Channelrhodopsin 2 (ChR2) is a microbial-type rhodopsin with a putative heptahelical structure that binds all-trans-retinal. Blue light illumination of ChR2 activates an intrinsic leak channel conductive for cations. Sequence comparison of ChR2 with the related ChR1 protein revealed a cluster of charged amino acids within the predicted transmembrane domain 2 (TM2), which includes glutamates E90, E97 and E101. Charge inversion substitutions of these residues significantly altered ChR2 function as revealed by two-electrode voltage-clamp recordings of light-induced currents from Xenopus laevis oocytes expressing the respective mutant proteins. Specifically, replacement of E90 by lysine or alanine resulted in differential effects on H+- and Na+-mediated currents. Our results are consistent with this glutamate side chain within the proposed TM2 contributing to ion flux through and the cation selectivity of ChR2. (C) 2011 Elsevier Inc. All rights reserved.