CATSPERε extracellular domains are essential for sperm calcium channel assembly and activity modulation

The flagellar-specific Ca²⁺ channel CatSper is a multiprotein complex that is critical for successful fertilization by controlling the sperm Ca²⁺ signaling in space and time. Large extracellular domains (ECDs) of four single-pass transmembrane subunits, CATSPERβ, γ, δ, and ε, form a unique canopy structure over the pore-forming channel. However, the molecular mechanisms of canopy assembly during development and its physiological function in mature sperm remain unknown. Here, using two genetic mouse models and the biochemical isolation of a bioactive CATSPERε fragment, we report that CATSPERε ECDs are essential for assembling the CatSper canopy, and thus the entire channel complex, and for modulating CatSper function for sperm hyperactivation and fertilization. CATSPERε-deficient males are sterile because their sperm fail to develop hyperactivated motility due to the absence of the entire channel. In transgenic mice overexpressing CATSPERε with truncated ECDs in testicular germ cells, truncated CATSPERε is unable to interact with native CatSper subunits and incorporate into the complex, thus failing to rescue the defective sperm hyperactivation and infertility of Catspere-null males. These findings provide insight into the underlying molecular and developmental mechanisms of CatSper complex assembly and how CatSper channels can be modulated in physiological settings and by therapeutic intervention.