Many details about the structure of a key membrane ion channel required for normal sperm function have long remained mysterious. Writing in Nature, Lin et al.1 present data that fill some of the gaps in our knowledge.
Sperm and eggs are the vessels of our past and future, and they must overcome daunting challenges for the exchange of their genes to occur. A sperm cell enters a competitive race with millions of other sperm that are also trying to fertilize an egg cell. Each sperm has a long way to swim in the female reproductive tract, and this journey requires dramatic changes in the type of motion used, including a vigorous type of swimming movement that is triggered on encountering high pH. The fusion of a single sperm and egg during fertilization is another complex process that, after countless remarkable transformations, propagates a new genome in every cell of what will become an amazing, trillion-cell baby.
Not unlike anxious parents expecting their first child, scientists have also anticipated the arrival of structural insights into a sperm ion channel called CatSper (Fig. 1), which governs the hyperactive motility of sperm and is necessary for fertility2. Since its discovery 20 years ago3,4, efforts to determine the composition and structure of CatSper have been evolving. Early attempts at structure determination using high-resolution X-ray crystallography were stymied by the large number of protein subunits that CatSper contains (more than ten), and by problems encountered when, in structural investigations, these proteins are expressed in cells in which they are not normally found.
An ancestral version of this channel first arose in single-celled organisms called uniflagellates5. The strong evolutionary pressures exerted on CatSper over time have driven its complexity — the CatSper complex is arguably the most elaborate ion channel known. The proteins CatSper1, CatSper2, CatSper3 and CatSper4 form a tetramer that is the ion channel’s pore, and at least six other proteins are known to be associated with this complex (CatSperβ, CatSperγ, CatSperδ, CatSperε, CatSperζ and EFCAB9)2,6. CatSper responds to high pH, and perhaps to other factors as well, when sperm reach the upper cervix, or utero-tubal junction, of the female reproductive tract. The activation of CatSper, which is associated with the entry of calcium ions into sperm7, completely transforms the motion of the sperm’s ‘tail’ (flagellum), generating a robust, asymmetrical beating pattern called hyperactivated motility. CatSper activity has a weak voltage dependence7 (sensitivity to the voltage across the cell membrane), perhaps due to the different number of positively charged amino acids in each of the pore-forming unit’s voltage sensors.
Lin and colleagues now report structural data for CatSper. The authors generated mice that had a version of CatSper1 tagged with green fluorescent protein, together with copies of a peptide called FLAG. They used this tagged version of CatSper1 to isolate the CatSper complex from mouse sperm cells, and now present the first structure of CatSper to be generated using cryogenic electron microscopy.
The structure reveals that four accessory transmembrane proteins (CatSperβ, CatSperγ, CatSperδ and CatSperε) form a tent-like ‘pavilion’ over the pore (Fig. 1), with their pole-like, transmembrane-spanning supports positioned outside the pore, flanking the voltage sensors of the adjacent pore subunit. CatSperβ and CatSperε are in contact with their voltage sensors, CatSper4 and CatSper2, respectively. The pavilion’s large interface, and particularly the presence of certain characteristic protein domains (for example, seven-bladed β-propeller domains), suggests regions that might bind to soluble ligand molecules. Notably, the multiple accessory components perturb the symmetry of the core tetrameric pore-forming subunits. This differs from the symmetry typically observed for related ion channels, and the discovery might reveal insights into the mechanisms involved in channel function.
The structure of CatSper brings several surprises. Structural modelling (and supporting evidence obtained using mass spectrometry) reveals that a protein called SLCO6C1, which is a type of transporter known as an organic anion transporter, links to CatSperε and to the voltage-sensing domain of CatSper2. This observation is consistent with other work8,9. Another newly discovered protein association with CatSper is that of the transmembrane protein CatSperη — another previously unknown interactor — with the protein TMEM249 and an as-yet-unidentified cytoplasmic protein.
The cytoplasmic components are the least clearly visualized part of the structure, with the subcomplex of CatSperζ and EFCAB9 revealing an unknown protein (consisting of α-helices) and another unknown entity sandwiched between the elongated S6 segments of CatSper2 and CatSper3. Two lobes of EFCAB9 containing calcium-binding motifs (termed EF-hands) fit into the region where EFCAB9 and CatSperζ interact. These domains might bind to calcium8, thereby linking calcium entry into sperm by means of intermediates to downstream proteins, such as the motor protein dynein. A major unanswered question is how the calcium signal results in an increased frequency and degree of bending of the sperm flagellar tail during swimming motions.
Exciting complementary data from other research groups, obtained using cryo-electron tomography and microscopy, show that the CatSper complex is assembled in a zigzag pattern9 in each of the four quadrilateral ‘racing stripes’ along the sperm tail10. An EFCAB9–CatSperζ pair bridges two staggered channel units (rotated by 180°) as a building block for the zigzag assembly, and it was proposed9 that this longitudinal nanodomain allows the simultaneous opening of the array of CatSper channels along the flagellum to allow rapid signalling in the sperm tail.
Lin and colleagues’ structural data, together with these contributions, greatly increase our knowledge of CatSper, and suggest potential answers to, or ways of addressing, long-standing questions. For example, to what, if anything, do the extracellular pavilion structures bind? If binding partners exist for these structures, does their binding affect CatSper voltage sensitivity or calcium entry, or cause structural changes that propagate along CatSper and affect cytoplasmic components of the complex? And does CatSper activation alter the activity of the anion transporter?
These seminal contributions will speed up the identification of molecules that could form targets for the development of male-specific contraceptives (and species-specific forms of such contraceptives). Finally, the discoveries relating to CatSper might point the way to the treatment of male infertility associated with mutations in the genes encoding components of this complex.
Nature 595, 654-655 (2021)
Lin, S., Ke, M., Zhang, Y., Yan, Z. & Wu, J. Nature 595, 746–750 (2021).
Qi, H. et al. Proc. Natl Acad. Sci. USA 104, 1219–1223 (2007).
Ren, D. et al. Nature 413, 603–609 (2001).
Quill, T. A., Ren, D., Clapham, D. E. & Garbers, D. L. Proc. Natl Acad. Sci. USA 98, 12527–12531 (2001).
Cai, X., Wang, X. & Clapham, D. E. Mol. Biol. Evol. 31, 2735–2740 (2014).
Wang, H., McGoldrick, L. L. & Chung, J.-J. Nature Rev. Urol. 18, 46–66 (2021).
Kirichok, Y., Navarro, B. & Clapham, D. E. Nature 439, 737–740 (2006).
Hwang, J. Y. et al. Cell 177, 1480–1494 (2019).
Zhao, Y. et al. Preprint at bioRxiv https://doi.org/10.1101/2021.06.19.448910 (2021).
Chung, J.-J. et al. Cell 157, 808–822 (2014).
The authors declare no competing interests