Zinc is a key regulator of the sperm-specific K+ channel (Slo3) function.

The voltage- and pH-gated Slo3 potassium channel is exclusively expressed in mammalian spermatozoa. Its sensitivity to both voltage and alkalization plays a crucial role in sperm fertility, which is tightly coupled to the capacitation process. Here, we show that sperm-enriched divalent cation Zn2+ undergoes dynamic alteration in spermatozoa during capacitation. We also found that intracellular Zn2+ regulates alkalinization-induced hyperpolarization in mouse spermatozoa, which is mediated by the Slo3 channel. Further examination of zinc regulation in mouse Slo3 (mSlo3) revealed that, in the Xenopus oocyte expression system, intracellular zinc directly inhibits mouse Slo3 currents in a dose-dependent manner at micromolar concentrations, with exceptionally slow dissociation. By combining MD simulations and electrophysiology, we also identified amino acid residues contributing to the Zn2+ slow dissociation from Slo3 channels. Our studies uncover the importance of intracellular zinc dynamics and its regulatory role in ion channels during sperm capacitation.