Abstract
Sperm-specific CatSper1 and CatSper2 proteins are critical to sperm-hyperactivated motility and male fertility. Although architecturally resembling voltage-gated ion channels, neither CatSper1 nor CatSper2 alone forms functional ion channels in heterologous expression systems, which may be related to the absence of yet unidentified accessory subunits. Here we isolated CatSper1- and CatSper2-associated protein(s) from human sperm and analyzed their identities by a multidimensional protein identification technology approach. We identified the T-type voltage-gated calcium channel Ca(v)3.3 as binding to both CatSper1 and CatSper2. The specificity of their interactions was verified by co-immunoprecipitation in transfected mammalian cells. Electrophysiological studies revealed that the co-expression of CatSper1 or CatSper2 specifically inhibited the amplitude of Ca(v)3.3-evoked T-type calcium current without altering other biophysical properties of Ca(v)3.3. Immunostaining studies revealed co-localization of CatSper1 and Ca(v)3.3 on the principal piece of human sperm tail. Furthermore, fluorescence resonance energy transfer analysis revealed close proximity and physical association of these two proteins on the sperm tail. These studies demonstrate that CatSper1 and CatSper2 can associate with and modulate the function of the Ca(v)3.3 channel, which might be important in the regulation of sperm function.
Highlights
Sperm must swim long distances in the female reproductive tract to reach the site of fertilization
In vitro fertilization assays revealed that CatSper1 mutant sperm could not fertilize eggs with an intact zona pellucida layer but could fertilize eggs whose outer layers had been enzymatically removed [3]
Targeted disruption of CatSper2 led to male sterile phenotypes, and the null sperm has identical loss-of-function phenotypes as does the CatSper1 null sperm [6, 7]
Summary
Sperm must swim long distances in the female reproductive tract to reach the site of fertilization. We adopted a GST pulldown approach to isolate CatSper-associated proteins from human sperm extracts, and we have identified the T-type calcium channel subunit Cav3.3 as an interacting protein. Considering that CatSper1 and Cav3.3 subunits are co-expressed and associated with each other on the tail of the human sperm, our observations suggest that CatSper-Cav3.3 interactions could play an important role in regulating sperm functions such as hyperactivated motility.
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