Two open states with progressive proton selectivities in the branched channelrhodopsin-2 photocycle

Channelrhodopsins are light-gated ion channels that mediate vision in phototactic green algae like Chlamydomonas. In neurosciences, channelrhodopsins are widely used to light-trigger action potentials in transfected cells. All known channelrhodopsins preferentially conduct H(+). Previous studies have indicated the existence of an early and a late conducting state within the channelrhodopsin photocycle. Here, we show that for channelrhodopsin-2 expressed in Xenopus oocytes and HEK cells, the two open states have different ion selectivities that cause changes in the channelrhodopsin-2 reversal voltage during a light pulse. An enzyme kinetic algorithm was applied to convert the reversal voltages in various ionic conditions to conductance ratios for H(+) and divalent cations (Ca(2+) and/or Mg(2+)), as compared to monovalent cations (Na(+) and/or K(+)). Compared to monovalent cation conductance, the H(+) conductance, alpha, is approximately 3 x 10(6) and the divalent cation conductance, beta, is approximately 0.01 in the early conducting state. In the stationary mixture of the early and late states, alpha is larger and beta smaller, both by a factor of approximately 2. The results suggest that the ionic basis of light perception in Chlamydomonas is relatively nonspecific in the beginning of a light pulse but becomes more selective for protons during longer light exposures.