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.

Figure 1

Multiple sequence alignment of mammalian PMATs and ENTs around the predicted TM1 region of hPMAT. I89 in hPMAT and its corresponding residues in PMAT orthologs and ENTs are highlighted in yellow for isoleucine (I) and red for methionine (M). I89 in hPMAT and corresponding residues in hENT1 and hENT2 are numbered. (r: rat; m: mouse; ca: canine; c: cattle; mac: macaques; h: human; rb: rabbit)

Figure 2

Concentration-dependent transport of MPP⁺ by WT PMAT and I89M mutant. EYFP-C1 vector-, WT PMAT- and I89M- stably transfected MDCK cells were incubated with varying concentrations of the substrates MPP⁺ for 1 min at 37°C. Specific uptake was calculated by subtracting the uptake values in vector-transfected cells. WT PMAT (●) and I89M (○) concentration-dependent uptake were shown. Each value represents the mean ± S.D. from three independent experiments (n=3) with different cell passages. For each experiment, uptake was carried out in triplicates in three different wells on the same plate.

Figure 3

(A) Confocal imaging of cellular localization of WT PMAT, I89M mutant and EYFP-C1 vector in stably transfected MDCK cells. (B) Plasma membrane expression (as detected by biotinylation) and intracellular expression (proteins not bound to the membrane-impermeable biotinylation reagent sulfo-NHS-SS-biotin) of WT PMAT, I89M mutant and EYFP-C1 vector followed by Western blot with an anti-yellow fluorescent protein (YFP) monoclonal antibody. Predicted molecular weight of YFP (~30 kDa) and YFP-tagged PMAT protein (~75 kDa) are indicated.

Figure 4

(A) Proposed secondary structure of hPMAT. Position of I89 residue is highlighted in red. Previously identified functionally important residues are highlighted in blue. (B) Sequence comparison between hPMAT and PfENT1 in the predicted TM1 region. Identical amino acids are shaded, conserved amino acids are boxed. The predicted TM1 for hPMAT and PfENT1 are shown by solid lines below the aligned sequences. (h: human; Pf: P. falciparum). (C) Helical wheel analysis of TM1 of PMAT. The transmembrane domain is assumed to be standard α-helix and each residue is plotted every 100° around the center of a circle. The figure shows the projection of the positions of the residues on a plane perpendicular to the helical axis. Hydrophobic residues are shown in white, and hydrophilic residues are shown in gray. Functionally important residues previously identified in PMAT, ENT1 and ENT2 with positions corresponding or close to I89 in PMAT are indicated in brackets.