Kuwabara Makoto F, Klemptner Joschua, Muth Julia, De Martino Emilia, Oliver Dominik, Berger Thomas K
Department of Neurophysiology, Institute of Physiology and Pathophysiology, Philipps University Marburg, Marburg, Germany.
Department of Neurophysiology, Institute of Physiology and Pathophysiology, Philipps University Marburg, Marburg, Germany.
Biophys J. 2024 Dec 17;123(24):4256-4265. doi: 10.1016/j.bpj.2024.08.018. Epub 2024 Aug 28.
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.
电压门控离子通道可响应膜电位变化，使离子穿过生物膜。HCNL1是最近发现的一种电压门控离子通道，它通过其电压感应结构域（VSD）选择性地传导质子，这让人联想到经过充分研究的去极化激活的Hv1质子通道。然而，HCNL1由超极化激活，允许质子内流，这导致斑马鱼精子细胞内酸化。锌离子（Zn）是许多蛋白质中的重要辅助因子，对精子生理至关重要。诸如Hv1和Otopetrin1等质子通道会受到Zn的抑制。我们使用电生理和荧光技术研究了Zn对异源表达的HCNL1通道的影响。细胞外Zn抑制HCNL1电流，其表观半数最大抑制浓度（IC）为26 μM。Zn减缓电压依赖性电流动力学，将电压依赖性激活转移到更负的电位，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Zn通过多种机制抑制HCNL1电流，包括对通道门控的调节。位于VSD细胞外侧的两个组氨酸残基可能对Zn配位作用较弱：任一组氨酸被丙氨酸取代的突变体显示IC值向更高浓度有适度偏移。有趣的是，Zn从细胞内侧以更低浓度抑制HCNL1（IC≈0.5 μM）。S1细胞内末端的一个组氨酸残基（第50位）对Zn结合很重要：抑制突变体HCNL1-H50A需要高得多的Zn浓度（IC≈106 μM）。我们预计Zn将成为研究HCNL1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。
