NLRP3 acts as a direct sensor of intracellular potassium ions

Summary The NLRP3 inflammasome is a sentinel of cellular homeostasis, and its activation triggers the assembly of a molecular machinery that drives inflammation in infection, cardiovascular, metabolic, and neurodegenerative diseases. The majority of the many triggers known to activate NLRP3 are believed to induce potassium ion (K + ) efflux from the cell as a fundamental danger signal for compromised cellular integrity. However, it has remained unclear how a reduction in intracellular K + concentration is mechanistically translated into conformational changes in NLRP3 that promote inflammasome assembly, interleukin (IL)-1 release, and cell death. Here, we provide evidence that alterations in K + levels directly regulate the conformation of the NLRP3 protein. In cell-free lysates derived from cell lines and primary blood immune cells high K + concentrations stabilized a compact, protease-resistant structure resembling inhibitor-bound NLRP3, whereas low K + conditions or the presence of a K + chelator favored an open, more flexible and protease-accessible conformation. Notably, human NLRP3 remained responsive to K + even when exogenously expressed in macrophage-like Drosophila cells or purified as recombinant protein. This indicates that K + sensing occurs independently of cellular co-factors and is consistent with direct ion coordination. Of note, stimulation with the K + -independent NLRP3 agonist CL097 failed to recapitulate the conformational transition caused by K + efflux inducer, nigericin. Moreover, pathogenic gain-of-function mutant variants of NLRP3 constitutively resembled the open and flexible protease-accessible conformation. Mapping K + -interactions by high-performance computation suggested that K + ions populate the nucleotide binding pocket of the FISNA-NACHT module of individual NLRP3 chains but also stabilize face-to-face interactions within inactive oligomeric ‘cage’ assemblies via the NACHT-adjacent acidic loop. Collectively, our findings enable us to propose a mechanistic model of how intracellular K + ions preclude NLRP3 activation prior to efflux and thus how NLRP3 responds to cellular danger as a direct K + sensing protein.