Voltage-gated proton (Hv1) channels are involved in many physiological processes, such as pH homeostasis and the innate immune response. Zn²⁺ is an important physiological inhibitor of Hv1. Sperm cells are quiescent in the male reproductive system due to Zn²⁺ inhibition of Hv1 channels, but become active once introduced into the low-Zn²⁺-concentration environment of the female reproductive tract. How Zn²⁺ inhibits Hv1 is not completely understood. In this study, we use the voltage clamp fluorometry technique to identify the molecular mechanism of Zn²⁺ inhibition of Hv1. We find that Zn²⁺ binds to both the activated closed and resting closed states of the Hv1 channel, thereby inhibiting both voltage sensor motion and gate opening. Mutations of some Hv1 residues affect only Zn²⁺ inhibition of the voltage sensor motion, whereas mutations of other residues also affect Zn²⁺ inhibition of gate opening. These effects are similar in monomeric and dimeric Hv1 channels, suggesting that the Zn²⁺-binding sites are localized within each subunit of the dimeric Hv1. We propose that Zn²⁺ binding has two major effects on Hv1: (i) at low concentrations, Zn²⁺ binds to one site and prevents the opening conformational change of the pore of Hv1, thereby inhibiting proton conduction; and (ii) at high concentrations, Zn²⁺, in addition, binds to a second site and inhibits the outward movement of the voltage sensor of Hv1. Elucidating the molecular mechanism of how Zn²⁺ inhibits Hv1 will further our understanding of Hv1 function and might provide valuable information for future drug development for Hv1 channels.

	Year	Citations

About