BPS2026 – Reconsidering native proton channel composition and function: Amyloid precursor protein (APP) is an accessory subunit in Hv1 channels in microglia

Microglia, resident immune cells of the central nervous system, are principal mediators of neuroinflammation in Alzheimer’s disease (AD) and ischemic stroke. Their activation is regulated by Hv1 voltage-gated proton channels, which sustain pH homeostasis and membrane potential to modulate cytokine secretion and production of reactive oxygen species (ROS). Hv1 has been thought to operate as a simple homodimer. Here, we identify APP as a direct binding partner. APP is a single pass transmembrane protein central to AD through secretase-mediated cleavage to yield a C-terminal transmembrane fragment of 99 residues (C99), which is further processed into amyloid-β (Aβ) peptides that aggregate into neurotoxic plaques. The roles of APP and C99 beyond Aβ production remain poorly defined. Using human induced pluripotent stem cell-derived microglia (iMG), we observed proton currents that were significantly different from those recorded on expression of Hv1 in HEK293T cells unless APP was co-expressed. In iMG, proton currents were markedly reduced following APP knockdown, accompanied by attenuated cytokine and ROS release. In HEK293T cells, co-expression of APP enhanced Hv1 currents and induced a hyperpolarizing shift in Hv1 activation; co-expression of C99 reproduced and amplified these effects. Co-immunoprecipitation confirmed stable assemblies of Hv1 and APP or C99 in HEK293T cells, while single-molecule total internal reflection fluorescence (TIRF) microscopy documented the complexes at the plasma membrane. Pharmacological profiling revealed decreased sensitivity of Hv1-APP/C99 complexes to both the C6 peptide and Zn 2+ inhibition. Moreover, early-onset AD-associated C99 point mutations (E682K and D694N) augmented Hv1 current amplitudes and exaggerated the hyperpolarizing shift in gating relative to wild-type C99. These findings fill a gap in our understanding of Hv1 channel physiology and provide a new foundation for development of interventions for Hv1-associated inflammatory disorders.