Importance of valine at position 152 for the substrate transport and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane binding of dopamine transporter.
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Human and bovine dopamine transporters (DAT) demonstrate discrete functional differences in dopamine (DA), 1-methyl-4-phenylpyridium (MPP(+)) transport, and cocaine analog binding. In a previous study, the functional analyses on the chimeras of human and bovine DAT have revealed that the region from residues 133 through 186 (encompassing the third transmembrane domain) is responsible for the substrate transport and cocaine analog binding. The present study has been carried out to determine the specific amino acid(s) conferring DAT functions by interchanging the amino acid residues in the corresponding region between human and bovine DAT. As described previously, the DA, MPP(+) transport, and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) binding almost disappeared in chimera hb3 in which the region from residues 133 through 186 of bovine DAT was substituted into human DAT. Replacement of isoleucine, residue 152 of chimera hb3 (bovine DAT sequence), with valine, the human DAT residue at the identical position, remarkably restored the substrate transport and CFT binding to 76% to 98% of the human DAT values. Similarly, substitution of isoleucine for valine at position 152 in the human DAT reduced the substrate transport and CFT binding by 57% to 97%. Among other amino acids tested at position 152 of the chimera hb3, only alanine resulted in small but significant increases in the DAT functions ranging from 16 to 34%. Thus, valine at position 152 plays a crucial role for molecular mechanisms underlying the interactions of DA, MPP(+), and CFT with human DAT.Keywords:
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Cocaine analogue, CFT (2β-carbomethoxy-3β-(4-fluorophenyl) tropane) binding to dopamine transporter (DAT) in different species is quite heterogeneous. CFT is scarcely detected in bovine DAT whereas it is conspicuous in humans. To examine the structural basis for this functional discrepancy, we analyzed transporter chimeras of these two DATs. The CFT binding activities are avid in all of the chimeric DATs of which both of the 3rd and the 6-8th transmembrane domain (TM) are composed of human DAT sequences. On the contrary, CFT binding activities were scarcely detected if either or both of two regions are replaced with bovine sequences. These findings indicate that the CFT binding absolutely requires human DAT sequences, at least, in the regions encompassing the 3rd and 6-8th transmembrane domain (TM), and that these regions might contribute to form the 3-dimensional pocket for CFT binding.
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Abstract : Although much is known about the effects of Na + , K + , and Cl ‐ on the functional activity of the neuronal dopamine transporter, little information is available on their role in the initial event in dopamine uptake, i.e., the recognition step. This was addressed here by studying the inhibition by dopamine of the binding of [ 3 H]WIN 35,428 {2β‐carbomethoxy‐3β‐(4‐fluorophenyl)[ 3 H]tropane}, a phenyltropane analogue of cocaine, to the cloned human dopamine transporter expressed in HEK‐293 cells. The decrease in the affinity of dopamine (or WIN 35,428) binding affinity with increasing [K + ] could be fitted to a competitive model involving an inhibitory cation site (1) overlapping with the dopamine (or WIN 35,428) domain. The K + IC 50 for inhibiting dopamine or WIN 35,428 binding increased linearly with [Na + ], indicating a K D,Na+ of 30‐44 m M and a K D,K+ of 13‐16 m M for this cation site. A second Na + site (2), distal from the WIN 35,428 domain but linked by positive allosterism, was indicated by model fitting of the WIN 35,428 binding affinities as a function of [Na + ]. No strong evidence for this second site was obtained for dopamine binding in the absence or presence of low (20 m M ) Cl ‐ and could not be acquired for high [Cl ‐ ] because of the lack of a suitable substitute ion for Na + . The K D but not B max of [ 3 H]WIN 35,428 binding increased as a function of the [K + ]/[Na + ] ratio regardless of total [Cl ‐ ] or ion tonicity. A similar plot was obtained for the K i of dopamine binding, with Cl ‐ at ≥ 140 m M decreasing the K i . At 290 m M Cl ‐ and 300 m M Na + the potency of K + in inhibiting dopamine binding was enhanced as compared with the absence of Cl ‐ in contrast to the lack of effect of Cl ‐ up to 140 m M (Na + up to 150 m M ). The results indicate that Cl ‐ at its extracellular level enhances dopamine binding through a mechanism not involving site 1. The observed correspondence between the WIN 35,428 and dopamine domains in their inclusion of the inhibitory cation site explains why many of the previously reported interrelated effects of Na + and K + on the binding site of radiolabeled blockers to the dopamine transporter are applicable to dopamine uptake in which dopamine recognition is the first step.
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Hxt2 and Hxt1 are high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae, respectively, that differ at 75 amino acid positions in their 12 transmembrane segments (TMs). Comprehensive analysis of chimeras of these two proteins has previously revealed that TMs 1, 5, 7, and 8 of Hxt2 are required for high affinity glucose transport activity and that leucine 201 in TM5 is the most important in this regard of the 20 amino acid residues in these regions that differ between Hxt2 and Hxt1. To evaluate the importance of the remaining residues, we systematically shuffled the amino acids at these positions and screened the resulting proteins for high affinity and high capacity glucose transport activity. In addition to leucine 201 (TM5), four residues of Hxt2 (leucine 59 and leucine 61 in TM1, asparagine 331 in TM7, and phenylalanine 366 in TM8) were found to be important for such activity. Furthermore, phenylalanine 198 (TM5), alanine 363 (TM8), and either valine 316 (TM7) or alanine 368 (TM8) were found to be supportive of maximal activity. Construction of a homology model suggested that asparagine 331 interacts directly with the substrate and that the other identified residues may contribute to maintenance of protein conformation. Hxt2 and Hxt1 are high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae, respectively, that differ at 75 amino acid positions in their 12 transmembrane segments (TMs). Comprehensive analysis of chimeras of these two proteins has previously revealed that TMs 1, 5, 7, and 8 of Hxt2 are required for high affinity glucose transport activity and that leucine 201 in TM5 is the most important in this regard of the 20 amino acid residues in these regions that differ between Hxt2 and Hxt1. To evaluate the importance of the remaining residues, we systematically shuffled the amino acids at these positions and screened the resulting proteins for high affinity and high capacity glucose transport activity. In addition to leucine 201 (TM5), four residues of Hxt2 (leucine 59 and leucine 61 in TM1, asparagine 331 in TM7, and phenylalanine 366 in TM8) were found to be important for such activity. Furthermore, phenylalanine 198 (TM5), alanine 363 (TM8), and either valine 316 (TM7) or alanine 368 (TM8) were found to be supportive of maximal activity. Construction of a homology model suggested that asparagine 331 interacts directly with the substrate and that the other identified residues may contribute to maintenance of protein conformation. Facilitated diffusion of glucose across the plasma membrane of the yeast Saccharomyces cerevisiae is mediated by a variety of hexose transporters (Hxt1–Hxt17, Gal2) (1Kruckeberg A.L. Arch. Microbiol. 1996; 166: 283-292Crossref PubMed Scopus (202) Google Scholar, 2Boles E. Hollenberg C.P. FEMS Microbiol. Rev. 1997; 21: 85-111Crossref PubMed Google Scholar) that belong to the major facilitator superfamily (MFS) 2The abbreviations used are: MFS, major facilitator superfamily; TM, transmembrane segment. (3Pao S.S. Paulsen I.T. Saier Jr. M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar). A common structural feature of members of this superfamily is the presence of 12 putative transmembrane segments (TMs), with both the NH2- and COOH-terminal domains being present on the cytoplasmic side of the membrane. Among the 18 hexose transporters of S. cerevisiae, Hxt2 is a major high affinity glucose transporter (Km = 3.6 mm), and Hxt1 is a low affinity glucose transporter (Km = 44 mm). The numbers of amino acid residues in each putative TM and inter-TM loop of Hxt2 are identical to those in the corresponding regions of Hxt1, and the two proteins share ∼70% sequence identity in these regions, with only 75 of the ∼250 residues in TMs differing between Hxt2 and Hxt1. Site-specific mutagenesis has been used extensively to determine the contribution of individual amino acid residues to the structure and function of transporters. In some instances, however, single point mutations have indirect effects on protein function through an extensive, rather than localized, distortion of protein structure. To circumvent this problem and to identify positively the contributing amino acid residues, we have adopted a comprehensive chimeric approach with two closely related paralogs that differ in substrate specificities (4Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 5Kasahara M. Shimoda E. Maeda M. FEBS Lett. 1996; 389: 174-178Crossref PubMed Scopus (22) Google Scholar, 6Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) or affinities (7Kasahara T. Kasahara M. Biochem. J. 2003; 372: 247-252Crossref PubMed Google Scholar, 8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). We previously investigated which TMs of Hxt2 are important for high affinity glucose transport with the use of a new procedure designated TM shuffling (7Kasahara T. Kasahara M. Biochem. J. 2003; 372: 247-252Crossref PubMed Google Scholar). We thus randomly replaced each of the 12 TMs of Hxt2 with the corresponding segments of Hxt1 at the DNA level. Clones encoding transporters with a high affinity for glucose were selected by plating the transformants on glucose-limited agar plates. Our results demonstrated that a minimal combination of TMs 1, 5, 7, and 8 of Hxt2 is necessary for high affinity glucose transport. The chimeric transporter C1578 (Fig. 1), in which all TMs but 1, 5, 7, and 8 of Hxt2 are replaced with the corresponding TMs of Hxt1, excluding a contribution of 55 of the 75 TM residues that differ between Hxt2 and Hxt1, thus exhibited high affinity and high capacity glucose transport activity similar to that of Hxt2. We subsequently showed that, among the 20 residues that differ between Hxt2 and Hxt1 in these four TMs, Leu201 in TM5 of Hxt2 is the most important for high affinity glucose transport (8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The crystal structures of the bacterial MFS transporters LacY (9Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1225) Google Scholar) and GlpT (10Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (854) Google Scholar) have been determined at a resolution of <4 Å, revealing that the configurations of the TMs in these two transporters are highly similar and that TMs 1, 5, 7, and 8 contribute to the central pore. Although alignment of the amino acid sequences of MFS transporters has revealed substantial sequence variability, these findings together with a low resolution crystal structure of the MFS transporter OxlT (11Hirai T. Heymann J.A. Maloney P.C. Subramaniam S. J. Bacteriol. 2003; 185: 1712-1718Crossref PubMed Scopus (95) Google Scholar) and molecular modeling (12Vardy E. Arkin I.T. Gottschalk K.E. Kaback H.R. Schuldiner S. Protein Sci. 2004; 13: 1832-1840Crossref PubMed Scopus (88) Google Scholar) suggest that MFS transporters share a similar conformation. We have now examined which amino acids among the 19 remaining residues in TMs 1, 5, 7, and 8 of Hxt2 that differ from those of Hxt1 are important for high affinity and high capacity glucose transport activity. Our results show that, in addition to Leu201, four of these residues are important for and three residues are supportive of such activity. Molecular modeling suggests that most of these residues do not directly interact with the substrate but rather have a structural role. Construction of Vectors—The plasmid Hxt2mnx-pVT, which comprises HXT2 under the control of the ADH1 promoter in the multicopy vector pVT102-U (YEp URA3 bla), was constructed as described previously (7Kasahara T. Kasahara M. Biochem. J. 2003; 372: 247-252Crossref PubMed Google Scholar). In brief, HXT2 was modified to be divisible into four regions by the introduction of MroI, NheI, XhoI, and ClaI sites into the nucleotide sequences corresponding to the NH2-terminal end of TM4, the loop between TM6 and TM7, and the loop between TM9 and TM10, as well as immediately after the sequence corresponding to the COOH terminus, respectively. The expression vector C1578-pVT, which encodes the chimeric transporter C1578 (in which all of the TMs of Hxt2, with the exception of 1, 5, 7, and 8, have been replaced with those of Hxt1), was also described previously (7Kasahara T. Kasahara M. Biochem. J. 2003; 372: 247-252Crossref PubMed Google Scholar) (Fig. 1). Mutagenesis—Site-directed mutants were prepared by replacing each target codon with modified sequences with the use of a PCR-based approach. Saturation mutagenesis of residues in TMs 1, 5, 7, and 8 was performed as described (8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In brief, mutation of 14 target residues in these TMs was achieved by PCR with each of the four regions of the modified HXT2 sequence divided by EcoRI, MroI, NheI, and XhoI sites and with degenerate primers. The PCR products for the EcoRI-MroI, MroI-NheI, and NheI-XhoI regions were connected first with the use of MroI and then with the use of NheI. The final product was used to replace the corresponding region of a modified version of C1578-pVT (C1578K-pVT (see “Results” and “Discussion”)). After amplification in Escherichia coli, plasmids were introduced into S. cerevisiae strain KY73 (MATα hxt1Δ::HIS3::Δhxt4 hxt5::LEU2 hxt2Δ::HIS3 hxt3Δ::LEU2::Δhxt6 hxt7Δ::HIS3 gal2Δ::DR ura3–52 MAL2 SUC2 MEL) (13Ye L. Kruckeberg A.L. Berden J.A. van Dam K. J. Bacteriol. 1999; 181: 4673-4675Crossref PubMed Google Scholar). Plate Selection—Transformants that possessed high affinity and high capacity glucose transport activity were selected after the incubation of yeast cells for 3 or 4 days at 30 °C on glucose-limited (glucose, 1 mg/ml) agar plates containing a synthetic medium supplemented with adenine and amino acids, but not with uracil (S0.1D plates) (7Kasahara T. Kasahara M. Biochem. J. 2003; 372: 247-252Crossref PubMed Google Scholar). To increase the number of clones encoding high affinity transporters examined, we sampled colonies with a size smaller than that of those expressing C1578 and that were found to encode transporters with a Vmax of 810–1280 pmol/107 cells/5 s, compared with the range of 1030–1500 pmol/107 cells/5 s in our previous study (8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). KY73 cells are not able to grow on S2D plates (glucose, 20 mg/ml), in which glucose is the only carbon source. We therefore also determined the total number of transformants that grew on S2Mal plates (maltose, 20 mg/ml). Modified portions of all clones selected in the present study were verified by DNA sequencing with an automated sequencer (model 310, Applied Biosystems). Transport Assay—Cells harboring plasmids were grown to log phase (optical density at 650 nm, 0.3–0.6) at 30 °C in S2Mal synthetic liquid medium. Glucose transport by the cells was measured at 30 °C for 5 s as described (4Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 14Kasahara T. Kasahara M. J. Biol. Chem. 2000; 275: 4422-4428Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Transport activities measured at a d-[14C]glucose concentration of 0.1 mm were expressed as picomoles of glucose/1 × 107 cells/5 s and were corrected for the background activity determined either in the presence of 0.5 mm HgCl2 or with 0.1 mm l-[14C]glucose. Construction of a Three-dimensional Model of Hxt2—The crystal structure of GlpT (Protein Data Bank 1PW4) formed the basis for construction of a structural model of Hxt2. CLUSTAL W (15Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55766) Google Scholar) was used to align residues in the putative TMs of Hxt1 to Hxt7, Gal2, GlpT, and LacY, and the alignment was modified manually. A working homology model of Hxt2 was generated with the Biopolymer module of Insight II (version 2000; Accelrys, San Diego, CA) as described (8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). Other Assays—A crude membrane fraction was prepared from cells as described (16Kasahara T. Kasahara M. Biochem. J. 1996; 315: 177-182Crossref PubMed Scopus (39) Google Scholar), and immunoblot analysis of this fraction was performed with rabbit polyclonal antibodies specific for the COOH-terminal region of Hxt2 (4Nishizawa K. Shimoda E. Kasahara M. J. Biol. Chem. 1995; 270: 2423-2426Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and with 125I-labeled protein A (GE Healthcare). The intensity of bands corresponding to immune complexes was measured with imaging plates (BAS 1800II; Fuji Film) (16Kasahara T. Kasahara M. Biochem. J. 1996; 315: 177-182Crossref PubMed Scopus (39) Google Scholar) within the range proportional to the amount of protein. Cell number was determined with a particle counter (Z2; Beckman Coulter). Protein concentration was measured with bicinchoninic acid (Pierce). Elimination of Five Residues and Construction of C1578K—We started our analysis with C1578, in which 19 amino acid residues were potential contributors to high affinity glucose transport. By saturation mutagenesis of TMs 1, 5, 7, and 8 in C1578, we previously found that five residues of Hxt2 (Ile57, Val69, Tyr215, Ile317, and Ile359) were present in ≤50% of the 60 clones isolated that encoded high affinity transporters (8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). These five residues were thus considered not to be required for high affinity glucose transport and were excluded from the present study. We therefore constructed a modified form of C1578 (C1578K) in which these five residues were replaced with the corresponding residues of Hxt1. Yeast cells expressing C1578K grew on selection plates (S0.1D plates) and showed glucose transport activity similar to that of those expressing C1578. The Km, Vmax, and the transport efficiency (Vmax/Km) of C1578K were 5.8 ± 0.6 mm, 1300 ± 140 pmol/107 cells/5 s, and 224 pmol/107 cells/5 s/mm (means ± S.E., n = 4), respectively, whereas the corresponding values for C1578 were 5.2 ± 0.1 mm, 1180 ± 70 pmol/107 cells/5 s, and 227 pmol/107 cells/5 s/mm (n = 12). Random Mutagenesis of 14 Residues—We then examined the remaining 14 amino acid residues that differ between Hxt2 and Hxt1, including Cys195 and Phe198, which we previously found to be supportive of high affinity transport activity (8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). We thus randomly replaced all of these 14 residues of C1578K simultaneously with the corresponding residues of Hxt1, generating 120,000 transformants corresponding to the 214 = 16,384 possible combinations of Hxt1 and Hxt2 residues at these sites. Selection on S0.1D plates yielded 1300 transformants, 40 of which were subjected to plasmid extraction and DNA sequencing. The 40 clones encoded 17 distinct proteins, all of which contained four residues of Hxt2 (Leu59 and Leu61 in TM1, Asn331 in TM7, and Phe366 in TM8) on the C1578K background (Table 1). We therefore considered these four residues to be important for high affinity and high capacity glucose transport activity. Five additional residues of Hxt2 (Cys58 and Ile63 in TM1, Cys195 in TM5, Leu357, and Val367 in TM8) were present in ≤59% of the 17 transporters and were therefore considered unimportant. We replaced these five residues of C1578K with the corresponding residues of Hxt1, thereby generating a transporter (H1) that contained a total of 10 Hxt2-derived residues in TMs 1,5, 7, and 8, and this transporter showed high affinity and high capacity glucose transport activity similar to that of C1578 (Table 2). The remaining five of the 14 residues examined (Phe198 in TM5, Val316 in TM7, and Gln352, Ala363, and Ala368 in TM8) were present in 71–94% of the 17 transporters and were subjected to further examination.TABLE 1Amino acid residues at 14 sites of C1578K-based chimeric transporters exhibiting high affinity and high capacity glucose transport activity Open table in a new tab TABLE 2Characterization of the 32 chimeras containing all possible combinations of Hxt2 and Hxt1 residues at positions 198, 316, 352, 363, and 368 KY73 cells expressing the chimeric transporters were subjected to plate assays with S2D and S0.1D media. Cell growth or no growth after incubation for 3–4 days at 30°C is indicated by + or – signs, respectively; + (s) indicates that the size of the colonies was smaller than that for cells expressing C1578. The level of transporter expression was determined by quantitative analysis of immunoblots. For assay of glucose transport activity, cells were grown to log phase at 30 °C in S2Mal synthetic liquid medium, after which activity was measured for 5 s at 30 °C with 0.1 mm d-glucose as the substrate. The Km and Vmax values were determined with 1–100 mm d-glucose and are expressed as mm and pmol/107 cells/5 s, respectively; the Vmax/Km ratio is expressed as pmol/107 cells/5 s/mm. All values are the means ± S.E. from at least three experiments. Clones encoding high affinity and high capacity glucose transporters are indicated in bold type.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Open table in a new tab Comprehensive Mutagenesis of the Remaining Five Residues—All 32 possible combinations of these five residues in the H1 background were generated (Table 2). We prepared a crude membrane fraction from cells expressing each of these 32 chimeric transporters and examined the extent of transporter expression by immunoblot analysis with antibodies to the COOH-terminal region of Hxt2 (Fig. 2). All 32 chimeras yielded a predominant immunoreactive band at a position corresponding to that of Hxt2 (47 kDa). Quantitative analysis of these bands revealed an expression level for the chimeras of 68–118% (n = 3 to 5) relative to the value for C1578 (Table 2). Eight (H1, H2, H3, H5, H6, H9, H13, and H17) of the 32 clones conferred the ability to grow on S0.1D plates, although the colony size varied. Whereas H1 possessed all five Hxt2-derived residues, H2, H3, H5, H9, and H17 possessed four and H6 and H13 possessed three Hxt2 residues at the five targeted positions. An assay of glucose transport activity revealed that the Km values of all eight mutants were similar to that of C1578, whereas the Vmax values varied (Table 2). No marked differences in substrate specificity were apparent among these eight transporters (Fig. 3).FIGURE 3Substrate specificities of chimeric transporters of the H series. The transport activities of eight H-series transporters (Table 2) and of C1578 were measured at 30 °C for 5 s with 0.1 mm d-[14C]glucose as substrate in the presence of a 200-fold excess of the indicated nonradioactive sugars. The data are the means ± S.E. of values from four to eight experiments and are expressed relative to the transport activity determined in the presence of 20 mm l-glucose. 2DG, 2-deoxy-d-glucose; 3OMG,3-O-methyl-d-glucose; 6DG, 6-deoxy-d-glucose.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Complementary Approach to Construct Chimeras That Mediate Low Affinity Transport—The reverse approach, determining which residues of Hxt1 are important for low affinity glucose transport, was problematic. A chimera in which all the TMs are derived from Hxt1 and the remainder of the molecule is derived from Hxt2, which was named C0 in a previous study (7Kasahara T. Kasahara M. Biochem. J. 2003; 372: 247-252Crossref PubMed Google Scholar), was found to be expressed but was virtually inactive, indicating the importance of structures adjacent to TMs. Efforts were continued with the chimera H5 generated in the present study. After replacement of the essential Leu201 of Hxt2 with Hxt1-derived Val, we performed saturation mutagenesis of eight residues (Leu59, Leu61, Phe198, Val316, Asn331, Ala363, Phe366, and Ala368). The clone that exhibited the lowest Km of 17 mm showed a reduced Vmax of 870 pmol/107 cells/5 s; the corresponding values for C1578 were 5.2 mm and 1180 pmol/107 cells/5 s, and those for Hxt1 were 44 mm and 2800 pmol/107 cells/5 s. The phenotype of this clone thus appears not to reflect that of Hxt1 but rather that of an impaired Hxt2. Given that Hxt2 and Hxt1 show similar substrate specificities and are both inhibited only by sulfhydryl reagents, further studies aimed at identifying residues necessary for low affinity transport will likely require the development of a new strategy. Location of Important Residues Inferred from a Homology Model—To evaluate the functional roles of the identified residues of Hxt2, we modified our previous homology model (8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar) on the basis of the crystal structure of GlpT, a glycerol 3-phosphate transporter of E. coli (10Huang Y. Lemieux M.J. Song J. Auer M. Wang D.N. Science. 2003; 301: 616-620Crossref PubMed Scopus (854) Google Scholar). The alignment of GlpT and Hxt2 adopted is shown in Fig. 4. The new model (Fig. 5) generally conforms with recent models of the MFS transporters ProP (17Wood J.M. Culham D.E. Hillar A. Vernikovska Y.I. Liu F. Boggs J.M. Keates R.A.B. Biochemistry. 2005; 44: 5634-5646Crossref PubMed Scopus (40) Google Scholar), TetAB (12Vardy E. Arkin I.T. Gottschalk K.E. Kaback H.R. Schuldiner S. Protein Sci. 2004; 13: 1832-1840Crossref PubMed Scopus (88) Google Scholar), GLUT1 (18Salas-Burgos A. Iserovich P. Zuniga F. Vera J.C. Fischbarg J. Biophys. J. 2004; 87: 2990-2999Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), GLUT7 (19Manolescu A. Salas-Burgos A.M. Fischbarg J. Cheeseman C.I. J. Biol. Chem. 2005; 280: 42978-42983Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), and rVMAT2 (12Vardy E. Arkin I.T. Gottschalk K.E. Kaback H.R. Schuldiner S. Protein Sci. 2004; 13: 1832-1840Crossref PubMed Scopus (88) Google Scholar). In the model, Asn331 is situated in the outer half of TM7 and facing the central pore, a favorable position for direct interaction with substrate. However, Leu59, Phe198, Val316, and Ala368 appear to interact with neighboring residues of other TMs. Leu201 in TM5, which is essential for high affinity transport, was positioned facing the central pore in our previous model (8Kasahara T. Ishiguro M. Kasahara M. J. Biol. Chem. 2004; 279: 30274-30278Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). In the present model, however, it faces TM8 and appears to interact with Phe362 at a van der Waals' distance; it may therefore act to maintain inter-TM stability. Many of the residues found to be important for high affinity transport are hydrophobic aliphatic amino acids and thus may contribute to conformational stability. All of the residues thought to contribute to protein structure, with the exception of Val316 in TM7, are clustered at one side of the cytoplasmic orifice of the central pore and facing outward (Fig. 5B). These Hxt2 conformational residues are also more hydrophobic or bulkier (or both) than the corresponding Hxt1 residues (Table 1). The implications of these observations warrant further investigation. A hydrophobic residue (Ile) in TM7 of GLUT7 was also shown to be important for substrate recognition (19Manolescu A. Salas-Burgos A.M. Fischbarg J. Cheeseman C.I. J. Biol. Chem. 2005; 280: 42978-42983Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar).FIGURE 5Stereograms showing the putative locations of residues in TMs 1, 5, 7, and 8 of Hxt2 identified as important or supportive for high affinity and high capacity glucose transport activity. For clarity, only TMs 1, 5, 7, 8, and 10 are shown in a lateral view (A) or in a perpendicular view from the cytoplasmic side (B). Asparagine 331, which was suggested to be important for substrate recognition, is shown in red, whereas other important residues (Leu59, Leu61, Leu201, and Phe366) that may play a structural role are shown in dark green, and four supportive residues (Phe198, Ala363, Val316, and Ala368) are shown in light green. Phenylalanine 431, which was identified previously as important for substrate recognition (6Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), is also shown in red.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Conclusions—Of the ∼250 amino acid residues in the 12 TMs of Hxt2, five have been found to be important for high affinity and high capacity glucose transport and three to be supportive of maximal activity. A working homology model suggests that among these eight residues, Asn331 interacts directly with the substrate and the others contribute to the protein conformation required for high affinity transport. Our present results suggest the possibility that fine structural tuning is important for construction of a high affinity and high capacity transporter. A subtle modification in structure may bring about a substantial change in transport characteristics. It also seems likely that most residues responsible for the direct recognition of glucose are common to both Hxt2 and Hxt1 and were not isolated in the present study. Previous results (6Kasahara M. Shimoda E. Maeda M. J. Biol. Chem. 1997; 272: 16721-16724Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) and our present data suggest that Asn331 (TM7) and Phe431 (TM10) are two candidates for substrate-interacting residues. The functional role of Asn331 should be the subject of further study. We thank A. L. Kruckeberg (Gothia Yeast Solutions, Gothenburg, Sweden) for yeast strain KY73 and M. Maeda for technical assistance.
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Cocaine blocks the normal role of the dopamine transporter (DAT) in terminating dopamine signaling through molecular interactions that are only partially understood. Cocaine analog structure-activity studies have suggested roles for both cationic and aromatic interactions among DAT, dopamine, and cocaine. We hypothesized that phenylalanine residues lying in putative DAT transmembrane (TM) domains were good candidates to contribute to aromatic and/or cationic interactions among DAT, dopamine, and cocaine. To test this idea, we characterized the influences of alanine substitution for each of 29 phenylalanine residues lying in or near a putative DAT TM domain. Cells express 22 mutants at near wild-type levels, manifest by DAT immunohistochemistry and binding of the radiolabeled cocaine analog [3H](−)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (CFT). Seven mutants fail to express at normal levels. Four mutations selectively reduce cocaine analog affinities. Alanine substitutions at Phe76, Phe98, Phe390, and Phe361 located in TM domains 1 and 2, the fourth extracellular loop near TM 4 and in TM 7, displayed normal affinities for dopamine but 3- to 8-fold reductions in affinities for CFT. One TM 3 mutation, F155A, selectively decreased dopamine affinity to less than 3% of wild-type levels while reducing CFT affinity less than 3-fold. In a current DAT structural model, each of the residues at which alanine substitution selectively reduces cocaine analog or dopamine affinities faces a central transporter cavity, whereas mutations that influence expression levels are more likely to lie at potential helix/helix interfaces. Specific, overlapping sets of phenylalanine residues contribute selectively to DAT recognition of dopamine and cocaine.
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Tropane
Tyramine
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Human and bovine dopamine transporters (DAT) demonstrate discrete functional differences in dopamine (DA), 1-methyl-4-phenylpyridium (MPP(+)) transport, and cocaine analog binding. In a previous study, the functional analyses on the chimeras of human and bovine DAT have revealed that the region from residues 133 through 186 (encompassing the third transmembrane domain) is responsible for the substrate transport and cocaine analog binding. The present study has been carried out to determine the specific amino acid(s) conferring DAT functions by interchanging the amino acid residues in the corresponding region between human and bovine DAT. As described previously, the DA, MPP(+) transport, and 2beta-carbomethoxy-3beta-(4-fluorophenyl)tropane (CFT) binding almost disappeared in chimera hb3 in which the region from residues 133 through 186 of bovine DAT was substituted into human DAT. Replacement of isoleucine, residue 152 of chimera hb3 (bovine DAT sequence), with valine, the human DAT residue at the identical position, remarkably restored the substrate transport and CFT binding to 76% to 98% of the human DAT values. Similarly, substitution of isoleucine for valine at position 152 in the human DAT reduced the substrate transport and CFT binding by 57% to 97%. Among other amino acids tested at position 152 of the chimera hb3, only alanine resulted in small but significant increases in the DAT functions ranging from 16 to 34%. Thus, valine at position 152 plays a crucial role for molecular mechanisms underlying the interactions of DA, MPP(+), and CFT with human DAT.
Tropane
Alanine
Isoleucine
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The structures of the leucine transporter, drosophila dopamine transporter, and human serotonin transporter show a secondary binding site (designated S2 ) for drugs and substrate in the extracellular vestibule toward the membrane exterior in relation to the primary substrate recognition site (S1 ). The present experiments are aimed at disrupting S2 by mutating Asp476 and Ile159 to Ala. Both mutants displayed a profound decrease in [(3) H]DA uptake compared with wild-type associated with a reduced turnover rate kcat . This was not caused by a conformational bias as the mutants responded to Zn(2+) (10 μM) similarly as WT. The dopamine transporters with either the D476A or I159A mutation both displayed a higher Ki for dopamine for the inhibition of [3H](-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane binding than did the WT transporter, in accordance with an allosteric interaction between the S1 and S2 sites. The results provide evidence in favor of a general applicability of the two-site allosteric model of the Javitch/Weinstein group from LeuT to dopamine transporter and possibly other monoamine transporters. X-ray structures of transporters closely related to the dopamine (DA) transporter show a secondary binding site S2 in the extracellular vestibule proximal to the primary binding site S1 which is closely linked to one of the Na(+) binding sites. This work examines the relationship between S2 and S1 sites. We found that S2 site impairment severely reduced DA transport and allosterically reduced S1 site affinity for the cocaine analog [(3) H]CFT. Our results are the first to lend direct support for the application of the two-site allosteric model, advanced for bacterial LeuT, to the human DA transporter. The model states that, after binding of the first DA molecule (DA1 ) to the primary S1 site (along with Na(+) ), binding of a second DA (DA2 ) to the S2 site triggers, through an allosteric interaction, the release of DA1 and Na(+) into the cytoplasm.
Neurotransmitter transporter
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Abstract: Little information is available on the role of Na + , K + , and Cl ‐ in the initial event of uptake of substrates by the dopamine transporter, i.e., the recognition step. In this study, substrate recognition was studied via the inhibition of binding of [ 3 H]WIN 35,428 [2β‐carbomethoxy‐3β‐(4‐fluorophenyl)[ 3 H]tropane], a cocaine analogue, to the human dopamine transporter in human embryonic kidney 293 cells. D‐Amphetamine was the most potent inhibitor, followed by p ‐tyramine and, finally, dl ‐octopamine; respective affinities at 150 m M Na + and 140 m M Cl ‐ were 5.5, 26, and 220 μ M . For each substrate, the decrease in the affinity with increasing [K + ] could be fitted to a competitive model involving the same inhibitory cation site (site 1) overlapping with the substrate domain as reported by us previously for dopamine. K + binds to this site with an apparent affinity, averaged across substrates, of 9, 24, 66, 99, and 134 m M at 2, 10, 60, 150, and 300 m M Na + , respectively. In general, increasing [Na + ] attenuated the inhibitory effect of K + in a manner that deviated from linearity, which could be modeled by a distal site for Na + , linked to site 1 by negative allosterism. The presence of Cl ‐ did not affect the binding of K + to site 1. Models assuming low binding of substrate in the absence of Na + did not provide fits as good as models in which substrate binds in the absence of Na + with appreciable affinity. The binding of dl ‐octopamine and p ‐tyramine was strongly inhibited by Na + , and stimulated by Cl ‐ only at high [Na + ] (300 m M ), consonant with a stimulatory action of Cl ‐ occurring through Na + disinhibition.
Octopamine (neurotransmitter)
Tyramine
Tropane
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Polar amino acids lying within three hydrophobic regions of the dopamine transporter (DAT) are analogous to those important for ligand recognition by catecholamine receptors. Possible functional significance of these amino acids was examined by expressing DAT cDNAs mutated in these polar residues. Replacement of aspartate at position 79 with alanine, glycine, or glutamate dramatically reduced uptake of [3H]dopamine and the tritium-labeled Parkinsonism-inducing neurotoxin 1-methyl-4-phenylpyridinium (MPP+) and reduced the mutants' affinity for the tritium-labeled cocaine analog (-)-2 beta-carbomethoxy-3 beta-(4-fluorophenyl)tropane (CFT) without affecting Bmax. Replacement of the serine residues at positions 356 and 359 in the seventh hydrophobic region by alanine or glycine caused reductions in [3H]dopamine and [3H]MPP+ uptake, whereas [3H]CFT binding was less affected. Substitution of two serines in the eighth hydrophobic region yielded wild-type values for [3H]dopamine and [3H]MPP+ uptake and [3H]CFT binding. These results demonstrate that aspartate and serine residues lying within the first and seventh hydrophobic putative transmembrane regions are crucial for DAT function and provide identification of residues differentially important for cocaine binding and for dopamine uptake.
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Alanine
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