Transmembrane PhoxID (tmPhoxID) exploits the membrane permeability of singlet oxygen to enable proximity labeling of intracellular proteins from extracellularly anchored photosensitizers, successfully mapping receptor-specific intracellular and extracellular interactomes of transmembrane proteins in living mouse brain with subsynaptic spatial resolution, and identifying a previously uncharacterized CAMKV-GABAAR interaction conserved in human brain tissue.

Transmembrane proteins perform essential roles in cellular transport, signalling, and communication. The function and dynamics of these proteins are precisely regulated by interactions on both sides of the plasma membrane; thus, mapping the composition of these interactomes is a fundamental challenge in molecular biology. Proximity labelling methods are powerful tools for this purpose; however, existing approaches that rely on membrane-impermeable reactive species are limited by the ability to detect only one side, either the extracellular or the intracellular region, of transmembrane proteins. Here, we capitalized on the cell permeability of singlet oxygen to carry out proximity labelling of the cytoplasmic side of transmembrane proteins using an extracellularly anchored photosensitizer. We applied this method, termed transmembrane PhoxID (tmPhoxID), to several receptors (GRID2, GABAAR, and GRM1) in the living mouse brain and successfully determined their specific intracellular interactomes. Notably, network analysis of the identified proteins revealed that this method can characterize the native components of transsynaptic nanocolumns formed at parallel fibre–Purkinje cell synapses. Furthermore, our study revealed a previously uncharacterized GABAAR-CAMKV interaction in mice and human brains. Our results provide a proof of concept for transmembrane and transcellular proximity labelling, providing a powerful platform for analysing the interactome of transmembrane proteins.