The specialization of mammalian cells is often achieved through the expression of distinct isoforms of a protein with unique molecular and functional properties. Na,K-ATPase (NKA), the ion transport system responsible for maintaining transmembrane Na⁺ and K⁺ gradients, is characterized by its remarkable molecular and functional diversity. Mammals, express four isoforms of the catalytic α-subunit of NKA (NKAα1, NKAα2, NKAα3, and NKAα4), each with distinct tissue distributions and physiological roles. NKAα4 is only expressed in male germ cells of the testis after meiosis and in sperm. NKAα4 is essential for sperm motility, hyperactivation, acrosome reaction and male fertility. Its deletion in mice results in a unique phenotype consisting in complete infertility of the male but not female mice. Sperm from NKAα4 null mice have compromised motility, lack hyperactive motility, have abnormal acrosome reaction, and show altered intracellular Na , Ca ², membrane potential, and pH. The unique expression pattern and critical physiological role of NKAα4 makes it an attractive target for the development of a non-hormonal male contraceptive. NKAα4 is characterized by a high affinity for the glycoside ouabain, which is reflected by its particular ouabain binding site, determined through cryo-EM technology. Taking advantage of this property of NKAα4, we developed a series of ouabain analogs which selectively target the high ouabain affinity site of NKAα4, inhibit NKAα4 activity, reduce sperm motility and interfere with sperm fertility in vitro and in vivo. These results validate NKAα4 as a critical ion transporter of sperm and show that ouabain analogues can be developed as effective and selective agents for male contraception. [Supported by NIH grant HD080423].

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