Channelrhodopsin C1C2: Photocycle kinetics and interactions near the central gate

Abstract

Channelrhodopsins (ChR) are cation channels that can be expressed heterologously in various living tissues, including cardiac and neuronal cells. To tune spatial and temporal control of potentials across ChR-enriched cell membranes, it is essential to understand how pore hydration impacts the ChR photocycle kinetics. Here, we measure channel opening and closing rates of channelrhodopsin chimera and selected variants (C1C2 wild type, C1C2-N297D, C1C2-N297V, and C1C2-V125L) and correlate them with changes in chemical interactions among functionally important residues in both closed and open states. Kinetic results substantiate that replacement of helices I and II in ChR2 with corresponding residues from ChR1, to make the chimera C1C2, affects the kinetics of channelrhodopsin pore gating significantly, making C1C2 a unique channel. As a prerequisite for studies of ion transport, detailed understanding of the water pathway within a ChR channel is important. Our atomistic simulations confirm that opening of the channel and initial hydration of the previously dry gating regions between helices I, II, III, and VII of the channel occurs with 1) the presence of 13-cis retinal; 2) deprotonation of a glutamic acid gating residue, E129; and 3) subsequent weakening of the central gate hydrogen bond between the same glutamic acid E129 and asparagine N282 in the central region of the pore. Also, an aspartate (D292) is the unambiguous primary proton acceptor for the retinal Schiff base in the hydrated channel. SIGNIFICANCEChannelrhodopsins (ChR) are light-sensitive ion channels used in optogenetics, a technique that applies light to selectively and non-invasively control cells (e.g., neurons) that have been modified genetically to express those channels. Using electrophysiology, we measured the opening and closing rates of a ChR chimera, and several variants, and correlated those rates with changes in chemical interactions determined from atomistic simulations. Significant new insights include correlation of single-point-mutations with four factors associated with pore hydration and cation conductance. Additionally, our work unambiguously identifies the primary proton acceptor for the retinal chromophore in the channel open state. These new insights add to mechanistic understanding of lightgated membrane transport and should facilitate future efforts to control membrane potentials spatially and temporally in optogenetics.

Hydrated anions: From clusters to bulk solution with quasi-chemical theory

“…20 For anions such as Cl − , regulation of ion concentration is achieved through membrane transport proteins of the CLC family and others, including channelrhodopsins, which facilitate the passage of Cl − through electro-chemical potential gradients. [25][26][27] While those structurally diverse proteins select for Cl − , other channel proteins discriminate against Cl − . An interesting example is FLUC, a family of fluoride-specific ion channels with dual-topology architecture.…”