Identification and Mutational Analysis of Amino Acid Residues Involved in Dipyridamole Interactions with Human and Caenorhabditis elegans Equilibrative Nucleoside Transporters

The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile429 in TM 11 of CeENT1 and Met33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport. The equilibrative nucleoside transporters, hENT1 and CeENT1 from humans and Caenorhabditis elegans, respectively, are inhibited by nanomolar concentrations of dipyridamole and share a common 11-transmembrane helix (TM) topology. Random mutagenesis and screening by functional complementation in yeast for clones with reduced sensitivities to dipyridamole yielded mutations at Ile429 in TM 11 of CeENT1 and Met33 in TM 1 of hENT1. Mutational analysis of the corresponding residues of both proteins suggested important roles for these residues in competitive inhibition of hENT1 and CeENT1 by dipyridamole. To verify the roles of these residues in dipyridamole interactions, hENT2, which naturally exhibits low dipyridamole sensitivity, was mutated to contain side chains favorable for high affinity dipyridamole binding (i.e. a Met at the TM 1 and/or an Ile at the TM 11 positions). The single mutants exhibited increased hENT2 sensitivity to inhibition by dipyridamole, and the double mutant was the most sensitive, with an IC50 value that was only 2% of that of wild type. Functional analysis of the TM 1 and 11 mutants of hENT1 and CeENT1 revealed that Ala and Thr in the TM 1 and 11 positions, respectively, impaired uridine and adenosine transport and that Leu442 of hENT1 was involved in permeant selectivity. Mechanistic and structural models of dipyridamole interactions with the TM 1 and 11 residues are proposed. This study demonstrated that the corresponding residues in TMs 1 and 11 of hENT1, hENT2, and CeENT1 are important for dipyridamole interactions and nucleoside transport. Nucleosides are hydrophilic molecules that require the presence of integral membrane transporter proteins to move across biological membranes (1Vickers M.F. Young J.D. Baldwin S.A. Cass C.E. Emerging Therapeutic Targets. 2000; 4: 515-539Crossref Scopus (14) Google Scholar, 2Cass C.E. Young J.D. Baldwin S.A. Cabrita M.A. Graham K.A. Griffiths M. Jennings L.L. Mackey J.R. Ng A.M.L. Ritzel M.W.L. Vickers M.F. Yao S.Y.M. Amidon G.L. Sadee W. Membrane Transporters as Drug Targets. 12. Kluwer Academic/Plenum Publishers, 1999: 313-332Google Scholar, 3Griffith D.A. Jarvis S.M. Biochim. Biophys. Acta. 1996; 1286: 153-181Crossref PubMed Scopus (453) Google Scholar). Nucleoside transporters are also responsible for the cellular uptake of many nucleoside analogs used in the treatment of solid tumors, hematologic malignancies, and viral diseases (4Baldwin S.A. Mackey J.R. Cass C.E. Young J.D. Mol. Med. Today. 1999; 5: 216-224Abstract Full Text PDF PubMed Scopus (303) Google Scholar, 5Cass C.E. Georgopapadakou N.H. Drug Transport in Antimicrobial and Anticancer Chemotherapy. Marcel Deker, New York1995: 403-451Google Scholar). Extracellular concentrations of adenosine, a signaling molecule that binds to G protein-coupled cell surface receptors, are regulated by nucleoside transporters (6Van Belle H. Cardiovasc. Res. 1993; 27: 68-76Crossref PubMed Scopus (114) Google Scholar). Members of the equilibrative nucleoside/nucleobase transporter (ENT) 1The abbreviations used are: ENT, equilibrative nucleoside transporter; NBMPR, nitrobenzylmercaptopurine ribonucleoside (6-[(4-nitrobenzyl)thiol]-9-β-d-ribofuranosyl purine); TM, transmembrane helix; Ce, C. elegans; h, human; r, rat; CMM, complete minimal medium; GLU, glucose; MTX, methotrexate; SAA, sulfanilamide; KTK, KY114 yeast-producing recombinant Herpes simplex thymidine kinase. family have been identified in many eukaryotes, and most mediate facilitated diffusion of nucleosides, although some members are proton-coupled (7Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar). All ENT family transporters appear to share a common membrane architecture with 11 transmembrane helices (TMs), a large cytoplasmic loop between TMs 6 and 7, and in many cases a large glycosylated loop between TMs 1 and 2 (8Sundaram M. Yao S.Y. Ingram J.C. Berry Z.A. Abidi F. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2001; 276: 45270-45275Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Four ENT family members have been identified by molecular cloning from human tissues. The potent transport inhibitor nitrobenzylmercaptopurine ribonucleoside (NBMPR) can be used to functionally distinguish human ENT1 (hENT1), which mediates equilibrative NBMPR-sensitive (es) transport activity, from hENT2, which mediates equilibrative NBMPR-insensitive (ei) transport activity (9Griffiths M. Beaumont N. Yao S.Y. Sundaram M. Boumah C.E. Davies A. Kwong F.Y. Coe I. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (357) Google Scholar, 10Griffiths M. Yao S.Y. Abidi F. Phillips S.E. Cass C.E. Young J.D. Baldwin S.A. Biochem. J. 1997; 328: 739-743Crossref PubMed Scopus (226) Google Scholar, 11Crawford C.R. Patel D.H. Naeve C. Belt J.A. J. Biol. Chem. 1998; 273: 5288-5293Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Moreover, hENT1 is 2–3 orders of magnitude more sensitive to inhibition by coronary vasodilator drugs such as dilazep and dipyridamole than hENT2 (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). No functional characteristics have been published for hENT3 or hENT4 (7Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar, 13Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. Pfluegers Arch. Eur. J. Physiol. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar, 14Acimovic Y. Coe I.R. Mol. Biol. Evol. 2002; 19: 2199-2210Crossref PubMed Scopus (76) Google Scholar). Five ENT family members have been identified by sequence homology in the genomic data base for Caenorhabditis elegans (15Appleford P.J. Griffiths M. Yao S.Y. Ng A.M. Chomey E.G. Isaac Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2004; PubMed Scopus Google Scholar). C. elegans ENT1 and 2 and share sequence and have been functionally and to be sensitive to inhibition by the coronary vasodilator dipyridamole to and dilazep (15Appleford P.J. Griffiths M. Yao S.Y. Ng A.M. Chomey E.G. Isaac Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2004; PubMed Scopus Google Scholar). have the of dipyridamole which to with and for binding to the of transporters at nanomolar although have been at concentrations S.M. Young J.D. J. Physiol. Scopus Google Scholar, S.M. Young J.D. Biochem. J. PubMed Scopus Google Scholar, S.M. Mol. Google Scholar, J.R. Mol. Google Scholar). The of dipyridamole binding to CeENT1 been of the residues involved in dipyridamole interactions with is a of hENT1 mutants for clones with reduced sensitivity to in the of of hENT1 and hENT2 as important of only dilazep also dipyridamole interactions (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The of study was to a to residues of hENT1 and CeENT1 involved in dipyridamole that dipyridamole was a competitive inhibitor of both uridine and adenosine by recombinant hENT1 and CeENT1 in the yeast a that a common structural for dipyridamole binding by the hENT1 and CeENT1 to mutagenesis and for clones by complementation in The hENT1 clones mutations in Met33 in TM the CeENT1 clones mutations in Ile429 in TM mutations of the corresponding residues of both proteins and for dipyridamole sensitivity and uridine transport The mutations that yielded the dipyridamole sensitivities for both proteins in TM 1 and Ile in TM hENT2 to in dipyridamole interactions with important ENT family The hENT1 and CeENT1 mutants also for to transport uridine and that dipyridamole sensitivity uridine to analysis to the competitive of inhibition been by the Mechanistic and structural models of TMs 1 and 11 with to dipyridamole and permeant interactions are proposed. The that the TM 1 and 11 residues important for dipyridamole interactions and uridine transport by hENT1 and and was the yeast used to KTK, which recombinant simplex thymidine Young J.D. Cass C.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google and which a in the the uridine M.F. Yao S.Y. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). by of the the 1993; PubMed Scopus Google and a A. Res. PubMed Scopus Google Scholar). in complete minimal yeast base to and 2% with and 2% in in with and Random the hENT1 or hENT2 to or as (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The CeENT1 was by the and and to Random mutagenesis of and was by the in the of for to 1 to the of A. M. Mol. Biol. 1996; Google Scholar). The mutations in the and and the and and the and in sequence Google Scholar). The hENT1 double mutants by the the or mutations with the corresponding in the the The CeENT1 double mutants by the the or with the corresponding in the the and of complementation was the of recombinant hENT1 or CeENT1 in yeast to thymidine of M.F. Sundaram M. Young J.D. Baldwin S.A. Cass C.E. Biochem. J. 1999; PubMed Google Scholar). with or a A. Res. PubMed Scopus Google at and at 6 at a of with an of at for in the presence of and complementation was dipyridamole was also was which with thymidine and dipyridamole and at for with to dipyridamole inhibition of complementation in 1 of for 2 and with thymidine and dipyridamole. The mutant CeENT1 from the yeast by and Nucleoside Transport in or of the mutant transporters in to in and to Transport at and and dipyridamole from and from The transport in as J. F. Vickers M.F. I. Baldwin S.A. Young J.D. Cass C.E. Mol. PubMed Scopus Google Scholar, M.F. J. F. I. Baldwin S.A. Young J.D. Cass C.E. Nucleosides 2004; PubMed Scopus Google Scholar). of yeast to of in the of uridine transport yeast recombinant for of (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google single of or used to of transport. uridine transport activity in yeast by the presence of the was by uptake in the presence of which with transport M. Eur. J. Biochem. PubMed Scopus Google Scholar). a and with The corresponding to of the from with and to for of by dipyridamole the yeast for with dipyridamole to of the inhibitor with binding the of permeant as (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar, S.M. Young J.D. Biochem. J. PubMed Scopus Google Scholar, Mol. Biol. Google Scholar, Mol. Google Scholar, M. Young J.D. Biochem. J. PubMed Scopus Google Scholar). of of hENT1 and dipyridamole inhibited hENT1 and CeENT1 by a common yeast recombinant hENT1 or CeENT1 with or in the or presence of concentrations of dipyridamole. analysis M. Biochem. J. PubMed Scopus Google of these data suggested that dipyridamole was a competitive inhibitor of adenosine transport for both with of 1 and respectively, for hENT1 and CeENT1 and and a yeast recombinant hENT1 or CeENT1 with or in the or presence of concentrations of dipyridamole. analysis of the data suggested that dipyridamole was also a competitive inhibitor of uridine transport for both hENT1 and CeENT1 with of and and and M. Biochem. J. PubMed Scopus Google and for dipyridamole inhibition of the hENT1, or hENT2 and 11 hENT2 in a Random and of of yeast in the presence of and in of and J. Biochem. PubMed Google Scholar). yeast membrane transport for thymidine M. Eur. J. Biochem. PubMed Scopus Google of hENT1 in transport of which is to by recombinant simplex thymidine in the Young J.D. Cass C.E. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M.F. Sundaram M. Young J.D. Baldwin S.A. Cass C.E. Biochem. J. 1999; PubMed Google Scholar, Vickers M. Cass C.E. Biochem. Biophys. Res. 1997; PubMed Scopus Google Scholar). The yeast and mutated by in the of and by complementation in yeast for functional mutants with reduced sensitivity to dipyridamole (i.e. for mutants that thymidine in the presence of of a hENT1 mutant for dipyridamole in the of of which mutations in which to a identified in of a hENT1 for to an inhibitor of hENT1 and hENT2 (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google that is to dipyridamole 2 for Four of the CeENT1 in the of of which contain mutations in the CeENT1 and of which a in in the of Ile429 in TM 11 to sequence of TM 1 of and C. elegans ENT1 and revealed that Met is at in hENT1, Ile is in the transporters and hENT2 The corresponding TM 1 in CeENT1 and (i.e. Ile was the as in the TM 1 also which are in transporters, that be involved in (7Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google these residues to and of hENT1. sequence of TM 11 of and C. elegans ENT1 and revealed that the transporters contain a both CeENT1 and contain an Ile at the corresponding (i.e. in hENT1 and in This suggested that the by or Ile responsible for the in dipyridamole sensitivities of the the of TM 11 is the of a (7Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar, M.A. C.E. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Mol. Biol. 2000; PubMed Scopus Google and although is of the mutations at the of TM 11 and of the for of hENT1, and the of mutations at Met33 and Leu442 of hENT1 and and Ile429 of mutagenesis was at these of the mutants and and and exhibited transport activity, and hENT1, or of the functionally mutants in the or presence of concentrations of dipyridamole and and The and mutants IC50 and than that of hENT1, and IC50 to that of hENT1. and IC50 that and respectively, than that of the of and respectively, to and of the hENT1 that the dipyridamole sensitivity of CeENT1 was by the TM 11 the TM 1 the sensitivity of hENT1 was by both TM 1 and 11 which is to dipyridamole S.Y. Ng A.M. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google an Ile at the TM 1 and a at the TM 11 The for dipyridamole inhibition of yielded an IC50 value of which was and than the for CeENT1 and hENT1, sensitive to dipyridamole inhibition than hENT1 or was of with dipyridamole. both hENT1 and contain a at the TM 11 suggested that to dipyridamole binding by both transporters than responsible for in both hENT1 and the presence of a Met at the TM 1 and an Ile at the TM 11 was with high sensitivities to dipyridamole. residues by the CeENT1 TM 11 mutants with reduced sensitivities and with the TM 1 mutant with high sensitivity and the for dipyridamole inhibition of uridine transport by the double mutants and in in I. an IC50 value that was of that of and than that of CeENT1 also an IC50 value that was of that of and to that of The and complete of sensitivity to dipyridamole in the mutants by the presence of a Met at suggested a functional between TMs 1 and 11 in binding of dipyridamole by To these the hENT1 TM 1 which sensitive to dipyridamole than wild hENT1, with the The for dipyridamole inhibition of uridine transport by and an IC50 value that was of that of and than that of hENT1 also an IC50 value that was than that of the and than that of hENT1. The that the of the to hENT1, and IC50 for dipyridamole inhibition a functional between TMs 1 and 11 in dipyridamole binding by hENT1, to the for the of dipyridamole sensitivity in ENT family members from suggested that both the TM 1 and 11 to dipyridamole for of hENT2 and the TM 1 and 11 also to the of hENT2 to dipyridamole, mutagenesis was to and The for dipyridamole inhibition of uridine transport by hENT2, or the hENT2 and and IC50 that 11 and respectively, of that of The double an IC50 value that was 2% of that of The of these mutations suggested that both the TM 1 and 11 to the of dipyridamole with of Transport for hENT1, and the of mutations at the TM 1 or 11 permeant transport the and of uridine transport by yeast hENT1, or of the mutants by transport at concentrations of and and uridine transport to and of 6 and respectively, and of and CeENT1 a affinity for uridine than hENT1 was with the by of these proteins in (15Appleford P.J. Griffiths M. Yao S.Y. Ng A.M. Chomey E.G. Isaac Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2004; PubMed Scopus Google Scholar). The value for hENT1 was than that (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google the uptake by the in study (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google was in the study by uptake in the presence of of uridine and adenosine transport for hENT1, and the and in a between the for hENT1 and a increased value and a reduced was for CeENT1 in that a increased value and a reduced value to of data suggested that the presence of an Ala at the TM 1 in reduced and transport of both hENT1 and the mutations in TM of the and Ile residues at the TM 11 in hENT1 and respectively, only uridine transport a value than hENT1, and and CeENT1 the of these residues by Thr reduced the of the transporters for a value that was than that of hENT1 and and a value that was than that of CeENT1 and The of the TM 11 double mutants of hENT1 and CeENT1 with of the corresponding single although the for and respectively, and than that of wild hENT1, the value of the double mutant was exhibited a to the of single to of wild the double mutant which the Thr at the TM 11 a value that was only than that of wild CeENT1 also than that of the corresponding single mutant (i.e. these that a Thr at the TM 11 the uridine transport of both hENT1 and of Transport for hENT1 and the of the hENT1 TM 1 and 11 mutations of adenosine the of adenosine transport was an value that was of that of wild hENT1 to of hENT1. an value that was than that of hENT1 transport by at concentrations that the affinity of mutant for adenosine was which was with the high value for uridine transport by mutant of of hENT1 and CeENT1 the of the TM 1 and 11 residues the competitive of dipyridamole inhibition of hENT1 and for uridine transport by and 7, and mutants dipyridamole sensitivities uridine transport and inhibited and with that to the corresponding IC50 in the that the of inhibition was by these the double mutants and also to analysis 7, and inhibited with dipyridamole sensitivity with the single mutants in and and also exhibited uridine transport that to of the corresponding wild transporters sequence hENT1 and CeENT1 both dipyridamole with high and with to the uridine and This common of inhibition suggested that the residues involved in dipyridamole binding by hENT1 and CeENT1 be and was with the of that suggested that the with the permeant binding of ENT transporters S.M. Young J.D. J. Physiol. Scopus Google Scholar, S.M. Mol. Google Scholar, Cass C.E. Mol. 18: Google Scholar). Random mutagenesis and screening for dipyridamole by functional complementation in yeast identified Met33 in TM 1 of hENT1 and Ile429 in TM 11 of CeENT1 as sequence of the high dipyridamole sensitivity transporters and the low dipyridamole sensitivity transporters and revealed that the TM 1 and 11 by residues with large side Met or Ile in TM 1 and or Ile in TM 11 although in the transporters a Met at is with high dipyridamole sensitivity (12Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google CeENT1 and contain an Ile at the corresponding although the transporters contain a at the TM 11 of dipyridamole the corresponding in CeENT1 and is The sequence suggested that binding of dipyridamole is and from residues from of ENT The of a in members of the ENT family is by the in study that only hENT1 and CeENT1 also hENT2 and exhibited dipyridamole sensitivity, to The with the of (9Griffiths M. Beaumont N. Yao S.Y. Sundaram M. Boumah C.E. Davies A. Kwong F.Y. Coe I. Cass C.E. Young J.D. Baldwin S.A. Nat. Med. 1997; 3: 89-93Crossref PubMed Scopus (357) Google Scholar, 10Griffiths M. Yao S.Y. Abidi F. Phillips S.E. Cass C.E. Young J.D. Baldwin S.A. Biochem. J. 1997; 328: 739-743Crossref PubMed Scopus (226) Google Scholar, P.J. Griffiths M. Yao S.Y. Ng A.M. Chomey E.G. Isaac Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2004; PubMed Scopus Google Scholar, S.Y. Ng A.M. Griffiths M. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, A. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the mutagenesis and dipyridamole and suggested that residues in both TM 1 and 11 to dipyridamole binding by hENT1, and a to the of the mutagenesis This is that TMs 1 and 11 are transmembrane helices (8Sundaram M. Yao S.Y. Ingram J.C. Berry Z.A. Abidi F. Cass C.E. Baldwin S.A. Young J.D. J. Biol. Chem. 2001; 276: 45270-45275Abstract Full Text Full Text PDF PubMed Scopus (124) Google and the that TMs 1 and 11 are in to a that be in only the of the TM 1 and 11 residues are in the is that dipyridamole interactions are from residues of the TMs 1 and 11 contain residues that be involved in (7Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google is that of the residues in and Leu442 of hENT1 and and Ile429 of the of the TM 1 and 11 The mutagenesis suggested that the TM 1 to Met and the TM 11 to Ile that the helices the TM 1 to and the TM 11 to or Thr that move the helices The mutagenesis also suggested that the TM 1 is a of of dipyridamole with wild hENT1, the TM 11 is to with dipyridamole The that the have dipyridamole binding to hENT1 and the TMs 1 and 11 such that both residues can to dipyridamole binding Met was Ile at the TM 1 is to high of and PubMed Scopus Google Scholar). in and the Ile in the TM 1 with dipyridamole, although the of the was the the of TM 1 is by the that mutant was functionally to wild TM 1 be from TM and the of dipyridamole with hENT1 TM 11 the that TMs 1 and 11 to to dipyridamole binding suggested that these helices are in CeENT1 than in hENT1 and hENT2 and that TM TM is a of dipyridamole in the wild The and mutations be to have the dipyridamole sensitivity of CeENT1 of the TM 11 to or Thr to move helix from TM 1 dipyridamole to the mutants with reduced The dipyridamole sensitivity of the CeENT1 TM 11 mutants was by the which a of inhibitor of uridine and adenosine transport by the TM 1 and 11 mutants of hENT1 and CeENT1 revealed for both an Ala at the TM 1 in a value and reduced reduced of transporter Thr at the TM 11 in increased for uridine and adenosine transport by both proteins and a common for the TM 1 and 11 in uridine transport by hENT1 and a value with that of hENT1 the impaired adenosine transport and uridine transport suggested that the of the permeant binding was to a favorable for adenosine transport and that the permeant a was for adenosine an Ile was for dipyridamole structural at for with adenosine and dipyridamole. is that the of wild hENT1 in is for adenosine the of in is for dipyridamole The analysis of of the mutants revealed that the competitive of dipyridamole inhibition was by the that the TM 1 and 11 residues identified in study or the that with the permeant binding the in is although dipyridamole be to TM 1 or 11 the helices be for dipyridamole to to the permeant binding To the that the TM 1 and 11 residues identified in study the permeant have models of TMs 1 and 11 of hENT1 of the of and in these helices in of ENT family (7Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google for the of these residues and a for the corresponding to Leu442 in hENT1 and Ile429 in CeENT1 is by a a of the TM 11 helix that is by hydrophilic residues the of the TM 11 helix is and is by residues is that of the helix is in with the of the the Leu442 and Ile429 the permeant of the The of Leu442 permeant in the permeant TM 1 is than TM with by hydrophilic residues the in which Met33 and are and many residues are in TM from TM 11 in that of these are hENT1 and which in the ENT family are or by residues (7Hyde R.J. Cass C.E. Young J.D. Baldwin S.A. Mol. Membr. Biol. 2001; 18: 53-63Crossref PubMed Google Scholar). are to be involved in and presence in TM 1 that TM 1 is a of helices than to the the of hENT1 Met33 for dipyridamole that the of TMs 1 and Met33 to This be in have identified in TMs 1 and 11 of hENT1 and that are functionally to dipyridamole binding and permeant transport. The of these residues in dipyridamole interactions with ENT family proteins was in analysis of The data are with the TM 1 and 11 residues of the dipyridamole binding and the permeant The of these require such as a analysis or of structural data the of the

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