Abstract
<jats:p> The sperm-specific CatSper channel is macromolecular Ca2+ channel complex essential for hyperactivated motility and male fertility. The pore-forming channel (CATSPER1-4) associates with a large extracellular domain-containing canopy (CATSPERβ—ε), a cytosolic Ca2+ sensing subcomplex (CATSPERζ—EFCAB9—ARMH2), the putative transporter SLCO6C1, and CATSPERθ—η, arranging into higher-order zigzag rows in the flagellar membrane. However, the molecular mechanism governing holo-complex assembly during spermatogenesis remains largely undefined. Here we demonstrate that the CATSPERβ—δ interaction represents an essential early step in CatSper biogenesis. CRISPR/Cas9 targeting of <jats:italic>Catsperb</jats:italic> exon 4 generated a frameshift knockout ( <jats:italic>Catsperb</jats:italic> <jats:sup>-/-</jats:sup> ) and an in-frame deletion mutant ( <jats:italic>Catsperb</jats:italic> <jats:sup>Δ/Δ</jats:sup> ) that specifically disrupts the β—δ interaction interface. Disrupting this interface reduced CATSPERδ among canopy subunits, impairing canopy assembly and destabilizing the core pore-forming channel. Consequently, mature spermatozoa completely lacked the entire CatSper complex, phenocopying the knockout. A transgenic line expressing extracellular-domain-truncated CATSPERδ lacking the β-binding region likewise phenocopied the Catsperb mutant, confirming the necessity of an intact β—δ interface. AlphaFold-Multimer modeling and alanine-substitution mutagenesis identified key hydrogen-bonding residues at this interface that mediate canopy subunit association. Consistent with complex loss, whole-sperm patch-clamp recordings revealed complete absence of CatSper conductance in mutant spermatozoa. Both mutant lines exhibited defective sperm hyperactivation and male infertility despite normal spermatogenesis and baseline motility. Together, these findings establish that canopy formation driven by CATSPERβ—δ interaction precedes and is required for pore-forming channel assembly, defining a hierarchical assembly pathway for the CatSper holo-complex and highlighting a key structural node for male fertility and contraceptive development. </jats:p>