{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T23:27:44Z","timestamp":1786404464575,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,7]]},"group-title":"Cell Biology","reference-count":56,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T00:00:00Z","timestamp":1786060800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"award":["R01HD096745"],"award-info":[{"award-number":["R01HD096745"]}],"id":[{"id":"https:\/\/ror.org\/01cwqze88","id-type":"ROR","asserted-by":"publisher"}]},{"award":["Francis G. Kingsley Fund"],"award-info":[{"award-number":["Francis G. Kingsley Fund"]}],"id":[{"id":"https:\/\/ror.org\/03v76x132","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  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 (CATSPER\u03b2\u2013\u03b5), a cytosolic Ca\u00b2\u207a sensing subcomplex (CATSPER\u03b6\u2013EFCAB9\u2013ARMH2), the putative transporter SLCO6C1, and CATSPER\u03b8\u2013\u03b7, 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 CATSPER\u03b2\u2013\u03b4 interaction represents an essential early step in CatSper biogenesis. CRISPR\/Cas9 targeting of\n                  <jats:italic>Catsperb<\/jats:italic>\n                  exon 4 generated a frameshift knockout (\n                  <jats:italic>Catsperb<\/jats:italic>\n                  <jats:sup>-\/-<\/jats:sup>\n                  ) and an in-frame deletion mutant (\n                  <jats:italic>Catsperb<\/jats:italic>\n                  <jats:sup>\u0394\/\u0394<\/jats:sup>\n                  ) that specifically disrupts the \u03b2\u2013\u03b4 interaction interface. Disrupting this interface reduced CATSPER\u03b4 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 CATSPER\u03b4 lacking the \u03b2-binding region likewise phenocopied the Catsperb mutant, confirming the necessity of an intact \u03b2\u2013\u03b4 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 CATSPER\u03b2\u2013\u03b4 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.\n                <\/jats:p>","DOI":"10.64898\/2026.08.06.743317","type":"posted-content","created":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T15:35:13Z","timestamp":1786116913000},"source":"Crossref","is-referenced-by-count":0,"title":["CATSPER\u03b2\u2013\u03b4 Interaction Governs Hierarchical CatSper Holo-complex Assembly and is Essential for Male Fertility"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9026-3725","authenticated-orcid":false,"given":"Jong-Nam","family":"Oh","sequence":"first","affiliation":[{"name":"Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7326-4379","authenticated-orcid":false,"given":"Huafeng","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA"},{"name":"Department of Pharmacology, Yale School of Medicine, New Haven, CT 06510, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tse-en","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1090-8349","authenticated-orcid":false,"given":"Xiaofang","family":"Huang","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5128-6326","authenticated-orcid":false,"given":"Leonard K.","family":"Kaczmarek","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Yale School of Medicine, New Haven, CT 06510, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8018-1355","authenticated-orcid":false,"given":"Jean-Ju","family":"Chung","sequence":"additional","affiliation":[{"name":"Department of Cellular and Molecular Physiology, Yale School of Medicine, New Haven, CT, USA"},{"name":"Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale School of Medicine, New Haven, CT, 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