Adjacent channelrhodopsin-2 residues within transmembranes 2 and 7 regulate cation selectivity and distribution of the two open states

Abstract

Channelrhodopsin-2 (ChR2) is a light-activated channel that can conduct cations of multiple valencies down the electrochemical gradient. Under continuous light exposure, ChR2 transitions from a high-conducting open state (O1) to a low-conducting open state (O2) with differing ion selectivity. The molecular basis for the O1 → O2 transition and how ChR2 modulates selectivity between states is currently unresolved. To this end, we used steered molecular dynamics, electrophysiology, and kinetic modeling to identify residues that contribute to gating and selectivity in discrete open states. Analysis of steered molecular dynamics experiments identified three transmembrane residues (Val-86, Lys-93, and Asn-258) that form a putative barrier to ion translocation. Kinetic modeling of photocurrents generated from ChR2 proteins with conservative mutations at these positions demonstrated that these residues contribute to cation selectivity (Val-86 and Asn-258), the transition between the two open states (Val-86), open channel stability, and the hydrogen-bonding network (K93I and K93N). These results suggest that this approach can be used to identify residues that contribute to the open-state transitions and the discrete ion selectivity within these states. With the rise of ChR2 use in optogenetics, it will be critical to identify residues that contribute to O1 or O2 selectivity and gating to minimize undesirable effects.

Channelrhodopsin C1C2: Photocycle kinetics and interactions near the central gate

“…Replacement of asparagine with valine in the analogous position of the ChR2-N258V variant in previous work led to protein expression in oocytes, but negligible photocurrents prevented functional studies of the variant. 16 In the present work, the introduction of a hydrophobic residue in the gating region of C1C2-N297V resulted in pore gating via E129(90)...E162(123) hydrogen bonding, an alternative central gate. The N297V mutation affected C1C2 photocycle kinetics minimally when compared with wild type C1C2.…”

“…E rev was found from the plot of photocurrent vs. membrane potential by linear interpolation, as previously described. 19,16 Data analysis was performed using Mathworks MATLAB R2017a.…”

“…1B). 16,21 Here, the model simulates channel photocurrents characterized by two closed states (C1 and C2) and two open states (O1 and O2) using the following set of rate equations,…”

“…This result contrasted with the analogous mutation in ChR2-N258V, which led to a complete cessation of measurable photocurrents despite a robust expression of protein. 16 The reversal potential, E rev , is the membrane potential at which the current flow through the channel reverses direction. Thus, E rev indicates the ease of ion passage through the pore.…”

“…How is gating achieved in the absence of the E129(90)...N297(258) hydrogen bond (C1C2-N297V)? Knowing that V86 contributes to ChR2 recovery to the ground state, 16 how does a conservative replacement of the analogous residue, which is spatially proximal to the central gate, affect chemical interactions in C1C2-V125L?…”