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Institute of Anatomy and Cell Biology, University of Wezburg, Wezburg, Germany
Abstract
The affinity of corticosterone to organic cation transporters (OCTs) is subtype- and species-dependent. For example, the IC50 values for corticosterone inhibition of cation uptake by transporters rOCT1 and rOCT2 are 150 and 4 e, respectively. By introducing domains and amino acids from rOCT2 into rOCT1, we found that the exchange of three amino acids in the presumed 10th transmembrane helix is sufficient to increase the affinity of rOCT1 for corticosterone to that of rOCT2. Replacement of these amino acids in rOCT2 decreased the affinity for corticosterone. These amino acids (Ala443, Leu447, and Gln448 in rOCT1 and Ile443, Tyr447, and Glu448 in rOCT2) are probably located within the substrate binding region because in rOCT1 mutants, the Km values for uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) were decreased in parallel with a decrease of the IC50 values for the inhibition of cation uptake by corticosterone. In mutant rOCT1(L447Y/Q448E), the IC50 value for the inhibition of [3H]MPP (0.1 e) uptake by corticosterone (24 ± 4 e) was significantly higher compared with the IC50 value for inhibition of [14C]TEA (10 e) uptake (5.3 ± 1.7 e). This finding suggests an allosteric interaction between transported cation and corticosterone. Because this substrate-specific effect cannot be explained by differential replacement of corticosterone by MPP versus TEA and was observed after point mutations within the presumed substrate region, the data suggest that MPP or TEA bind to the substrate binding region simultaneously with corticosterone and cause a short-range allosteric effect on the corticosterone binding site.
The polyspecific organic cation transporters OCT1 to -3 (SLC22A1eC3) are involved in the elimination and distribution of drugs, environmental toxins, and endogenous organic cations, including monoamine neurotransmitters (Koepsell et al., 2003; Koepsell, 2004). Together with the organic anion transporters and the sodium carnitine cotransporters OCTN1 and OCTN2, the OCTs belong to the SLC22 transporter family, which is a member of the major facilitator superfamily (Pao et al., 1998; Koepsell et al., 2003). The three OCT subtypes have overlapping substrate specificities but differ in tissue distribution, regulation, and selectivity for substrates and inhibitors (Koepsell et al., 2003). For example, steroid hormones inhibit organic cation transport by the three OCT subtypes, with different affinities showing distinct species difference (Koepsell et al., 2003). Steroids are also involved in the long-term regulation of OCT2 but not of OCT1 (Urakami et al., 2000; Shu et al., 2001). For the inhibition of OCTs by corticosterone, IC50 values are 10 e (human OCT1), 30 e (human OCT2), 0.2 e (human OCT3, also called EMT), 150 e (rat OCT1), 4 e (rat OCT2), and 5 e (rat OCT3) (Gredemann et al., 1998; Wu et al., 1998; Zhang et al., 1998; Arndt et al., 2001; Hayer-Zillgen et al., 2002). We recently reported the expression of rat OCT2 (rOCT2) in oocytes of Xenopus laevis and investigated the inhibition of cation-induced inwardly and outwardly directed currents after short-term addition (30 s) of corticosterone to the extracellular or intracellular side of the plasma membrane, respectively (Volk et al., 2003). From both sides, corticosterone inhibited cation-induced currents with different affinities and could be partially or totally competed away by transported cations. Our interpretation of these data was that corticosterone binds to the inwardly as well as the outwardly oriented substrate binding site of rOCT2. Because previous point mutations in the 11th transmembrane helix (TMH) of rOCT1 led to an affinity increase for some but not all transported substrates (Gorboulev et al., 1999), we raised the hypothesis that the substrate binding sites of the OCTs contain overlapping interaction domains for structurally different substrates (Gorboulev et al., 1999; Volk et al., 2003).
The aim of the present study was to identify amino acids of rOCT2 that are responsible for the higher affinity of corticosterone (IC50 4 e) compared with OCT1 (IC50 150 e). We introduced polypeptide stretches of rOCT2 into rOCT1, exchanged amino acids between both transporters, and measured IC50 values for the inhibition of uptake of tetraethylammonium (TEA) and 1-methyl-4-phenylpyridinium (MPP) by corticosterone as well as Km values for TEA and MPP. Our data indicate that amino acids Ala443, Leu447, and Gln448 in the 10th transmembrane helix form part of the substrate binding region of OCTs and are critically involved in corticosterone binding.
Materials and Methods
Construction of Chimeras and Point Mutants. The overlap extension method of polymerase chain reaction (PCR) (Ho et al., 1989) was used to construct chimeric transporters or to introduce point mutations. To generate chimeras, two PCR fragments of rOCT1 that flank the region to be inserted and a PCR-fragment of rOCT2 that contains the region to be inserted were amplified. The 5' ends of the primers used for the amplification of the rOCT2 fragment contained nucleotide sequences that corresponded to the respective flanking regions of rOCT1. Next, the three PCR products were combined and fused by PCR, which yielded the desired rOCT2 fragment flanked with parts of rOCT1. This PCR amplificate was digested with two unique restriction endonucleases and ligated to the rOCT1/RSSP plasmid (Gredemann et al., 1994). To introduce point mutations, two overlapping fragments of the respective transporter were amplified into which the desired point mutation was introduced at overlapping ends. The amplificates were fused by PCR, and the product was inserted into the rOCT1/RSSP plasmid. All chimeric and mutant proteins were sequenced partially to confirm the presence of the desired modifications and the absence of any PCR errors.
Expression of rOCT1, OCT2, and Mutants in Oocytes of X. laevis. For injection into X. laevis oocytes, m7G(5')ppp(5')G-capped cRNAs were prepared from the cDNAs of rOCT1 (Gredemann et al., 1994), rOCT2 (Okuda et al., 1996), and their mutants using the mMESSAGE mMACHINE kit (Ambion, Huntingdon, Cambridgeshire, UK). The restriction enzymes used for linearization of the respective cDNA vectors and the RNA polymerases used for transcription were described earlier (Arndt et al., 2001). Stage V to VI oocytes were defolliculated with collagenase A and stored for several hours in Ori buffer [5 mM 3-(N-morpholino)propanesulfonic acid-NaOH, pH 7.4, 100 mM NaCl, 3 mM KCl, 2 mM CaCl2, and 1 mM MgCl2] supplemented with 50 mg/l gentamicin. The oocytes were injected with 50 nl of H2O per oocyte containing 10 ng of the respective cRNA and incubated for 3 to 5 days at 16°C in Ori buffer supplemented with 50 mg/l gentamicin.
Tracer Uptake Measurements. X. laevis oocytes injected with cRNAs and noninjected control oocytes were incubated for 30 min at room temperature with Ori buffer containing [14C]TEA or [3H]MPP, washed in ice-cold Ori buffer supplemented with 100 e quinine, and analyzed by liquid scintillation counting (Arndt et al., 2001). Expressed uptake was calculated by subtracting the uptake measured in noninjected control oocytes. TEA and MPP uptake in noninjected oocytes was identical with the uptake in oocytes expressing the transporter that was measured in the presence of 100 e quinine or 10 e cyanine 863 (inhibitors of OCTs). To determine concentration-inhibition curves, we measured the uptake of 10 e [14C]TEA or 0.1 e [3H]MPP in the presence of various concentrations of corticosterone or of unlabeled MPP or TEA. For inhibition by corticosterone, oocytes were preincubated for 10 min with corticosterone at the respective concentrations. Corticosterone was first dissolved in ethanol and then added to Ori buffer, keeping the final ethanol concentration lower than 1%. Uptake with 2 mM TEA or 200 e MPP was taken as maximal transport velocity (Vmax).
Calculation and Statistics. For each substrate concentration or combination of substrate and inhibitor, uptake rates were calculated from 7 to 10 oocytes, and uptake in 7 to 10 noninjected oocytes was measured in parallel. From uptake measurements with 8 to 10 different concentrations of TEA or MPP, the Km values were determined by fitting the Michaelis-Menten equation to the data. IC50 values were determined from uptake with TEA or MPP and from 8 to 12 different concentrations of the nontransported inhibitor corticosterone or competing substrates. IC50 values were calculated by fitting the Hill equation for multisite inhibition to the data. To compare inhibition at a given concentration of corticosterone, IC50 values, or Km values between transporters, three to nine independent experiments were performed, and the respective degrees of inhibition, Km values, and IC50 values were calculated from the individual experiments. The data are presented as means ± S.E.M. One-way ANOVA with post hoc Tukey test was used to evaluate differences where indicated. For comparison of two values, unpaired two-sided Student's t test was used. Statistical calculations were done with Prism 4.0 (GraphPad Software Inc., San Diego, CA).
Materials. [14C]TEA (1.9 GBq/mmol) and [3H]MPP (3.1 TBq/mmol) were obtained from Biotrend (Kln, Germany). All other chemicals were obtained as described earlier (Gorboulev et al., 1999; Arndt et al., 2001).
Results
Functional Characterization of Chimeras Containing rOCT1 Backbone with Inserted Domains of rOCT2. We replaced individually 16 successive amino acid stretches by the respective regions of rOCT2 (Fig. 1a), and tested for transport of 10 e [14C]TEA (Fig. 1b), Km value for TEA uptake (Fig. 1c), and inhibition of TEA uptake by 4 e corticosterone (Fig. 1d). Eight chimeras (N, 3, 5, 6, iL, 8, 12, and C) had uptake rates of 10 e [14C]TEA similar to the wild type. Six chimeras (1, eL, 2, 7, 9, 10) had uptake rates between 13 and 49% of the rOCT1 wild type. With chimera 11, the expressed uptake rate was 4.5% of the wild type, and chimera 4 showed no significant TEA uptake at all (Fig. 1b). The low transport activity expressed by chimera 11 and the absence of transport observed with chimera 4 suggest that the exchanged domains exhibit interactions with other domains of rOCT2 wild type. These interactions may be disturbed when they are inserted into rOCT1, resulting in inactivation or defective membrane targeting (Koepsell et al., 2003). For the other chimeras, we measured the Km values for TEA uptake (Fig. 1c). They were similar to rOCT1 and rOCT2 wild types in chimeras N, 1, eL, 2, 3, 6, iL, 8, 9, 10, 12, and C and were approximately two times lower in chimeras 5 and 7. The data suggest functional integrity of the chimeras with the exception of chimeras 4 and 11.
For the functionally active chimeras, we measured the uptake of 10 e [14C]TEA with and without 4 e corticosterone present (Fig. 1d). Corticosterone (4 e) represents the previously determined Ki value for the inhibition of rOCT2 (Arndt et al., 2001; Volk et al., 2003). With the exception of chimera 10, 4 e corticosterone did not significantly inhibit TEA uptake by wild-type rOCT1 or by the chimeras. TEA uptake by chimera 10 was inhibited by the same degree as rOCT2 wild type (63 ± 6% versus 66 ± 6%). The inhibition observed with chimera 11 (34 ± 5%, n = 3) was not statistical significant according to ANOVA with post-hoc Tukey's test. Further investigations focused on chimera 10.
The concentration-dependence for corticosterone inhibition of [14C]TEA (10 e) uptake by rOCT1, rOCT2, and chimera 10 confirmed that high affinity for corticosterone was conveyed to rOCT1 by transferring the presumed 10th TMH of rOCT2 to rOCT1 (Fig. 2a and Table 1). The IC50 values for corticosterone inhibition of TEA uptake were 198 ± 10 e for rOCT1 (n = 9), 5.9 ± 1.4 e for rOCT2 (n = 5), and 4.5 ± 0.8 e for chimera 10 (n = 4). The IC50 values are similar to the respective Ki values because the substrate concentration (S) used of 10 e TEA is seven times (rOCT1, rOCT2) or five times lower (chimera 10) than the respective Km values (Fig. 1c and Table 2). Assuming competitive inhibition and Michaelis-MenteneCtype substrate dependence, the determined IC50 values are 13% (rOCT1, rOCT2) or 17% (chimera 10) higher than the respective Ki values [Ki = IC50/(1 + S/Km)].
Uptake of 10 e [14C]TEA or 0.1 e [3H]MPP by the indicated transporters and mutants was measured in the presence of 8 to 10 different corticosterone concentrations. IC50 values are indicated that were calculated by fitting the Hill equation to the data (mean ± S.E.M. from three to nine independent experiments). Hill coefficients for inhibition of TEA uptake by corticosterone were not significantly different from 1. Hill coefficients for inhibition of MPP uptake by corticosterone were 1.1 ± 0.3 (rOCT1), 0.6 ± 0.1 (rOCT2), 1.0 ± 0.2 (rOCT1-ch10), 2.2 ± 0.3 (rOCT1-L447Y/Q448E), 1.1 ± 0.1 (rOCT1-A443I/L447Y/Q448E), 1.5 ± 0.1 (rOCT2- Y447L/E448Q), and 1.5 ± 0.1 (rOCT2-I443A,Y447L,E448Q).
The transporters were expressed in X. laevis oocytes and uptake was measured at various concentrations of TEA or MPP. In addition, uptake of 0.1 e [3H]MPP was measured in the presence of various concentrations of TEA, uptake of 10 e [14C]TEA was measured in the presence of various concentrations of MPP. Km and IC50 values were determined by fitting the Michaelis-Menten equation or the Hill equation to the data, respectively. Hill coefficient varied between 0.75 and 1.25 but was not significantly different between different groups. Mean ± S.E.M. from three to seven independent experiments are indicated.
Corticosterone Inhibition of rOCT1 Mutants Containing Selected Amino Acids from rOCT2. To identify the amino acids that are responsible for the higher affinity of corticosterone in rOCT2 compared with rOCT1, we exchanged individual amino acids in the presumed 10th TMD of rOCT1 for the amino acids in the respective positions of rOCT2. With mutants L447Y and Q448E, we observed significantly lower IC50 values for corticosterone inhibition of TEA (10 e) uptake than with rOCT1 wild type (Fig. 2b and Table 1). The IC50 values obtained with mutants L447Y (42 ± 9) and Q448E (40 ± 11) were, however, still significantly higher than with rOCT2. When both amino acids were replaced simultaneously [rOCT1(L447Y/Q448E)], the IC50 value for corticosterone inhibition of TEA uptake decreased further to 5.3 ± 1.7 e, which is significantly lower (P < 0.01) than in the mutants with a single amino acid exchange but not different from rOCT2 (Fig. 2b and Table 1).
Recent experiments indicated that rOCT2 contains a substrate binding region with overlapping binding sites for structurally different substrates which may be exposed to the extracellular or intracellular side of the plasma membrane and that corticosterone binds to either conformation of the substrate binding region (Gorboulev et al., 1999; Koepsell et al., 2003; Volk et al., 2003). In such a model, substrates and corticosterone may interact within the binding region, either directly via partial replacement or indirectly via short-range allosteric effects.
To investigate putative differential effects of substrates on the affinity of the inhibitor corticosterone, we measured the inhibition of MPP uptake by corticosterone (Fig. 3 and Table 1). MPP was selected because it has little structural similarity to TEA. The measurements were performed with 0.1 e MPP, a concentration that is at least 10 times lower than the respective Km values of rOCT1, rOCT2, or the mutants (for Km values, see Table 2). At this concentration, a potential replacement of corticosterone by MPP should not increase the IC50 by more than 10%. In the measurements using 10 e TEA, a replacement of corticosterone by TEA should not increase the IC50 values of rOCT1, rOCT2, or rOCT1(L447Y/Q448E) mutant by more than 15% (for Km values, see Table 2). For rOCT1, rOCT2, and chimera 10, the IC50 values for inhibition of TEA uptake by corticosterone versus inhibition of MPP uptake by corticosterone were not significantly different (Figs. 2 and 3a; Table 1). With the double mutant rOCT1(L447Y/Q448E), however, the IC50 value of corticosterone for inhibition of MPP uptake was remarkably higher than for inhibition of TEA uptake (5.3 ± 1.7, n = 3, versus 24 ± 3.8, n = 4, respectively; P < 0.01 for difference). These data suggest that the affinity for corticosterone is modulated differentially by transport and/or binding of TEA or MPP. Because the substrate effects cannot be explained by a differential replacement of corticosterone by each of the two substrates, they imply a different allosteric interaction of TEA versus MPP with corticosterone.
Trying to reconstitute in rOCT1 the same high-affinity inhibition by corticosterone of both TEA and MPP uptake as exhibited by rOCT2, we studied triple mutants of rOCT1 carrying additional amino acids from the 10th TMH of rOCT2 (Fig. 3b and Table 1). In one of these (A443I/L447Y/Q448E), the IC50 value for corticosterone inhibition of MPP uptake was similar to that in rOCT2. In this triple mutant, the IC50 values for corticosterone obtained with TEA and MPP were not significantly different from each other.
Interaction of Cationic Substrates with rOCT1 Mutants Exhibiting High Affinity for Corticosterone. We observed previously for rOCT2 that choline-induced currents were inhibited after brief application of corticosterone from either the extracellular or intracellular side of the plasma membrane (Volk et al., 2003) and that the presence of choline prevented inhibition from either side, partially or totally. We thus hypothesized that corticosterone binds to the substrate binding region of rOCT2, which can exist in an extracellularly and an intracellularly oriented conformation. To investigate whether corticosterone binds to the substrate binding region, we examined whether the rOCT1 point mutants with increased corticosterone affinity also exhibit altered affinities for transported substrates.
We determined the Km values for MPP and TEA in rOCT1 wild-type and different mutants, as well as the IC50 values for inhibition of [14C]TEA (10 e) uptake by MPP and the IC50 values for inhibition of [3H]MPP (0.1 e) uptake by TEA (Figs. 4 and 5; Table 2). The Km values measured for MPP uptake were not significantly different from the IC50 values measured for inhibition of TEA uptake by MPP, and the Km values for TEA uptake were not significantly different from the IC50 values for inhibition of MPP uptake by TEA (Table 2). Compared with rOCT1 wild type, the Km value for MPP uptake and the IC50 for inhibition of TEA uptake by MPP were significantly decreased in both the rOCT1(L447Y/Q448E) double mutant and the rOCT1(A443I/L447Y/Q448E) triple mutant. At variance, the Km values for TEA uptake and the IC50 values for inhibition of MPP uptake by TEA were not significantly different between rOCT1 wild type and rOCT1(L447Y/Q448E). In the rOCT1(A443I/L447Y/Q448E) triple mutant; however, the Km value and the IC50 value were significantly decreased. Because combined mutation of L447Y, Q448E, and/or A443I leads to increased affinity for corticosterone and for either TEA or both TEA and MPP, our data suggest that Ala443, Leu447, and Gln448 are localized within the substrate binding region of rOCT1 and that corticosterone binds to the same region.
We also measured substrate uptake by rOCT1 wild type, double mutant L447Y/Q448E, and triple mutant A443I/L447Y/Q448E at saturating concentrations of TEA (2 mM) versus MPP (0.2 mM). The measurements with TEA and MPP were performed simultaneously (within 1 h) using identical batches of oocytes and the same cRNA preparations. The ratios between maximal transport velocities for TEA and MPP were the following: rOCT1, 11.6 ± 3.5, n = 3; rOCT1(L447Y/Q448E), 9.8 ± 2.2, n = 3; and rOCT1(A443I/L447Y/Q448E), 11.7 ± 2.6, n = 3. Taken together, the data indicate that in the double mutant rOCT1(L447Y/Q448E), the apparent selectivity for MPP versus TEA is increased, whereas the maximal velocity for MPP versus TEA is not changed.
Corticosterone Inhibition of rOCT2 Mutants Containing Individual Amino Acids from rOCT1. To confirm the critical role of amino acids Ile443, Tyr447, and Glu448 in rOCT2 for the higher corticosterone affinity of rOCT2 versus rOCT1, we tested whether the high affinity of rOCT2 can be switched to low affinity by replacing these amino acids with the corresponding amino acids of rOCT1. We generated the double mutant rOCT2(Y447L/E448Q) and the triple mutant rOCT2(I443A/Y447L/E448Q) and measured the inhibition of MPP uptake by corticosterone (Table 1 and Fig. 6). In the double mutant rOCT2(Y447L/E448Q), the IC50 value for corticosterone inhibition (IC50 = 36 ± 6.1 e, n = 3) was significantly increased compared with wild-type rOCT2 (5.2 ± 1.9, n = 4, P < 0.01 for difference). For corticosterone inhibition of MPP uptake expressed by the triple mutant rOCT2 (I443A/Y447L/E448Q), an IC50 of 382 ± 100 e was determined. This value was not significantly different from rOCT1.
Discussion
In this work, we identified three amino acids in the presumed 10th TMH (A443I, L447Y, and Q448E) of rOCT2 that are responsible for the higher affinity of corticosterone compared with rOCT1. We present evidence that these amino acids are located within the substrate region of OCTs by demonstrating that the affinity of transported cations was increased together with the affinity of corticosterone. In one mutant, an allosteric interaction between transported substrate and corticosterone was detected, suggesting that more than one compound can bind simultaneously to the substrate binding region.
Our interpretation that the identified amino acids are localized within the substrate binding region of rOCT1 or rOCT2 is made on the basis of two arguments. First, we consider it highly improbable that a combined mutation of three amino acids within one helix (Ala443, Leu447, and Gln448) can increase the affinity of transported substrates if they are not localized within and/or close to the substrate binding region. Second, using the crystal structure of the lactose permease from Escherichia coli (Abramson et al., 2003) that belongs to the major facilitator superfamily like the OCTs (Pao et al., 1998), we modeled the presumed 12 transmembrane domains of rOCT1 (Popp et al., 2005). In this model seven amino acid residues that are critical for substrate binding and/or substrate specificity are localized in one region surrounding a large cleft that is formed by the 1st, 2nd, 4th, 5th, 7th, 8th, 10th, and 11th TMH and are accessible from the aqueous phase. These seven amino acids are Asp475 in the 11th TMH (Gorboulev et al., 1999), three amino acids on one side of the presumed 4th TMH (Trp218, Tyr222, and Thr226) (Popp et al., 2005), and Ala443, Leu447, and Gln448 that are described in the present work. In the three-dimensional model of rOCT1 the 10th helix protrudes into the cleft, allowing an interaction of corticosterone with the two succeeding amino acids Leu447 and Gln448.
In Fig. 7, we aligned the presumed 10th TMH and parts of the flanking loops of rOCT1, rOCT2, rOCT3, and human OCT3 (hOCT3) and indicated the IC50 values that were determined for inhibition of the OCTs by corticosterone (Gredemann et al., 1998; Wu et al., 1998; Arndt et al., 2001). Gly439, Glu456, Leu457, Tyr458, Pro459, and Thr460 (rOCT1 numbering) are conserved in nearly all SLC22 members, whereas Trp430, Arg440, Gly442, Thr444, Val453, and Asn454 are conserved within the OCTs. Note that rOCT3 has approximately the same affinity for corticosterone as rOCT2. In the three positions critical for the higher affinity of corticosterone to rOCT2 versus rOCT1, rOCT3 contains the same amino acids (Ile443 and Glu448) or a similar amino acid to rOCT2 (Phe443 in rOCT3 versus Tyr443 in rOCT2). This supports the critical role of the amino acids in positions 443, 447, and 448 for the affinity of corticosterone. The comparison suggests that the amino acids in positions 432, 433, 435, 437, 441, 449, 451, and 455 that are different between rOCT2 and rOCT3 are less critical for corticosterone binding. Note that the amino acid sequence in the 10th TMH and the flanking loops are identical between rOCT3 and hOCT3, whereas the IC50 value for corticosterone inhibition of cation uptake by hOCT3 versus rOCT3 is more than 20 times lower. We conclude that corticosterone interacts with a second domain of the OCT transporters and that differences between hOCT3 versus rOCT3 within this second domain are responsible for the difference in affinity.
Previous short-term inhibition experiments using electrical measurements with intact oocytes and inside-outeCoriented giant patches indicated that the substrate binding region of OCTs can be exposed to the extracellular or intracellular side of the plasma membrane (Volk et al., 2003). These experiments showed that the affinities of corticosterone and tetrabutylammonium were different from both sides. Because the inhibition by corticosterone from either side of the plasma membrane was dependent on the membrane potential similar to the Km values determined for the uptake and efflux of cations (Budiman et al., 2000; Volk et al., 2003), we hypothesized that corticosterone binds to extracellularly and intracellularly oriented conformations of the substrate binding region. The inhibition experiments performed in the present study do not allow us to distinguish whether the mutations changed the affinity of corticosterone at the extracellular or intracellular orientation of the substrate binding region or whether the affinity to both orientations is changed. Because we used a 30-min incubation period for the uptake measurements in the presence of corticosterone and preincubated the oocytes with the respective corticosterone concentrations, corticosterone was equilibrated across the plasma membrane. Most probably, our measurements characterized the corticosterone binding site at the inwardly directed conformation of the substrate binding region because this site has a higher affinity for corticosterone (Arndt et al., 2001; Volk et al., 2003). Allowing the equilibration of corticosterone across the plasma membrane, we previously measured an IC50 value for the inhibition of cation uptake by corticosterone that was identical with the IC50 value obtained after a short application of corticosterone to the intracellular side of the plasma membrane.
The rOCT1(L447Y/Q448E) double mutant exhibited functional properties that indicated allosteric interaction between transported cation (TEA and/or MPP) and the inhibitor corticosterone, which requires simultaneous binding of substrate and inhibitor. The IC50 value for the inhibition of TEA uptake by corticosterone was 4.5 times lower than the IC50 value for the inhibition of MPP uptake (Table 1). Because the concentration of 10 e [14C]TEA used for the uptake measurements was 1/7 of the Km values of rOCT1 wild type and rOCT1(L447Y/Q448E) (Table 2), and because the concentration of 0.1 e [3H]MPP used for the uptake measurements was 1/50 of the Km value of rOCT1 wild type and 1/10 of the Km value of rOCT1(L447Y/Q448E), differences in competitive replacement of corticosterone by TEA and MPP cannot explain the observed differences in the IC50 values. Competitive replacement of corticosterone by TEA or MPP would increase the IC50 values for inhibition of TEA uptake by approximately 14% and the IC50 values measured with MPP by approximately 2% (rOCT1 wild type) or 10% rOCT1(L447Y/Q448E). In conclusion, the different IC50 values determined for corticosterone inhibition of TEA uptake versus MPP uptake in the rOCT1(L447Y/Q448E) mutant indicate differential allosteric effects of TEA versus MPP on corticosterone binding or an allosteric effect by only one of the two substrates. We cannot distinguish whether the allosteric effect is triggered by substrate binding or by substrate-dependent conformational change during the transport cycle.
The above-described allosteric effect can be caused by the interaction between substrate binding regions in monomers of a dimeric transporter, by interaction between coexisting substrate binding regions in a monomeric transporter, or by short-range interaction between TEA (and/or MPP) and corticosterone within one substrate binding region. Notwithstanding that additional experiments are necessary to make an unequivocal distinction between these possibilities, we believe that a short-range allosteric interaction within one substrate binding region is the most probable explanation. The three-dimensional model of rOCT1 supports the existence of only one substrate binding region in the rOCT1 monomer and shows that the amino acids in the binding region that are critical for the binding of TEA and corticosterone are distant enough to allow simultaneous binding of both compounds. An allosteric interaction between cation binding to one rOCT1 monomer and corticosterone binding to another rOCT1 monomer in a dimer or oligomer is less probable because the interaction was only observed after a mutation within the binding region of rOCT1 [i.e., in the rOCT1(L447Y/Q448E) mutant]. In the present study, an allosteric effect between two ligands was observed in the rOCT1(L447Y/Q448E) mutant but not in the rOCT1 wild type. This indicates that some cooperation between 447Y and 448E with the rest of the rOCT1 structure was required for the observed allosteric interaction. However, because we observed allosteric interactions of MPP and other substrates also in wild types of human OCT1 and human OCT3 (U. Roth and H. Koepsell, unpublished data) we interpret the allosteric interaction observed in the rOCT1(L447Y/Q448E) mutant as a demonstration of principle (i.e., that ligands of OCT binding regions can exhibit allosteric interactions).
Mapping the surface of substrate binding regions in OCTs by crystallization of ligand transporter complexes and by characterization of point mutations will help to design drugs that are transported by specific OCT subtypes, inhibit specific OCTs, or do not interact with them. This will allow for the influence of the absorption of drugs in small intestine and their renal and hepatic excretion and thereby modulate physiological functions that are controlled by the OCTs. The interaction of glucocorticoids with OCTs can be of clinical importance. For example, OCT1 and OCT2 are responsible for the luminal release of acetylcholine from bronchial epithelia in the lung, and aerosols containing the glucocorticoids budesonide probably inhibit this function (K. S. Lips, C. Volk, B. M. Schmitt, U. Pfeil, P. Arndt, D. Miska, L. Ermert, W. Kummer, H. Koepsell, submitted). The interaction of corticosteroids with hOCT3 may be most relevant because this transporter has the highest affinity to corticosterone among the OCTs. Like other OCTs, hOCT3 translocates monoamine neurotransmitters. Among many others, hOCT3 is expressed in smooth muscle cells of blood vessels and neurons throughout the brain (Slitt et al., 2002; Horvath et al., 2003; Schmitt et al., 2003; Vialou et al., 2004). Inhibition of hOCT3 may lead to an increase of blood pressure and to alterations in behavior (Vialou et al., 2004).
This study was supported by Deutsche Forschungsgemeinschaft SFB 487/A4.
V.G. and N.S. contributed equally to this work.
doi:10.1124/mol.104.008821.
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