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.

中文翻译：

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锌抑制电压门控质子通道 HCNL1


电压门控离子通道允许离子通量穿过生物膜，以响应膜电位的变化。HCNL1 是最近发现的电压门控离子通道，可选择性地通过其电压感应域 （VSD） 传导质子，让人想起经过充分研究的去极化激活的 Hv1 质子通道。然而，HCNL1 被超极化激活，允许质子流入，从而导致斑马鱼精子的细胞内酸化。锌离子 （Zn2+） 是许多蛋白质中的重要辅助因子，对精子生理学至关重要。Hv1 和 Otopetrin1 等质子通道被 Zn2+ 抑制。我们使用电生理学和荧光技术研究了 Zn 2 + 对异源表达的 HCNL1 通道的影响。细胞外 Zn2 + 抑制 HCNL1 电流，明显的半最大抑制 （IC50） 为 26 μM。Zn2+ 减慢电压依赖性电流动力学，将电压依赖性激活转移到更多的负电位，并改变超极化诱导的电压传感器构象变化。我们的数据表明，细胞外 Zn2 + 通过多种机制抑制 HCNL1 电流，包括通道门控的调节。位于 VSD 细胞外侧的两个组氨酸残基可能对 Zn 2 + 配位有微弱的贡献：用丙氨酸取代组氨酸的突变体显示 IC50 值向更高浓度的适度变化。有趣的是，Zn2 + 在细胞内侧以更低的浓度抑制 HCNL1 （IC50 ≈ 0.5 μM）。S1 细胞内末端（位置 50）的组氨酸残基对 Zn2 + 结合很重要：需要更高的 Zn 2 + 浓度来抑制突变体 HCNL1-H50A （IC50 ≈ 106 μM）。 我们预计 Zn2+ 将成为研究 HCNL1 的结构-功能关系以及 HCNL1 在斑马鱼精子中的生理作用的有用离子。