Abstract
Transient receptor potential (TRP) channels are regulated by a diverse network of intracellular partners that govern their trafficking, stability, and functional expression at the plasma membrane. Here, we present a comprehensive and integrative characterization of 14-3-3 proteins as conserved binding partners of TRP channels. Leveraging the extensive structural repertoire of 14-3-3 complexes resolved to date, we combined large-scale sequence and structural analyses with molecular docking, coevolutionary inference, machine learning-based predictions, atomistic simulations, and targeted experimental validation to elucidate the molecular principles underlying TRP-14-3-3 recognition. Integration of these approaches into a unified consensus scoring framework revealed recurrent, solvent-exposed cytoplasmic motifs across the TRP channel family with a high propensity for 14-3-3 binding. Focusing on the TRPM4-14-3-3γ interaction, we identified an N-terminal cytoplasmic region of the channel as the primary 14-3-3 binding hotspot. Structural modeling and molecular dynamics simulations revealed a stable electrostatically driven interface, which was experimentally validated by fluorescence anisotropy assays. Moreover, biochemical and functional analyses demonstrated that TRPM4 interacts not only with 14-3-3γ but also with 14-3-3η, leading to a reduced channel-mediated sodium influx. Together, these findings establish 14-3-3 proteins as general and evolutionarily conserved regulators of TRP channels and provide a broadly applicable framework for identifying transient protein-protein interactions relevant to TRP channel dysregulation in disease.