title: Residue Ile89 in human plasma membrane monoamine transporter influences its organic cation transport activity and sensitivity to inhibition by dilazep.
abstract: Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter belonging to the equilibrative nucleoside transporter (ENT) family. Despite its distinct substrate specificity from the classic nucleoside transporters ENT1 and 2, PMAT appears to share similar protein architecture with ENT1/2 and retains low affinity binding to classic ENT inhibitors such as nitrobenzylmercaptopurine riboside (NBMPR) and the coronary vasodilators dilazep and dipyridamole. Here we investigated the role of residue Ile89, a position known to be important for ENT interaction with dilazep, dipyridamole, and nucleoside substrates, in PMAT transport function and its interaction with classic ENT inhibitors using Madin-Darby canine kidney (MDCK) cells stably expressing human PMAT. Substitution of Ile89 in PMAT with Met, the counterpart residue in ENT1, resulted in normal plasma membrane localization and protein expression. Transport kinetic analysis revealed that I89M mutant had a 2.7-fold reduction in maximal transport velocity (V(max)) with no significant change in apparent binding affinity (K(m)) towards the prototype PMAT substrate 1-methyl-4-phenylpyridinium (MPP+), suggesting that I89 is an important determinant for the catalytic activity of PMAT. Dose-dependent inhibition studies further showed that the I89M mutation significantly increased PMAT's sensitivity to dilazep by 2.5-fold without affecting its sensitivity to dipyridamole and NBMPR. Located at the extracellular end of transmembrane domain 1 of PMAT, I89 may occupy an important position close to the substrate permeation pathway and may be involved in direct interaction with the vasodilator dilazep.
authors.affiliation: Department of Pharmaceutics, University of Washington, Seattle, WA 98195, USA. horaceho@u.washington.edu
authors.forename: Horace T B
authors.initials: HT
authors.lastname: Ho
authors.forename: Li
authors.initials: L
authors.lastname: Xia
authors.forename: Joanne
authors.initials: J
authors.lastname: Wang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D004109:Dilazep / Q000494:pharmacology*; D004285:Dogs; D033705:Equilibrative Nucleoside Transport Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport / Q000235:genetics; D007532:Isoleucine / Q000235:genetics / Q000502:physiology*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation
chemical_list: D033705:Equilibrative Nucleoside Transport Proteins; D026901:Membrane Transport Proteins; C495988:SLC29A4 protein, human; D007532:Isoleucine; D004109:Dilazep