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 (Zn) are important cofactors in many proteins and essential for sperm physiology. Proton channels such as Hv1 and Otopetrin1 are inhibited by Zn. We investigated the effect of Zn on heterologously expressed HCNL1 channels using electrophysiological and fluorometric techniques. Extracellular Zn inhibits HCNL1 currents with an apparent half-maximal inhibition (IC) of 26 μM. Zn 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 Zn 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 Zn coordination: mutants with either histidine replaced with alanine show modest shifts of the IC values to higher concentrations. Interestingly, Zn inhibits HCNL1 at even lower concentrations from the intracellular side (IC ≈ 0.5 μM). A histidine residue at the intracellular end of S1 (position 50) is important for Zn binding: much higher Zn concentrations are required to inhibit the mutant HCNL1-H50A (IC ≈ 106 μM). We anticipate that Zn will be a useful ion to study the structure-function relationship of HCNL1 as well as the physiological role of HCNL1 in zebrafish sperm.