Structural mechanism of Necrocide 1 activation of human TRPM4 that triggers necrosis by sodium overload

The small molecule Necrocide 1 (NC1) constitutively activates human TRPM4, triggering Na influx and leading to necrotic cell death, a process termed Necrosis by Sodium Overload (NECSO). NC1 activation is specific to human TRPM4 and does not affect most of the other mammalian TRPM4 orthologs. Here, we elucidate the molecular mechanism underlying NC1 activation and its species-specific selectivity for human TRPM4 using a combination of single-particle cryo-EM, electrophysiology, and cell death assays. We demonstrate that NC1 functions as a non-competitive surrogate of the endogenous Ca{superscript 2} ligand - it binds to a pocket within the S1-S4 domain adjacent to the Ca{superscript 2} site and induces the same conformational changes as those triggered by Ca{superscript 2}. Like Ca{superscript 2}-mediated activation, NC1-induced channel opening also requires membrane PI(4,5)P2 to stabilize the open state. Through comparative mutagenesis and structural analysis of human and mouse TRPM4, we identify the molecular determinants of NC1 specificity. Our results reveal that the insensitivity of mouse TRPM4 to NC1 arises not from a lack of binding, but from drug-induced conformational changes that destabilize the selectivity filter and inactivate the channel. We identify three critical residues that confer NC1 sensitivity, and their substitution renders mouse TRPM4 responsive to NC1, akin to the human channel. Given the upregulation of TRPM4 in various human cancers, our mechanistic insights into NC1 activation and specificity provide a framework for the potential development of cancer therapeutics targeting TRPM4-mediated necrosis.