How to Perform a Tracer Displacement BRET Assay for the TRPML1 Ion Channel

The transient receptor mucolipin subtype 1 (TRPML1) is a ubiquitously expressed ion channel involved in lysosomal homeostasis. Recent pharmaceutical interest in developing agonist ligands has emerged due to beneficial effects in neurodegenerative diseases. The major high-throughput screening techniques to investigate this ion channel involve fluorescent calcium imaging and electrophysiology. Despite their high capacity for screening compounds, it is well known that both methods face hurdles, such as the need for expensive, specialized equipment. Here, we present a novel technique to screen for ligands of TRPML1 using a bioluminescence resonance energy transfer (BRET) assay. This assay consists of a target engagement assay in live cells, which permits the determination of binding constants between ligands and the target of interest in equilibrium or time-dependently. We employ a full-length TRPML1 C-terminally tagged with the small bioluminescent protein nanoluciferase. This ensures the correct localization of the ion channel in the lysosomal membrane and an optimal placement of the luciferase in the cytoplasm. We also developed a cell- and lysosome-permeable fluorescent BRET tracer that gives a BRET signal only when bound to the ion channel. This new protocol allows researchers worldwide to screen compounds that would interact with TRPML1 by using any plate reader with luminescent and fluorescence filters. Key features • This assay enables high-throughput screening of ligands for the TRPML1 ion channel without the need for a kinetic plate reader. • With the tracer displacement assay, it is possible to derive the apparent ligand affinity (K_dA) for TRPML1. • The assay uses full-length TRPML1, preserving the physiological context of lysosomal TRPML1.