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 (Vmax) with no significant change in apparent binding affinity (Km) 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.

The plasma membrane monoamine transporter (PMAT): Structure, function, and role in organic cation disposition

“…Helical wheel analysis revealed a distinct amphipathic pattern of residue distribution on TM5 with E206 and T220 clustered in the center of a hydrophilic face, suggesting a critical role of TM5 in forming part of the substrate permeation pathway . Several additional residues (Y85, Y112, I89) on TM1 and TM2 were also identified to be important for PMAT to interact with its substrates or inhibitors …”

“…Moreover, PMAT appears to retain several features reminiscent of the ENTs. It transports the purine nucleoside adenosine and preserves low‐affinity binding to the classic ENT inhibitors NBMPR, dipyridamole, and dilazep . Based on these observations, we hypothesized that PMAT maintains the overall protein architecture of the ENTs but has diverged from the ENT lineage to handle structurally diverse OCs via changes of key amino acid residues in its substrate binding pocket.…”

“…These data suggest that, although the C‐terminal half of PMAT may participate in the formation of the substrate permeation pathway, its major OC recognition sites are located within the N‐terminal half ( Figure ), a region also known to be important for the ENTs in their interaction with nucleoside substrates. Within TM1‐6, site‐directed mutagenesis analyses further identified a number of key residues involved in substrate recognition and interaction . A negatively charged glutamate residue (E206) on TM5 is critical for the cation selectivity of PMAT .…”

“…It transports the purine nucleoside adenosine and preserves lowaffinity binding to the classic ENT inhibitors NBMPR, dipyridamole, and dilazep. 10,36 Based on these observations, we hypothesized that PMAT maintains the overall protein architecture of the ENTs but has diverged from the ENT lineage to handle structurally diverse OCs via changes of key amino acid residues in its substrate binding pocket. Indeed, a chimeric transporter consisting of the N-terminal (TM1-6) half of PMAT and the C-terminal half (TM7-11) of ENT1 is functional and behaved like PMAT, transporting MPP 1 but not uridine.…”

“…Within TM1-6, site-directed mutagenesis analyses further identified a number of key residues involved in substrate recognition and interaction. [36][37][38] A negatively charged glutamate residue (E206) on TM5 is critical for the cation selectivity of PMAT. 37 Replacement of this residue by charge reversal (E206R) or neutralization (E206Q) abolished OC transport activity, whereas conserving the negative charge (E206D) restored transporter function.…”