Structural Insights into the Integration of Temperature and pH by Sperm Calcium Channel CatSper

The cation channel of sperm (CatSper) is a sperm-specific calcium channel essential for male fertility across metazoans. Its activation is tightly restricted to defined physiological contexts, including intracellular alkalinization, membrane depolarization, and elevated temperature. The structural and evolutionary mechanisms underlying this polymodal integration remain poorly understood, in part due to the architectural complexity of CatSper, a [~]15-subunit assembly organized in zigzag arrays along the sperm flagellum. Here we combine comparative genomics across 47 species with AlphaFold3-based modeling and evolutionary sequence-structure analyses to uncover a mechanism for temperature and pH integration. We identify the pore-forming subunit CatSper1 as an evolutionary hotspot exhibiting exceptional divergence in its N-terminal domain. Phylogenetic analysis shows that N-terminal length and histidine enrichment scale with species-specific fertilization temperatures, suggesting adaptive tuning of physicochemical sensitivity. Structural modeling indicates that conserved surface-exposed histidine clusters form inter-complex coupling interfaces between adjacent CatSper assemblies positioned near the dominant voltage-sensing module. Functional validation using electrophysiology and calcium imaging in mouse sperm shows that capacitation-associated partial removal of the CatSper1 N-terminus selectively impairs temperature-dependent activation. Together, these findings support a model in which temperature-dependent histidine deprotonation modulates supramolecular CatSper assembly to coordinate channel activation.