Direct detection of electrogenic polyamine transport

Polyamines are cationic metabolites that support critical cellular processes including transcription, translation, and cell proliferation. Although polyamine synthesis and transport are emerging therapeutic targets in human disease, the polyamine transport system remains poorly understood. ATP13A2-4 were recently identified as endolysosomal polyamine exporters, transporting putrescine (PUT), spermidine (SPD), and spermine (SPM) from the vesicular lumen to the cytosol. Despite interest in pharmacologically targeting these transporters, existing assays of transporter function rely on indirect or endpoint measures that cannot resolve transport as it occurs. Here, we report the first voltage-clamp recordings of polyamine transporter activity, directly detecting ATP13A2- and ATP13A3-mediated charge movement across cell membranes. When expressed in Xenopus oocytes, these transporters localized to the plasma membrane, enabling electrophysiological measurements. Polyamines induced inward currents dependent on transporter catalytic activity, and their dose-dependence correlated with ATPase assays. ATP13A2 currents were largest in response to SPM, whereas ATP13A3 currents were largest with PUT, consistent with their established substrate preferences. Polyamine-induced currents were blocked by the polyamine transport inhibitor AMXT-1501. These findings establish ATP13A2 and ATP13A3 as electrogenic polyamine pumps and demonstrate the utility of two-electrode voltage clamp as a label-free, transporter-resolved assay to complement other approaches in polyamine transporter-targeted drug discovery.