Abstract
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(2+)) are important cofactors in many proteins and essential for sperm physiology. Proton channels such as Hv1 and Otopetrin1 are inhibited by Zn(2+). We investigated the effect of Zn(2+) on heterologously expressed HCNL1 channels using electrophysiological and fluorometric techniques. Extracellular Zn(2+) inhibits HCNL1 currents with an apparent half-maximal inhibition (IC(50)) of 26 μM. Zn(2+) 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(2+) 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(2+) coordination: mutants with either histidine replaced with alanine show modest shifts of the IC(50) values to higher concentrations. Interestingly, Zn(2+) inhibits HCNL1 at even lower concentrations from the intracellular side (IC(50) ≈ 0.5 μM). A histidine residue at the intracellular end of S1 (position 50) is important for Zn(2+) binding: much higher Zn(2+) concentrations are required to inhibit the mutant HCNL1-H50A (IC(50) ≈ 106 μM). We anticipate that Zn(2+) will be a useful ion to study the structure-function relationship of HCNL1 as well as the physiological role of HCNL1 in zebrafish sperm.