Ruse Christiane, Liewald Jana Fiona, Seidenthal Marius, Tillert Linda, Vierock Johannes, Gottschalk Alexander
Institute of Biophysical Chemistry, Goethe University, Max-von-Laue-Strasse 9, D-60438 Frankfurt, Germany.
Buchmann Institute of Molecular Life Sciences, Goethe University, Max-von-Laue-Strasse 15, D-60438 Frankfurt, Germany.
Genetics. 2025 Jul 9;230(3). doi: 10.1093/genetics/iyaf083.
Among the most frequent applications of optogenetic tools are those for depolarization of excitable cells such as neurons and muscles. Equally important are inhibitory tools that act through cellular hyperpolarization, which often rely on chloride conductance. Yet, in vivo, re- and hyperpolarization is typically mediated by potassium. Recently, light-gated ion channels with high potassium selectivity were identified (Kalium channelrhodopsins, KCRs), and their use described in different organisms. Here, we characterized HcKCR1 and WiChR in cholinergic neurons and muscles of the nematode Caenorhabditis elegans. Hyperpolarization of both cell types induces muscle relaxation and elongation of the animals. We analyzed body length changes during illumination, to benchmark stimulation parameters like light intensity and duration. For HcKCR1 in cholinergic neurons, continuous illumination at high light intensities (1-4.5 mW/mm²) evoked only a transient elongation, while stimulation at 0.1 mW/mm² maintained inhibition for the duration of the stimulus in some transgenic strains. Electrophysiological characterization verified that initial hyperpolarization quickly changed to depolarization, and current-voltage relationships indicate that the initial channel selectivity for potassium became progressively tolerant for sodium during the light pulse, scaling with light intensity. For animals expressing WiChR, we again observed brief hyperpolarization during continuous illumination; however, still during the stimulus, this changed to depolarization. This effect was long-lasting, requiring dozens of seconds for reversion, but could be reduced by pulsed illumination and fully avoided by less efficient channel activation using green or orange light. Hence, KCRs can be applied to hyperpolarize C. elegans cells, but require optimized expression and illumination parameters.
光遗传学工具最常见的应用之一是用于使可兴奋细胞（如神经元和肌肉）去极化。同样重要的是通过细胞超极化起作用的抑制性工具，这通常依赖于氯离子电导。然而，在体内，复极化和超极化通常由钾介导。最近，人们发现了具有高钾选择性的光门控离子通道（钾离子通道视紫红质，KCRs），并描述了它们在不同生物体中的应用。在这里，我们对线虫秀丽隐杆线虫的胆碱能神经元和肌肉中的HcKCR1和WiChR进行了表征。两种细胞类型的超极化都会导致动物肌肉松弛和身体伸长。我们分析了光照期间的体长变化，以确定光强度和持续时间等刺激参数。对于胆碱能神经元中的HcKCR1，在高光强度（1-4.5 mW/mm²）下持续光照仅引起短暂的伸长，而在0.1 mW/mm²下刺激在一些转基因品系中在刺激持续时间内维持抑制作用。电生理表征证实，初始超极化迅速转变为去极化，电流-电压关系表明，在光脉冲期间，初始对钾的通道选择性逐渐变得对钠具有耐受性，并与光强度成比例。对于表达WiChR的动物，我们在持续光照期间再次观察到短暂的超极化；然而，仍在刺激期间，这转变为去极化。这种效应是持久的，需要几十秒才能恢复，但可以通过脉冲光照减少，并通过使用绿色或橙色光进行效率较低的通道激活完全避免。因此，KCRs可用于使秀丽隐杆线虫细胞超极化，但需要优化表达和光照参数。
