Involvement of Proton-Coupled SLC49A4-Mediated Transport in the Export of Lysosomally Trapped Pyrilamine

Our recent study has revealed that SLC49A4, known as disrupted in renal carcinoma 2, is an H+ -coupled lysosomal exporter for pyridoxine (vitamin B6), a cationic compound. We examined in the present study the possibility that this transporter might also transport cationic amphiphilic drugs (CADs) that are known to undergo lysosomal trapping, using pyrilamine, an H1 -antagonist, as a model CAD. The COS -7 cell line was used as a model cell system for transient introduction of human SLC49A4 and a recombinant SLC49A4 protein (SLC49A4-AA), in which the N -terminal dileucine motif involved in lysosomal localization was removed by replacing with dialanine for redirected localization to the plasma membrane. The introduction of SLC49A4 into COS -7 cells induced a significant decrease in the accumulation of pyrilamine in the intracellular compartments in the cells treated with digitonin for permeabilization of plasma membranes, suggesting its operation for lysosomal pyrilamine export. Accordingly, functional analysis using the SLC49A4-AA mutant, which operates for cellular uptake at the plasma membrane in transiently transfected COS -7 cells, demonstrated its H+ -coupled operation for pyrilamine transport, which was saturable with a Michaelis constant of 132 lM at pH 5.5. In addition, many CADs that may potentially undergo lysosomal trapping, which include imipramine, propranolol, verapamil, and some others, were found to inhibit SLC49A4-AA-mediated pyrilamine transport, suggesting their affinity for SLC49A4. These results suggest the involvement SLC49A4 in the lysosomal trapping of pyrilamine as a lysosomal exporter. The CADs that inhibited SLC49A4-AA-mediated pyrilamine transport could also be SLC49A4 substrate candidates.