A novel BRET-based assay to investigate binding and residence time of unmodified ligands to the human lysosomal ion channel TRPML1 in intact cells

Micael R Cunha; Carolina M C Catta-Preta; Jéssica E Takarada; Gabriela A Moreira; Katlin B Massirer; Rafael M Couñago

doi:10.1016/j.jbc.2023.104807

A novel BRET-based assay to investigate binding and residence time of unmodified ligands to the human lysosomal ion channel TRPML1 in intact cells

J Biol Chem. 2023 Jun;299(6):104807. doi: 10.1016/j.jbc.2023.104807. Epub 2023 May 11.

Authors

Micael R Cunha¹, Carolina M C Catta-Preta², Jéssica E Takarada², Gabriela A Moreira², Katlin B Massirer³, Rafael M Couñago⁴

Affiliations

¹ Center of Medicinal Chemistry (CQMED), Center for Molecular Biology and Genetic Engineering (CBMEG), University of Campinas, Campinas, Brazil. Electronic address: micaelrc@unicamp.br.
² Center of Medicinal Chemistry (CQMED), Center for Molecular Biology and Genetic Engineering (CBMEG), University of Campinas, Campinas, Brazil.
³ Center of Medicinal Chemistry (CQMED), Center for Molecular Biology and Genetic Engineering (CBMEG), University of Campinas, Campinas, Brazil. Electronic address: kmassire@unicamp.br.
⁴ Center of Medicinal Chemistry (CQMED), Center for Molecular Biology and Genetic Engineering (CBMEG), University of Campinas, Campinas, Brazil; Structural Genomics Consortium and Division of Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina, USA. Electronic address: rafael.counago@unc.edu.

Abstract

Here, we report a bioluminescence resonance energy transfer (BRET) assay as a novel way to investigate the binding of unlabeled ligands to the human transient receptor potential mucolipin 1 (hTRPML1), a lysosomal ion channel involved in several genetic diseases and cancer progression. This novel BRET assay can be used to determine equilibrium and kinetic binding parameters of unlabeled compounds to hTRPML1 using intact human-derived cells, thus complementing the information obtained using functional assays based on ion channel activation. We expect this new BRET assay to expedite the identification and optimization of cell-permeable ligands that interact with hTRPML1 within the physiologically relevant environment of lysosomes.

Keywords: bioluminescence resonance energy transfer; calcium channel; lysosome; membrane protein; transient receptor potential channels.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bioluminescence Resonance Energy Transfer Techniques* / methods
Humans
Ligands
Lysosomes / metabolism
Transient Receptor Potential Channels* / metabolism

Substances

Ligands
Transient Receptor Potential Channels
MCOLN1 protein, human