Molecular determinants of acidic pH-dependent transport of human equilibrative nucleoside transporter 3
2017; Elsevier BV; Volume: 292; Issue: 36 Linguagem: Inglês
10.1074/jbc.m117.787952
ISSN1083-351X
AutoresMd Fazlur Rahman, Candice C. Askwith, Rajgopal Govindarajan,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoEquilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [3H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5–7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity. Equilibrative nucleoside transporters (ENTs) translocate hydrophilic nucleosides across cellular membranes and are essential for salvage nucleotide synthesis and purinergic signaling. Unlike the prototypic human ENT members hENT1 and hENT2, which mediate plasma membrane nucleoside transport at pH 7.4, hENT3 is an acidic pH-activated lysosomal transporter partially localized to mitochondria. Recent studies demonstrate that hENT3 is indispensable for lysosomal homeostasis, and that mutations in hENT3 can result in a spectrum of lysosomal storage-like disorders. However, despite hENT3's prominent role in lysosome pathophysiology, the molecular basis of hENT3-mediated transport is unknown. Therefore, we sought to examine the mechanistic basis of acidic pH-driven hENT3 nucleoside transport with site-directed mutagenesis, homology modeling, and [3H]adenosine flux measurements in mutant RNA-injected Xenopus oocytes. Scanning mutagenesis of putative residues responsible for pH-dependent transport via hENT3 revealed that the ionization states of Asp-219 and Glu-447, and not His, strongly determined the pH-dependent transport permissible-impermissible states of the transporter. Except for substitution with certain isosteric and polar residues, substitution of either Asp-219 or Glu-447 with any other residues resulted in robust activity that was pH-independent. Dual substitution of Asp-219 and Glu-447 to Ala sustained pH-independent activity over a broad range of physiological pH (pH 5.5–7.4), which also maintained stringent substrate selectivity toward endogenous nucleosides and clinically used nucleoside drugs. Our results suggest a putative pH-sensing role for Asp-219 and Glu-447 in hENT3 and that the size, ionization state, or electronegative polarity at these positions is crucial for obligate acidic pH-dependent activity. Two human nucleoside transporter gene families encoding the human concentrative nucleoside transporters (SLC28; hCNTs) 2The abbreviations used are: (h)CNT, (human) concentrative nucleoside transporter; hENT, (human) equilibrative nucleoside transporter; PHID, pigmented hypertrichosis with insulin-dependent diabetes mellitus syndrome; SHML, sinus histiocytosis with massive lymphadenopathy; RDD, Rosai-Dorfman disease. 2The abbreviations used are: (h)CNT, (human) concentrative nucleoside transporter; hENT, (human) equilibrative nucleoside transporter; PHID, pigmented hypertrichosis with insulin-dependent diabetes mellitus syndrome; SHML, sinus histiocytosis with massive lymphadenopathy; RDD, Rosai-Dorfman disease. and equilibrative nucleoside transporters (SLC29; hENTs) are required for the membrane transport of hydrophilic nucleosides (1.Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. The equilibrative nucleoside transporter family, SLC29.Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar2.Gray J.H. Owen R.P. Giacomini K.M. The concentrative nucleoside transporter family, SLC28.Pflugers Arch. 2004; 447: 728-734Crossref PubMed Scopus (347) Google Scholar, 3.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar4.Pastor-Anglada M. Cano-Soldado P. Errasti-Murugarren E. Casado F.J. SLC28 genes and concentrative nucleoside transporter (CNT) proteins.Xenobiotica. 2008; 38: 972-994Crossref PubMed Scopus (65) Google Scholar). hCNTs are unidirectional, Na+-driven, high-affinity transporters with a defined substrate selectivity (2.Gray J.H. Owen R.P. Giacomini K.M. The concentrative nucleoside transporter family, SLC28.Pflugers Arch. 2004; 447: 728-734Crossref PubMed Scopus (347) Google Scholar, 4.Pastor-Anglada M. Cano-Soldado P. Errasti-Murugarren E. Casado F.J. SLC28 genes and concentrative nucleoside transporter (CNT) proteins.Xenobiotica. 2008; 38: 972-994Crossref PubMed Scopus (65) Google Scholar), i.e. hCNT1 transports pyrimidine nucleosides, hCNT2 transports purine nucleosides, and hCNT3 transports both nucleosides. hENTs are bidirectional, Na+-independent, high-capacity transporters with a wider substrate selectivity (1.Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. The equilibrative nucleoside transporter family, SLC29.Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar, 3.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar), i.e. all hENTs transport both purine and pyrimidine nucleosides with some ENTs even transporting nucleobases (ENT2 and ENT3), nucleotides (ENT3), and cyclic nucleotides (ENT2) (1.Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. The equilibrative nucleoside transporter family, SLC29.Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar, 3.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar). Expression of hCNTs is largely restricted to differentiated epithelial tissues, e.g. liver, kidney, brain, and intestine (5.Felipe A. Valdes R. Santo B. Lloberas J. Casado J. Pastor-Anglada M. Na+-dependent nucleoside transport in liver: two different isoforms from the same gene family are expressed in liver cells.Biochem. J. 1998; 330: 997-1001Crossref PubMed Scopus (65) Google Scholar6.Ngo L.Y. Patil S.D. Unadkat J.D. Ontogenic and longitudinal activity of Na+-nucleoside transporters in the human intestine.Am. J. Physiol. Gastrointest. Liver Physiol. 2001; 280: G475-G481Crossref PubMed Google Scholar, 7.Pennycooke M. Chaudary N. Shuralyova I. Zhang Y. Coe I.R. Differential expression of human nucleoside transporters in normal and tumor tissue.Biochem. Biophys. Res. Commun. 2001; 280: 951-959Crossref PubMed Scopus (159) Google Scholar, 8.Ritzel M.W. Yao S.Y. Huang M.Y. Elliott J.F. Cass C.E. Young J.D. Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1).Am. J. Physiol. 1997; 272: C707-C714Crossref PubMed Google Scholar9.Govindarajan R. Bakken A.H. Hudkins K.L. Lai Y. Casado F.J. Pastor-Anglada M. Tse C.M. Hayashi J. Unadkat J.D. In situ hybridization and immunolocalization of concentrative and equilibrative nucleoside transporters in the human intestine, liver, kidneys, and placenta.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2007; 293: R1809-R1822Crossref PubMed Scopus (122) Google Scholar), whereas hENTs are expressed ubiquitously (1.Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. The equilibrative nucleoside transporter family, SLC29.Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar, 3.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar, 10.Baldwin S.A. Yao S.Y. Hyde R.J. Ng A.M. Foppolo S. Barnes K. Ritzel M.W. Cass C.E. Young J.D. Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.J. Biol. Chem. 2005; 280: 15880-15887Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Studies to date suggest that both hCNTs and hENTs are essential for the salvage synthesis of nucleotides and purinergic signaling and determining the clinical efficacy of therapeutic nucleoside drugs (1.Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. The equilibrative nucleoside transporter family, SLC29.Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar2.Gray J.H. Owen R.P. Giacomini K.M. The concentrative nucleoside transporter family, SLC28.Pflugers Arch. 2004; 447: 728-734Crossref PubMed Scopus (347) Google Scholar, 3.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar4.Pastor-Anglada M. Cano-Soldado P. Errasti-Murugarren E. Casado F.J. SLC28 genes and concentrative nucleoside transporter (CNT) proteins.Xenobiotica. 2008; 38: 972-994Crossref PubMed Scopus (65) Google Scholar). In the past decade considerable knowledge has been gained into understanding the molecular determinants of hCNT transport particularly with respect to nucleoside and sodium binding (11.Cano-Soldado P. Gorraitz E. Errasti-Murugarren E. Casado F.J. Lostao M.P. Pastor-Anglada M. Functional analysis of the human concentrative nucleoside transporter-1 variant hCNT1S546P provides insight into the sodium-binding pocket.Am. J. Physiol. Cell Physiol. 2012; 302: C257-C266Crossref PubMed Scopus (12) Google Scholar, 12.Slugoski M.D. Ng A.M. Yao S.Y. Smith K.M. Lin C.C. Zhang J. Karpinski E. Cass C.E. Baldwin S.A. Young J.D. A proton-mediated conformational shift identifies a mobile pore-lining cysteine residue (Cys-561) in human concentrative nucleoside transporter 3.J. Biol. Chem. 2008; 283: 8496-8507Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar13.Slugoski M.D. Ng A.M. Yao S.Y. Lin C.C. Mulinta R. Cass C.E. Baldwin S.A. Young J.D. Substituted cysteine accessibility method analysis of human concentrative nucleoside transporter hCNT3 reveals a novel discontinuous region of functional importance within the CNT family motif (G/A)XKX3NEFVA(Y/M/F).J. Biol. Chem. 2009; 284: 17281-17292Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), and recently, the crystal structure of a CNT from Vibrio cholerae was elucidated (14.Johnson Z.L. Cheong C.G. Lee S.Y. Crystal structure of a concentrative nucleoside transporter from Vibrio cholerae at 2.4 Å.Nature. 2012; 483: 489-493Crossref PubMed Scopus (90) Google Scholar). However, hENTs are predominantly evaluated for functional roles using in vitro systems (e.g. Xenopus oocytes, lipid vesicles, and cultured cells) and mouse models with limited knowledge available on the structural basis of transport (1.Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. The equilibrative nucleoside transporter family, SLC29.Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar, 3.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar). Although site-directed mutagenesis studies and chimeric ENT analysis are beginning to identify specific transmembrane regions and residues involved in the substrate selectivity (endogenous nucleosides and therapeutic nucleoside analogs) and inhibitor (e.g. cardiovascular drugs)-binding characteristics of ENTs (15.Endres C.J. Unadkat J.D. Residues Met89 and Ser160 in the human equilibrative nucleoside transporter 1 affect its affinity for adenosine, guanosine, S6-(4-nitrobenzyl)-mercaptopurine riboside, and dipyridamole.Mol. Pharmacol. 2005; 67: 837-844Crossref PubMed Scopus (35) Google Scholar, 16.Visser F. Vickers M.F. Ng A.M. Baldwin S.A. Young J.D. Cass C.E. Mutation of residue 33 of human equilibrative nucleoside transporters 1 and 2 alters sensitivity to inhibition of transport by dilazep and dipyridamole.J. Biol. Chem. 2002; 277: 395-401Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar17.Visser F. Sun L. Damaraju V. Tackaberry T. Peng Y. Robins M.J. Baldwin S.A. Young J.D. Cass C.E. Residues 334 and 338 in transmembrane segment 8 of human equilibrative nucleoside transporter 1 are important determinants of inhibitor sensitivity, protein folding, and catalytic turnover.J. Biol. Chem. 2007; 282: 14148-14157Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), no crystal structures are available for any member of this family. Because hENTs and hCNTs are structurally unrelated and differ in the number of predicted transmembrane domains (11 versus 13), transport directionalities (bidirectional versus unidirectional), and cotransport (none versus sodium) requirements, it makes extrapolation of hCNT structural information to hENTs difficult. Furthermore, all hENTs (hENT1, -2, -3, and -4) have a very similar membrane topology as well as substrate and inhibitor-binding characteristics (1.Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. The equilibrative nucleoside transporter family, SLC29.Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar, 3.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar), yet individual hENTs drastically differ in their functional activity at various physiological pH as well as their cellular and subcellular distributions. In this regard, hENT1 and hENT2 function at pH 7.4, hENT3 functions at pH 5.5, and hENT4 functions at pH 5.5–6.5 (10.Baldwin S.A. Yao S.Y. Hyde R.J. Ng A.M. Foppolo S. Barnes K. Ritzel M.W. Cass C.E. Young J.D. Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.J. Biol. Chem. 2005; 280: 15880-15887Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 18.Barnes K. Dobrzynski H. Foppolo S. Beal P.R. Ismat F. Scullion E.R. Sun L. Tellez J. Ritzel M.W. Claycomb W.C. Cass C.E. Young J.D. Billeter-Clark R. Boyett M.R. Baldwin S.A. Distribution and functional characterization of equilibrative nucleoside transporter-4, a novel cardiac adenosine transporter activated at acidic pH.Circ. Res. 2006; 99: 510-519Crossref PubMed Scopus (152) Google Scholar). Consistent with their apparent transport maxima, hENT1 and hENT2 are primarily localized to the cell surface (1.Baldwin S.A. Beal P.R. Yao S.Y. King A.E. Cass C.E. Young J.D. The equilibrative nucleoside transporter family, SLC29.Pflugers Arch. 2004; 447: 735-743Crossref PubMed Scopus (594) Google Scholar, 3.Young J.D. Yao S.Y. Sun L. Cass C.E. Baldwin S.A. Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins.Xenobiotica. 2008; 38: 995-1021Crossref PubMed Scopus (159) Google Scholar), whereas hENT3 is localized to vesicular and tubular intracellular organelles including the late endosome/lysosome and mitochondria (10.Baldwin S.A. Yao S.Y. Hyde R.J. Ng A.M. Foppolo S. Barnes K. Ritzel M.W. Cass C.E. Young J.D. Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.J. Biol. Chem. 2005; 280: 15880-15887Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 19.Govindarajan R. Leung G.P. Zhou M. Tse C.M. Wang J. Unadkat J.D. Facilitated mitochondrial import of antiviral and anticancer nucleoside drugs by human equilibrative nucleoside transporter-3.Am. J. Physiol. Gastrointest. Liver Physiol. 2009; 296: G910-G922Crossref PubMed Scopus (112) Google Scholar). hENT4 is found in the plasma membrane of ventricular myocytes and vascular endothelial cells and transports adenosine in an acidic pH-dependent manner. However, hENT4 has been renamed as the plasma membrane monoamine transporter due to its relatively higher affinity to monoamines (e.g. serotonin) than adenosine (18.Barnes K. Dobrzynski H. Foppolo S. Beal P.R. Ismat F. Scullion E.R. Sun L. Tellez J. Ritzel M.W. Claycomb W.C. Cass C.E. Young J.D. Billeter-Clark R. Boyett M.R. Baldwin S.A. Distribution and functional characterization of equilibrative nucleoside transporter-4, a novel cardiac adenosine transporter activated at acidic pH.Circ. Res. 2006; 99: 510-519Crossref PubMed Scopus (152) Google Scholar). Recently, numerous reports from independent laboratories have identified exclusive mutations in hENT3 that cause a wide range of human clinical disorders (H syndrome, PHID (pigmented hypertrichosis with insulin-dependent diabetes mellitus syndrome) syndrome, SHML (sinus histiocytosis with massive lymphadenopathy)/RDD (Rosai-Dorfman Disease) syndrome, dysosteosclerosis, etc.) with intriguing resemblances to lysosome storage-like and mitochondrial disorders (20.Kang N. Jun A.H. Bhutia Y.D. Kannan N. Unadkat J.D. Govindarajan R. Human equilibrative nucleoside transporter-3 (hENT3) spectrum disorder mutations impair nucleoside transport, protein localization, and stability.J. Biol. Chem. 2010; 285: 28343-28352Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar21.Morgan N.V. Morris M.R. Cangul H. Gleeson D. Straatman-Iwanowska A. Davies N. Keenan S. Pasha S. Rahman F. Gentle D. Vreeswijk M.P. Devilee P. Knowles M.A. Ceylaner S. Trembath R.C. Dalence C. et al.Mutations in SLC29A3, encoding an equilibrative nucleoside transporter ENT3, cause a familial histiocytosis syndrome (Faisalabad histiocytosis) and familial Rosai-Dorfman disease.PLoS Genet. 2010; 6: e1000833Crossref PubMed Scopus (145) Google Scholar, 22.Molho-Pessach V. Lerer I. Abeliovich D. Agha Z. Abu Libdeh A. Broshtilova V. Elpeleg O. Zlotogorski A. The H syndrome is caused by mutations in the nucleoside transporter hENT3.Am. J. Hum. Genet. 2008; 83: 529-534Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 23.Campeau P.M. Lu J.T. Sule G. Jiang M.M. Bae Y. Madan S. Högler W. Shaw N.J. Mumm S. Gibbs R.A. Whyte M.P. Lee B.H. Whole-exome sequencing identifies mutations in the nucleoside transporter gene SLC29A3 in dysosteosclerosis, a form of osteopetrosis.Hum. Mol. Genet. 2012; 21: 4904-4909Crossref PubMed Scopus (72) Google Scholar, 24.Cliffe S.T. Kramer J.M. Hussain K. Robben J.H. de Jong E.K. de Brouwer A.P. Nibbeling E. Kamsteeg E.J. Wong M. Prendiville J. James C. Padidela R. Becknell C. van Bokhoven H. Deen P.M. et al.SLC29A3 gene is mutated in pigmented hypertrichosis with insulin-dependent diabetes mellitus syndrome and interacts with the insulin signaling pathway.Hum. Mol. Genet. 2009; 18: 2257-2265Crossref PubMed Scopus (84) Google Scholar, 25.Priya T.P. Philip N. Molho-Pessach V. Busa T. Dalal A. Zlotogorski A. H syndrome: novel and recurrent mutations in SLC29A3.Br. J. Dermatol. 2010; 162: 1132-1134Crossref PubMed Scopus (31) Google Scholar, 26.Ramot Y. Sayama K. Sheffer R. Doviner V. Hiller N. Kaufmann-Yehezkely M. Zlotogorski A. Early-onset sensorineural hearing loss is a prominent feature of H syndrome.Int. J. Pediatr. Otorhinolaryngol. 2010; 74: 825-827Crossref PubMed Scopus (17) Google Scholar, 27.Colmenero I. Molho-Pessach V. Torrelo A. Zlotogorski A. Requena L. Emperipolesis: an additional common histopathologic finding in H syndrome and Rosai-Dorfman disease.Am. J. Dermatopathol. 2012; 34: 315-320Crossref PubMed Scopus (24) Google Scholar, 28.Farooq M. Moustafa R.M. Fujimoto A. Fujikawa H. Abbas O. Kibbi A.G. Kurban M. Shimomura Y. Identification of two novel mutations in SLC29A3 encoding an equilibrative nucleoside transporter (hENT3) in two distinct Syrian families with H syndrome: expression studies of SLC29A3 (hENT3) in human skin.Dermatology. 2012; 224: 277-284Crossref PubMed Scopus (18) Google Scholar, 29.de Jesus J. Imane Z. Senée V. Romero S. Guillausseau P.J. Balafrej A. Julier C. SLC29A3 mutation in a patient with syndromic diabetes with features of pigmented hypertrichotic dermatosis with insulin-dependent diabetes, H syndrome and Faisalabad histiocytosis.Diabetes Metab. 2013; 39: 281-285Crossref PubMed Scopus (19) Google Scholar, 30.Elbarbary N.S. Tjora E. Molnes J. Lie B.A. Habib M.A. Salem M.A. Njølstad P.R. An Egyptian family with H syndrome due to a novel mutation in SLC29A3 illustrating overlapping features with pigmented hypertrichotic dermatosis with insulin-dependent diabetes and Faisalabad histiocytosis.Pediatr. Diabetes. 2013; 14: 466-472Crossref PubMed Scopus (15) Google Scholar31.Al-Haggar M. Salem N. Wahba Y. Ahmad N. Jonard L. Abdel-Hady D. El-Hawary A. El-Sharkawy A. Eid A.R. El-Hawary A. Novel homozygous SLC29A3 mutations among two unrelated Egyptian families with spectral features of H-syndrome.Pediatr. Diabetes. 2015; 16: 305-316Crossref PubMed Scopus (9) Google Scholar). However, the precise mechanism of disease pathogeneses and possible treatment interventions for these disorders is not understood largely due to a lack of information on the hENT3 structural and functional characteristics. We undertook this project to determine the mechanistic basis of acidic pH-dependent activity of hENT3 to better understand the subcellular transport characteristics of nucleosides. Here, we report the ionization states of Asp-219 and Glu-447 in hENT3 to determine the pH-dependent transport permissible-impermissible conformational states of the transporter by acting as pH sensors of the transport microenvironment. Transportability of hENT1 and -2 is generally tested at the physiological blood pH (pH 7.4), which is representative of the pH at the cell surface and within the interstitium. However, secondary localization in intracellular organelles is reported for many hENTs including hENT1 and hENT2, where the pH could vary significantly from pH 7.4 (10.Baldwin S.A. Yao S.Y. Hyde R.J. Ng A.M. Foppolo S. Barnes K. Ritzel M.W. Cass C.E. Young J.D. Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.J. Biol. Chem. 2005; 280: 15880-15887Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 19.Govindarajan R. Leung G.P. Zhou M. Tse C.M. Wang J. Unadkat J.D. Facilitated mitochondrial import of antiviral and anticancer nucleoside drugs by human equilibrative nucleoside transporter-3.Am. J. Physiol. Gastrointest. Liver Physiol. 2009; 296: G910-G922Crossref PubMed Scopus (112) Google Scholar, 32.Lee E.W. Lai Y. Zhang H. Unadkat J.D. Identification of the mitochondrial targeting signal of the human equilibrative nucleoside transporter 1 (hENT1): implications for interspecies differences in mitochondrial toxicity of fialuridine.J. Biol. Chem. 2006; 281: 16700-16706Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Conversely, hENT3 is primarily identified intracellularly with partial localization in lysosomes and mitochondria (10.Baldwin S.A. Yao S.Y. Hyde R.J. Ng A.M. Foppolo S. Barnes K. Ritzel M.W. Cass C.E. Young J.D. Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.J. Biol. Chem. 2005; 280: 15880-15887Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 19.Govindarajan R. Leung G.P. Zhou M. Tse C.M. Wang J. Unadkat J.D. Facilitated mitochondrial import of antiviral and anticancer nucleoside drugs by human equilibrative nucleoside transporter-3.Am. J. Physiol. Gastrointest. Liver Physiol. 2009; 296: G910-G922Crossref PubMed Scopus (112) Google Scholar). To test the functionality of hENTs in a wide range of physiologically relevant pH values, [3H]adenosine transport levels of hENT cRNA injected Xenopus oocytes were analyzed using extracellular solutions buffered between pH 4.5 and 8.5 (Fig. 1A). Surprisingly, the total flux of tritiated [3H]adenosine of hENT1- or hENT2-injected oocytes was robust even at a low pH. In fact, transport was not significantly different throughout the entire pH range tested (pH 4.5 to pH 8.5) (Fig. 1A). To investigate transport of hENT3, we utilized the Δ36hENT3 construct in which the N-terminal 36 amino acids of hENT3 are deleted to enable cell-surface localization of an otherwise intracellular transporter (10.Baldwin S.A. Yao S.Y. Hyde R.J. Ng A.M. Foppolo S. Barnes K. Ritzel M.W. Cass C.E. Young J.D. Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.J. Biol. Chem. 2005; 280: 15880-15887Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 20.Kang N. Jun A.H. Bhutia Y.D. Kannan N. Unadkat J.D. Govindarajan R. Human equilibrative nucleoside transporter-3 (hENT3) spectrum disorder mutations impair nucleoside transport, protein localization, and stability.J. Biol. Chem. 2010; 285: 28343-28352Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Conversely to the results with hENT1 and hENT2, the [3H]adenosine transport maxima of Δ36hENT3 was seen at pH 5.5 and was reduced drastically in transport solutions with pH values >6.5 (Fig. 1A). The transport maximum for hENT4 was observed at pH 5.5–6.5 as reported earlier (Fig. 1A) (18.Barnes K. Dobrzynski H. Foppolo S. Beal P.R. Ismat F. Scullion E.R. Sun L. Tellez J. Ritzel M.W. Claycomb W.C. Cass C.E. Young J.D. Billeter-Clark R. Boyett M.R. Baldwin S.A. Distribution and functional characterization of equilibrative nucleoside transporter-4, a novel cardiac adenosine transporter activated at acidic pH.Circ. Res. 2006; 99: 510-519Crossref PubMed Scopus (152) Google Scholar). The maximal flux of adenosine in oocytes varied significantly among hENTs, which is likely due to differences in affinities of hENTs to adenosine (Km effect) and their expression levels on the oocyte cell surface (Vmax effect) (Fig. 1A). To relate to the pH-dependent activities of hENTs, we utilized an R-value (defined as the ratio of the transport flux at pH 7.4 to that of at pH 5.5) as reported earlier for an Escherichia coli acidic pH-dependent arginine-agmatine antiporter (33.Wang S. Yan R. Zhang X. Chu Q. Shi Y. Molecular mechanism of pH-dependent substrate transport by an arginine-agmatine antiporter.Proc. Natl. Acad. Sci. U.S.A. 2014; 111: 12734-12739Crossref PubMed Scopus (15) Google Scholar). If the R value is close to 1, the transporter is completely pH independent, and if the R value is close to 0 the transporter is completely pH-dependent. The estimated R values (in parenthesis) for hENTs demonstrated a rank order of hENT3 (0.07) > hENT4 (0.63) > hENT2 (1.02) > hENT1 (1.02). This indicates that hENT3 is the strongest pH-dependent member of the family followed by hENT4, and hENT1 and hENT2 are pH-independent members (Fig. 1B). Similar to hENT3, the mouse ortholog, mENT3, is also an acidic pH-driven transporter (10.Baldwin S.A. Yao S.Y. Hyde R.J. Ng A.M. Foppolo S. Barnes K. Ritzel M.W. Cass C.E. Young J.D. Functional characterization of novel human and mouse equilibrative nucleoside transporters (hENT3 and mENT3) located in intracellular membranes.J. Biol. Chem. 2005; 280: 15880-15887Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). However, the fungal ortholog of ENT3, Fun26, which localizes to acidic vacuoles analogous to lysosomes in mammals, was reported to be a pH-independent transporter (34.Boswell-Casteel R.C. Johnson J.M. Duggan K.D. Roe-Žurž Z. Schmitz H. Burleson C. Hays F.A. FUN26 (function unknown now 26) protein from saccharomyces cerevisiae is a broad selectivity, high affinity, nucleoside and nucleobase transporter.J. Biol. Chem. 2014; 289: 24440-24451Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). These findings suggested a possible functional divergence of the pH-dependent transport activity within the mammalian ENT families. In search of the amino acid changes responsible for this divergence, we performed sequence identity and similarity comparisons among the human, mouse, and rat ENT family members using Clustal Omega and constructed a phylogenetic tree using Genebee Services (Fig. 1C). These analyses showed hENT4 has the highest sequence identity (20.175) and similarity (165) with hENT3, the only other pH-sensitive member of the family (supplemental Fig. S1). The analysis also suggested that the amino acids conferring pH dependence would be conserved in more similar members (hENT3 and hENT4) than in distant (hENT1 an
Referência(s)