A NOX2-independent mechanism of Hv1 channel activation promotes inflammatory cytokine release from BV-2 microglia via intracellular Ca2+ mobilisation

Microglia are the primary immune cells to sense and respond to all the pathological events in the brain. Voltage-gated proton channels (Hv₁) are specifically expressed in the microglia to regulate their intracellular pH and contribute to redox homeostasis. Our previous work identified that S-023-0515 is a novel activator of microglial Hv₁ channels, inducing neuroinflammation through unknown mechanisms. In this study, we demonstrate the direct binding of S-023-0515 onto the Hv₁ channel using molecular docking, molecular dynamics (MD) simulations, and Bio-layer interferometry (BLI) techniques. Treatment with S-023-0515 induced a sustained intracellular alkalization, resulting in a gradual increase in cytosolic CA²⁺ levels. None of the major plasma membrane CA²⁺ ion channels, such as TRPV1, Cav1.2, ASIC and P2X7, nor intracellular CA²⁺ release channels were found to be involved in S-023-0515-induced CA²⁺ increase. This mobilisation of CA²⁺ occurred through the sarco/endoplasmic reticulum CA²⁺-ATPase (SERCA), as its inhibition annulled the S-023-0515-mediated CA²⁺ rise. Hv₁-mediated Ca²⁺ signaling further promoted NF-κB activation leading to a steady increase in proinflammatory cytokines, such as TNF-α and IL-1β, in BV-2 microglial cells, representing an inflammatory microglial phenotype. Notably, the proinflammatory response was solely attributable to Hv₁ channel activation because neither NOX2 stimulation nor local cellular pH was altered following S-023-0515 treatment. Together, these findings suggest that NOX2-independent activation of Hv₁ channels triggers a Hv₁-SERCA-Ca²⁺-NF-κΒ signalling cascade, disrupting intracellular CA²⁺ homeostasis and leading to microglial neurotoxicity.