Channelrhodopsins (ChRs) are microbial rhodopsins that function as light-gated ion channels. When expressed in heterologous systems, they enable optical control of membrane potential and are widely used as optogenetic actuators. Cation- and anion-conducting ChRs allow either depolarization or hyperpolarization of the membrane, and more than 400 variants are currently known. Systematic electrophysiological characterization is essential to understand their properties and guide their application in neuroscience and cell biology. Here, we studied channelrhodopsin-2 (ChR2) expressed together with the voltage-gated sodium channel Nav1.5 in HEK293 cells, using the SyncroPatch 384 automated patch clamp platform with integrated optical stimulation. Photocurrents were recorded across a range of irradiances, from near-threshold activation to saturating intensities. Under voltage-clamp conditions with sodium channels blocked by tetracaine, we quantified peak and steady-state photocurrents, activation and deactivation kinetics, and desensitization during illumination. To examine temperature effects, kinetics were compared at 20°C and 30°C. Ion selectivity was further assessed by systematically varying external (bath) and internal (pipette) ionic compositions. In current-clamp recordings without tetracaine, increasing light intensities produced graded membrane depolarizations, and strong illumination reliably activated Nav1.5, resulting in rapid additional depolarization. These experiments demonstrate how ChR2-mediated conductances interact withinfluence voltage-gated sodium currents channel activity and how environmental factors such as temperature and ionic composition influence functional properties. This study provides a comprehensive analysis of ChR2 in a heterologous system, combining optical stimulation and automated patch-clamp to evaluate kinetics, desensitization, ion selectivity, and channel interplay under defined experimental conditions.
Strassmaier et al. (2026) studied this question.