT1, was used as a means to demonstrate that basolateral TEA transport in the S2 segment of rabbit RPT is dominated by OCT2, whereas OCT1's high affinity for tyramine was used to implicate OCT1 as the principal OC transporter in the S1 segment of rabbit RPT (45, 54). [This latter observation belies that fact that the expression of the mRNA for OCT1 in outer cortex of rabbit kidney is <10% of that for OCT2 (45), thus prompting caution about drawing conclusions from the comparatively high expression of MATE2-K mRNA, compared with MATE1, in rabbit renal cortex (Fig. 2).] For an apical transporter like MATE1 and MATE2-K, we took the approach that the comparable comparison with that described above, i.e., selectivity of individual rabbit OCT orthologs in heterologous expression systems vs. the selectivity of total OC uptake in isolated rabbit tubule segments, is the comparison of the selectivity of rabbit MATEs with that of OC/H+ exchange in rabbit renal BBMV. For most of the test compounds used in this study, rabbit MATE1 and MATE2-K displayed very similar IC50 values (Fig. 9A, inset) that, in turn, correlated closely with the IC50 values these agents displayed when inhibiting OC/H+ exchange in rabbit renal BBMV (Fig. 9A). Two compounds, cimetidine and choline, did show marked differences in their interaction with MATE1 and MATE2-K, differing in IC50 values by factors of 8 (choline) to 80 (cimetidine; Fig. 9B). Interestingly, the IC50 values for inhibition of OC/H+ exchange in renal BBMV produced by these two compounds lie between the extremes observed for the two individual MATEs. These data, in conjunction with the substantial level of mRNA expression for both transporters in rabbit renal cortex (and the fact that both homologs have been shown to be expressed in the apical membrane of human RPT; see Refs. 21 and 31), support the hypotheses that apical OC/H+ exchange involves the combined activity of MATE1 and MATE2-K.

In conclusion, rabbit kidney expresses orthologs to the OC transporters expressed in the apical membrane of human RPT cells, MATE1 and MATE2-K. Both the rabbit and human MATE proteins appear to have secondary structures comprised of 13 TMH and an extracellular COOH terminus. These transporters support the pH gradient-sensitive transport of a structurally diverse range of OCs and have kinetic and selectivity characteristics that correlate closely with those displayed by OC/H+ exchange as expressed in native renal brush-border membranes. These data suggest that the apical exit step in the multispecific OC secretion of RPT cells involves the combined activity of MATE1 and MATE2-K.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
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 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants DK-058251 and ES-06694.


   ACKNOWLEDGMENTS
 
We thank Dr. R. M. Pelis for helpful discussions.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. H. Wright, Dept. of Physiology, College of Medicine, Univ. of Arizona, Tucson, AZ 85724 (e-mail: shwright{at}u.arizona.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.


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