Zinc inhibits the voltage-gated proton channel HCNL1

Voltage-gated ion channels allow ion flux across biological membranes in response to changes in the membrane potential. HCNL1 is a recently discovered voltage-gated ion channel that selectively conducts protons through its voltage-sensing domain (VSD), reminiscent of the well-studied depolarization-activated Hv1 proton channel. However, HCNL1 is activated by hyperpolarization, allowing the influx of protons, which leads to an intracellular acidification in zebrafish sperm. Zinc ions (Zn²⁺) are important cofactors in many proteins and essential for sperm physiology. Proton channels such as Hv1 and Otopetrin1 are inhibited by Zn²⁺. We investigated the effect of Zn²⁺ on heterologously expressed HCNL1 channels using electrophysiological and fluorometric techniques. Extracellular Zn²⁺ inhibits HCNL1 currents with an apparent half-maximal inhibition (IC₅₀) of 26 μM. Zn²⁺ slows voltage-dependent current kinetics, shifts the voltage-dependent activation to more negative potentials, and alters hyperpolarization-induced conformational changes of the voltage sensor. Our data suggest that extracellular Zn²⁺ inhibits HCNL1 currents by multiple mechanisms, including modulation of channel gating. Two histidine residues located at the extracellular side of the VSD might weakly contribute to Zn²⁺ coordination: mutants with either histidine replaced with alanine show modest shifts of the IC₅₀ values to higher concentrations. Interestingly, Zn²⁺ inhibits HCNL1 at even lower concentrations from the intracellular side (IC₅₀ ≈ 0.5 μM). A histidine residue at the intracellular end of S1 (position 50) is important for Zn²⁺ binding: much higher Zn²⁺ concentrations are required to inhibit the mutant HCNL1-H50A (IC₅₀ ≈ 106 μM). We anticipate that Zn²⁺ will be a useful ion to study the structure-function relationship of HCNL1 as well as the physiological role of HCNL1 in zebrafish sperm.