Potassium-selective channelrhodopsins can exert hyper- or depolarizing effects in excitable cells of <i>Caenorhabditis elegans</i>, depending on experimental conditions

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.