Abstract

Significance Channelrhodopsins are membrane proteins that enable cellular regulation of transmembrane ion conductance through light-gated pores; these proteins have found application in optogenetics. This paper tests the hypothesis that selectivity of channelrhodopsins is determined by surface potential of the pore region: Cations are conducted by a negatively charged pore, and chloride ions are conducted by a pore that has neutral and positively charged residues. In confirming this hypothesis and applying the resulting principles, we engineer improved chloride-conducting channels with higher chloride selectivity and conductivity. We also provide insights into the distinct mechanisms underlying inhibition mediated by higher-efficiency chloride channels compared with ion pumps. Finally, we demonstrate initial utility of light-gated microbial opsin-based chloride channels in controlling behavior of freely moving animals.