Unitary currents through K ⁺ selective channelrhodopsins reveal multiple conductance states within an activated state complex

The analysis of how sensation, cognition, and behavior arise from neuronal activity has been enormously facilitated by optogenetics, using algae-derived light sensitive channelrhodopsins, ChRs, expressed in brain cells. To disclose the unitary conductance and the details of the gating mechanism, we recorded unitary currents of the potentially inhibitory K ⁺ selective ChRs HcKCR1 and WiChR1 at femtosiemens resolution. Both ChRs open in bursts with a large dominating conductance of 1.6 pS for KCR1 and 2.4 pS for WiChR1 but they are accompanied by widely distributed conductance levels covering a continuum of between 0.3 and 3.0 pS. In WiChR1, these bursts are approximately four times longer than in KCR1. The photocycle and the closed-open gating in an activated state complex seem to be two orthogonal processes. The single-channel currents of the two ChRs explain all characteristic features of the respective whole-cell currents. Our data provide insight into exceptionally small and heterogeneous conductance levels of single channelrhodopsin molecules and open the door to tailor specific tools for optogenetic control in neurons of brain networks.