Molecular Determinants of Substrate Selectivity of a Novel Organic Cation Transporter (PMAT) in the SLC29 Family

Abstract

Plasma membrane monoamine transporter (PMAT or ENT4) is a newly cloned transporter assigned to the equilibrative nucleoside transporter (ENT) family (SLC29). Unlike ENT1–3, PMAT mainly functions as a polyspecific organic cation transporter. In this study, we investigated the molecular mechanisms underlying the unique substrate selectivity of PMAT. By constructing chimeras between human PMAT and ENT1, we showed that a chimera consisting of transmembrane domains (TM) 1–6 of PMAT and TM7–11 of hENT1 behaved like PMAT, transporting 1-methyl-4-phenylpyridinium (MPP+, an organic cation) but not uridine (a nucleoside), suggesting that TM1–6 contains critical domains responsible for substrate recognition. To identify residues important for the cation selectivity of PMAT, 10 negatively charged residues were chosen and substituted with alanine. Five of the alanine mutants retained PMAT activity, and four were non-functional due to impaired targeting to the plasma membrane. However, alanine substitution at Glu206 in TM5 abolished PMAT activity without affecting cell surface expression. Eliminating the at Glu206 in of organic cation activity, the transporter the in ENT1, uridine in showed an PMAT of TM5 a with Glu206 and in the of the In that Glu206 functions as a critical for and TM5 of the substrate in PMAT. Plasma membrane monoamine transporter (PMAT or ENT4) is a newly cloned transporter assigned to the equilibrative nucleoside transporter (ENT) family (SLC29). Unlike ENT1–3, PMAT mainly functions as a polyspecific organic cation transporter. In this study, we investigated the molecular mechanisms underlying the unique substrate selectivity of PMAT. By constructing chimeras between human PMAT and ENT1, we showed that a chimera consisting of transmembrane domains (TM) 1–6 of PMAT and TM7–11 of hENT1 behaved like PMAT, transporting 1-methyl-4-phenylpyridinium (MPP+, an organic cation) but not uridine (a nucleoside), suggesting that TM1–6 contains critical domains responsible for substrate recognition. To identify residues important for the cation selectivity of PMAT, 10 negatively charged residues were chosen and substituted with alanine. Five of the alanine mutants retained PMAT activity, and four were non-functional due to impaired targeting to the plasma membrane. However, alanine substitution at Glu206 in TM5 abolished PMAT activity without affecting cell surface expression. Eliminating the at Glu206 in of organic cation activity, the transporter the in ENT1, uridine in showed an PMAT of TM5 a with Glu206 and in the of the In that Glu206 functions as a critical for and TM5 of the substrate in PMAT. in the of PMAT, plasma membrane monoamine equilibrative nucleoside organic cation PMAT, plasma membrane monoamine equilibrative nucleoside organic cation a of membrane consisting of in in the family at and to a is that the family to of with the transporter not but for and an in the equilibrative nucleoside transporter (ENT) human and four as ENT1–3, nucleoside that and and important in nucleoside of and of and nucleoside is an nucleoside a in of as and to as a nucleoside transporter However, and showed that functions as a polyspecific organic cation transporter that organic 1-methyl-4-phenylpyridinium and but with or nucleoside monoamine we a plasma membrane monoamine transporter for substrate selectivity of PMAT is to that of the organic cation in the family In PMAT a in of monoamine as as in of organic unique substrate selectivity of PMAT in the family PMAT the and is PMAT to the or to the the human and a to the is the of a transporter a However, in the between PMAT and the to and PMAT not to to the is PMAT and an showed that PMAT at an in between PMAT and the In this study, we investigated the molecular and underlying the unique substrate selectivity of PMAT in the and of PMAT with human and in a and of and human were with the hENT1 PMAT and and were for and of and the of membrane chimeras and mutants were PMAT and hENT1 as PMAT and hENT1 were the the and To chimeras and an of hENT1 in the between and PMAT and chimeras were a and To chimeras with an human PMAT and in chimeras were to of the chimera mutants were the as of chimera and in the of at the of in chimera and were a as cell were in and to the cell were a at the at the of were and in the of and were in and with a with a as the were at and at in were in and to for were at in a and were and were the with were and the in a and the in to In with an were as a the in of Plasma were with of with of a with and were with of were with the for to of the were in of for of to the to cell membrane to a with the as transmembrane is to a in is the of a of the of the residues a to the and assigned to the of were in and were as and the were as and of PMAT and hENT1 human PMAT a but to that PMAT a membrane to the with transmembrane a and an we that the in substrate PMAT and a and a of domains for substrate and that the of and contains critical domains for and To we chimeras and between human PMAT and the human equilibrative nucleoside hENT1 of the of PMAT and the of chimera in a To membrane of to the of and the substrate of the chimeras were and uridine for the PMAT and hENT1 PMAT and hENT1 were the plasma in of substrate selectivity and of not PMAT showed activity for but not for uridine In hENT1 uridine but not showed plasma membrane not and behaved like PMAT, transporting but not uridine substrate selectivity of chimera that TM1–6 contains the domains for substrate and in PMAT. chimera not due to in as not to chimeras with were not as chimeras were not in not of for identify and residues important for the substrate selectivity of PMAT, we PMAT to a of organic we that negatively charged residues important for cation in PMAT but substituted a or charged residues in the the and residues in human and residues were not in the or or the and the negatively charged 10 showed and were for an of residues in and In we of with human and the between and the in the to in the of organic the in PMAT, for and were chosen as in of the residues in the and in the in of of with in and TM5 residues and Glu206 in but substituted with residues in the of residues for alanine of substitution to the of the PMAT mutants and were and in of alanine mutants PMAT and in of and to alanine not in a in and were and a and activity that of the PMAT. In mutants and activity of organic PMAT alanine mutants in of 10 and at for in as of in in the of in the activity of an alanine due to a in activity or an of cell surface of the transporter. To to the of alanine mutants mutants and retained or activity, a plasma membrane and not in and were to membrane that with alanine PMAT to the plasma membrane. and showed the to the of in and not suggesting that the alanine at showed of transporter activity plasma membrane that and the of in the is not or but is due to a in of of PMAT, and PMAT alanine mutants in of Glu206 in the with and the of Glu206 in PMAT with and were and in mutants plasma membrane to that of the PMAT surface with membrane of the in Glu206 cell showed that of the or to a charged in of transporter activity In substitution of Glu206 with a a transporter with that the at is for the of PMAT, and organic cation to PMAT with membrane of Glu206 and mutants as or with an of organic Glu206 and mutants in of 10 and at for in as of in in the of PMAT mutants were in and were as to the transporter of PMAT in a PMAT the of Glu206 in PMAT, hENT1 a is Glu206 mutants of PMAT showed activity the substrate an mutants and activity organic and in uridine as with PMAT In the retained PMAT activity showed activity uridine In plasma PMAT mutants and the showed activity uridine not that Glu206 an for the cation selectivity of PMAT, and this negatively charged as a to as the However, in ENT1, with or the mutants behaved like ENT1, transporting uridine but not not that Glu206 is for the cation selectivity in PMAT, the in ENT1, to a negatively charged is not to an organic cation as a and of PMAT and hENT1 of at for the in the of at that Glu206 in TM5 is for the cation selectivity of PMAT. alanine showed activity organic is in with PMAT, the plasma membrane of not as and cell surface and and the activity of this that in with To the of we and the of but the the and the in mutants plasma membrane with cell surface with that of PMAT a activity of and mutants showed that the activity mainly due to a in suggesting that substitution of the of the transporter. plasma membrane but activity suggesting that substitution of a abolished transporter activity the of in Glu206 and a of in residues Glu206 and in the of the a not for that TM5 in the of the substrate in of TM5 in PMAT. transmembrane is to a and is the of a the of the of the residues a to the residues in residues in and assigned to the of in this PMAT and hENT1 chimera that the critical in PMAT in a negatively charged in as a molecular for the organic cation selectivity of PMAT. that TM5 is a of the substrate in that chimera the substrate of PMAT that TM1–6 contains the domains for substrate is that in the of PMAT in the of the substrate the of chimera that the the organic cation selectivity is of this is that residues for substrate in with of the showed that the of and contains critical domains for that the substrate selectivity and and the a In a of the that the of the a important in substrate selectivity the in substrate selectivity PMAT and the mechanisms organic cation functions identify residues in PMAT, we a mainly negatively charged residues or we that a is for a to PMAT the negatively charged or in PMAT, 10 residues were for with the 10 Glu206 to for the organic cation activity of PMAT. residues were not for the activity and or to membrane and or and In Glu206 to or to a of organic cation activity without affecting plasma membrane Glu206 to a organic cation activity the in ENT1, activity the to the uridine that Glu206 is for the cation selectivity of PMAT and as the for that PMAT is to a at at the in the at this to and is that Glu206 not with the substrate but is in a as of the human organic transporter that a negatively charged in is critical for activity the of the transporter However, the that mutants and the to uridine that Glu206 is in substrate selectivity at Glu206 the of PMAT, that mutants and activity mutants showed plasma membrane that were to the and that not of the a for Glu206 in the cation selectivity of to in TM5 an important in transporter of to residues or PMAT activity, and with a abolished activity without affecting cell surface expression. that the of the at is critical for PMAT the of is with substrate or to the of an of the Glu206 and suggesting that this is a of the substrate of TM5 is a and that of TM5 an for substrate and Glu206 and in the of the and the and with the However, we the to the substrate the of TM5 in substrate and the and molecular that the of PMAT. the unique the and of in the in the of PMAT, plasma membrane monoamine equilibrative nucleoside organic cation PMAT, plasma membrane monoamine equilibrative nucleoside organic cation a of membrane consisting of in in the family at and to a is that the family to of with the transporter not but for and an in the equilibrative nucleoside transporter (ENT) human and four as ENT1–3, nucleoside that and and important in nucleoside of and of and nucleoside is an nucleoside a in of as and to as a nucleoside transporter However, and showed that functions as a polyspecific organic cation transporter that organic 1-methyl-4-phenylpyridinium and but with or nucleoside monoamine we a plasma membrane monoamine transporter for substrate selectivity of PMAT is to that of the organic cation in the family In PMAT a in of monoamine as as in of organic unique substrate selectivity of PMAT in the family PMAT the and is PMAT to the or to the the human and a to the is the of a transporter a However, in the between PMAT and the to and PMAT not to to the is PMAT and an showed that PMAT at an in between PMAT and the In this study, we investigated the molecular and underlying the unique substrate selectivity of PMAT in the and of and human were with the hENT1 PMAT and and were for and of and the of membrane chimeras and mutants were PMAT and hENT1 as PMAT and hENT1 were the the and To chimeras and an of hENT1 in the between and PMAT and chimeras were a and To chimeras with an human PMAT and in chimeras were to of the chimera mutants were the as of chimera and in the of at the of in chimera and were a as cell were in and to the cell were a at the at the of were and in the of and were in and with a with a as the were at and at in were in and to for were at in a and were and were the with were and the in a and the in to In with an were as a the in of Plasma were with of with of a with and were with of were with the for to of the were in of for of to the to cell membrane to a with the as transmembrane is to a in is the of a of the of the residues a to the and assigned to the of were in and were as and the were as and of and human were with the hENT1 PMAT and and were for and of and the of membrane chimeras and mutants were PMAT and hENT1 as PMAT and hENT1 were the the and To chimeras and an of hENT1 in the between and PMAT and chimeras were a and To chimeras with an human PMAT and in chimeras were to of the chimera mutants were the as of chimera and in the of at the of in chimera and were a as cell were in and to the cell were a at the at the of were and in the of and were in and with a with a as the were at and at in were in and to for were at in a and were and were the with were and the in a and the in to In with an were as a the in of Plasma were with of with of a with and were with of were with the for to of the were in of for of to the to cell membrane to a with the as transmembrane is to a in is the of a of the of the residues a to the and assigned to the of were in and were as and the were as and of PMAT and hENT1 human PMAT a but to that PMAT a membrane to the with transmembrane a and an we that the in substrate PMAT and a and a of domains for substrate and that the of and contains critical domains for and To we chimeras and between human PMAT and the human equilibrative nucleoside hENT1 of the of PMAT and the of chimera in a To membrane of to the of and the substrate of the chimeras were and uridine for the PMAT and hENT1 PMAT and hENT1 were the plasma in of substrate selectivity and of not PMAT showed activity for but not for uridine In hENT1 uridine but not showed plasma membrane not and behaved like PMAT, transporting but not uridine substrate selectivity of chimera that TM1–6 contains the domains for substrate and in PMAT. chimera not due to in as not to chimeras with were not as chimeras were not in not of for identify and residues important for the substrate selectivity of PMAT, we PMAT to a of organic we that negatively charged residues important for cation in PMAT but substituted a or charged residues in the the and residues in human and residues were not in the or or the and the negatively charged 10 showed and were for an of residues in and In we of with human and the between and the in the to in the of organic the in PMAT, for and were chosen as in of the residues in the and in the in of substitution to the of the PMAT mutants and were and in of alanine mutants PMAT and in of and to alanine not in a in and were and a and activity that of the PMAT. In mutants and activity of organic PMAT alanine mutants in of 10 and at for in as of in in the of in the activity of an alanine due to a in activity or an of cell surface of the transporter. To to the of alanine mutants mutants and retained or activity, a plasma membrane and not in and were to membrane that with alanine PMAT to the plasma membrane. and showed the to the of in and not suggesting that the alanine at showed of transporter activity plasma membrane that and the of in the is not or but is due to a in of of PMAT, and PMAT alanine mutants in of Glu206 in the with and the of Glu206 in PMAT with and were and in mutants plasma membrane to that of the PMAT surface with membrane of the in Glu206 cell showed that of the or to a charged in of transporter activity In substitution of Glu206 with a a transporter with that the at is for the of PMAT, and organic cation to PMAT with membrane of Glu206 and mutants as or with an of organic Glu206 and mutants in of 10 and at for in as of in in the of PMAT mutants were in and were as to the transporter of PMAT in a PMAT the of Glu206 in PMAT, hENT1 a is Glu206 mutants of PMAT showed activity the substrate an mutants and activity organic and in uridine as with PMAT In the retained PMAT activity showed activity uridine In plasma PMAT mutants and the showed activity uridine not that Glu206 an for the cation selectivity of PMAT, and this negatively charged as a to as the However, in ENT1, with or the mutants behaved like ENT1, transporting uridine but not not that Glu206 is for the cation selectivity in PMAT, the in ENT1, to a negatively charged is not to an organic cation as a and of PMAT and hENT1 of at for the in the of at that Glu206 in TM5 is for the cation selectivity of PMAT. alanine showed activity organic is in with PMAT, the plasma membrane of not as and cell surface and and the activity of this that in with To the of we and the of but the the and the in mutants plasma membrane with cell surface with that of PMAT a activity of and mutants showed that the activity mainly due to a in suggesting that substitution of the of the transporter. plasma membrane but activity suggesting that substitution of a abolished transporter activity the of in Glu206 and a of in residues Glu206 and in the of the a not for that TM5 in the of the substrate in of TM5 in PMAT. transmembrane is to a and is the of a the of the of the residues a to the residues in residues in and assigned to the of and of PMAT and hENT1 human PMAT a but to that PMAT a membrane to the with transmembrane a and an we that the in substrate PMAT and a and a of domains for substrate and that the of and contains critical domains for and To we chimeras and between human PMAT and the human equilibrative nucleoside hENT1 of the of PMAT and the of chimera in a To membrane of to the of and the substrate of the chimeras were and uridine for the PMAT and hENT1 PMAT and hENT1 were the plasma in of substrate selectivity and of not PMAT showed activity for but not for uridine In hENT1 uridine but not showed plasma membrane not and behaved like PMAT, transporting but not uridine substrate selectivity of chimera that TM1–6 contains the domains for substrate and in PMAT. chimera not due to in as not to chimeras with were not as chimeras were not in not of for identify and residues important for the substrate selectivity of PMAT, we PMAT to a of organic we that negatively charged residues important for cation in PMAT but substituted a or charged residues in the the and residues in human and residues were not in the or or the and the negatively charged 10 showed and were for an of residues in and In we of with human and the between and the in the to in the of organic the in PMAT, for and were chosen as in of the residues in the and in the in of substitution to the of the PMAT mutants and were and in of alanine mutants PMAT and in of and to alanine not in a in and were and a and activity that of the PMAT. In mutants and activity of in the activity of an alanine due to a in activity or an of cell surface of the transporter. To to the of alanine mutants mutants and retained or activity, a plasma membrane and not in and were to membrane that with alanine PMAT to the plasma membrane. and showed the to the of in and not suggesting that the alanine at showed of transporter activity plasma membrane that and the of in the is not or but is due to a in of Glu206 in the with and the of Glu206 in PMAT with and were and in mutants plasma membrane to that of the PMAT surface with membrane of the in Glu206 cell showed that of the or to a charged in of transporter activity In substitution of Glu206 with a a transporter with that the at is for the of PMAT, and organic cation to PMAT with PMAT the of Glu206 in PMAT, hENT1 a is Glu206 mutants of PMAT showed activity the substrate an mutants and activity organic and in uridine as with PMAT In the retained PMAT activity showed activity uridine In plasma PMAT mutants and the showed activity uridine not that Glu206 an for the cation selectivity of PMAT, and this negatively charged as a to as the However, in ENT1, with or the mutants behaved like ENT1, transporting uridine but not not that Glu206 is for the cation selectivity in PMAT, the in ENT1, to a negatively charged is not to an organic cation as a of at that Glu206 in TM5 is for the cation selectivity of PMAT. alanine showed activity organic is in with PMAT, the plasma membrane of not as and cell surface and and the activity of this that in with To the of we and the of but the the and the in mutants plasma membrane with cell surface with that of PMAT a activity of and mutants showed that the activity mainly due to a in suggesting that substitution of the of the transporter. plasma membrane but activity suggesting that substitution of a abolished transporter activity the of in Glu206 and a of in residues Glu206 and in the of the a not for that TM5 in the of the substrate in PMAT. in this PMAT and hENT1 chimera that the critical in PMAT in a negatively charged in as a molecular for the organic cation selectivity of PMAT. that TM5 is a of the substrate in that chimera the substrate of PMAT that TM1–6 contains the domains for substrate is that in the of PMAT in the of the substrate the of chimera that the the organic cation selectivity is of this is that residues for substrate in with of the showed that the of and contains critical domains for that the substrate selectivity and and the a In a of the that the of the a important in substrate selectivity the in substrate selectivity PMAT and the mechanisms organic cation functions identify residues in PMAT, we a mainly negatively charged residues or we that a is for a to PMAT the negatively charged or in PMAT, 10 residues were for with the 10 Glu206 to for the organic cation activity of PMAT. residues were not for the activity and or to membrane and or and In Glu206 to or to a of organic cation activity without affecting plasma membrane Glu206 to a organic cation activity the in ENT1, activity the to the uridine that Glu206 is for the cation selectivity of PMAT and as the for that PMAT is to a at at the in the at this to and is that Glu206 not with the substrate but is in a as of the human organic transporter that a negatively charged in is critical for activity the of the transporter However, the that mutants and the to uridine that Glu206 is in substrate selectivity at Glu206 the of PMAT, that mutants and activity mutants showed plasma membrane that were to the and that not of the a for Glu206 in the cation selectivity of to in TM5 an important in transporter of to residues or PMAT activity, and with a abolished activity without affecting cell surface expression. that the of the at is critical for PMAT the of is with substrate or to the of an of the Glu206 and suggesting that this is a of the substrate of TM5 is a and that of TM5 an for substrate and Glu206 and in the of the and the and with the However, we the to the substrate the of TM5 in substrate and the and molecular that the of PMAT. the unique the and of in the in this PMAT and hENT1 chimera that the critical in PMAT in a negatively charged in as a molecular for the organic cation selectivity of PMAT. that TM5 is a of the substrate in PMAT. that chimera the substrate of PMAT that TM1–6 contains the domains for substrate is that in the of PMAT in the of the substrate the of chimera that the the organic cation selectivity is of this is that residues for substrate in with of the showed that the of and contains critical domains for that the substrate selectivity and and the a In a of the that the of the a important in substrate selectivity the in substrate selectivity PMAT and the mechanisms organic cation functions To identify residues in PMAT, we a mainly negatively charged residues or we that a is for a to PMAT the negatively charged or in PMAT, 10 residues were for with the 10 Glu206 to for the organic cation activity of PMAT. residues were not for the activity and or to membrane and or and In Glu206 to or to a of organic cation activity without affecting plasma membrane Glu206 to a organic cation activity the in ENT1, activity the to the uridine that Glu206 is for the cation selectivity of PMAT and as the for that PMAT is to a at at the in the at this to and is that Glu206 not with the substrate but is in a as of the human organic transporter that a negatively charged in is critical for activity the of the transporter However, the that mutants and the to uridine that Glu206 is in substrate selectivity at Glu206 the of PMAT, that mutants and activity mutants showed plasma membrane that were to the and that not of the a for Glu206 in the cation selectivity of PMAT. In to in TM5 an important in transporter of to residues or PMAT activity, and with a abolished activity without affecting cell surface expression. that the of the at is critical for PMAT the of is with substrate or to the of an of the Glu206 and suggesting that this is a of the substrate of TM5 is a and that of TM5 an for substrate and Glu206 and in the of the and the and with the However, we the to the substrate the of TM5 in substrate and In the and molecular that the of PMAT. the unique the and of in the