Kinetics of proton release and uptake by channelrhodopsin-2

Electrophysiological experiments showed that the light-activated cation channel channelrhodopsin-2 (ChR2) pumps protons in the absence of a membrane potential. We determined here the kinetics of transient pH change using a water-soluble pH-indicator. It is shown that ChR2 released protons prior to uptake with a stoichiometry of 0.3 protons per ChR2. Comparison to the photocycle kinetics revealed that proton release and uptake match rise and decay of the P3520 intermediate. As the P3520 state also represents the conductive state of cation channeling, the concurrence of proton pumping and channel gating implies an intimate mechanistic link of the two functional modes. Studies on the E123T and S245E mutants show that these residues are not critically involved in proton translocation.

► Transient proton translocation by channelrhodopsin-2 was detected by an optical pH indicator.
► Proton release and uptake proceed with time constants of 1.7 ms and 11 ms, respectively.
► The response time of the pH indicator is not limited by surface-to-bulk diffusion.
► Residues E123 and S245 do not participate in proton release.
► The concurrence of proton release and ion channeling points to a mechanistic link.