Proton transfer reactions in the red light-activatable channelrhodopsin variant ReaChR and their relevance for its function

Joel C.D Kaufmann,Benjamin S Krause,Christiane Grimm,Eglof Ritter,Peter Hegemann,Franz J Bartl

doi:10.1074/jbc.m117.779629

Abstract

Channelrhodopsins (ChRs) are light-gated ion channels widely used for activating selected cells in large cellular networks. ChR variants with a red-shifted absorption maximum, such as the modified Volvox carteri ChR1 red-activatable channelrhodopsin ("ReaChR," λmax = 527 nm), are of particular interest because longer wavelengths allow optical excitation of cells in deeper layers of organic tissue. In all ChRs investigated so far, proton transfer reactions and hydrogen bond changes are crucial for the formation of the ion-conducting pore and the selectivity for protons versus cations, such as Na+, K+, and Ca2+ (1). By using a combination of electrophysiological measurements and UV-visible and FTIR spectroscopy, we characterized the proton transfer events in the photocycle of ReaChR and describe their relevance for its function. 1) The central gate residue Glu130 (Glu90 in Chlamydomonas reinhardtii (Cr) ChR2) (i) undergoes a hydrogen bond change in D → K transition and (ii) deprotonates in K → M transition. Its negative charge in the open state is decisive for proton selectivity. 2) The counter-ion Asp293 (Asp253 in CrChR2) receives the retinal Schiff base proton during M-state formation. Starting from M, a photocycle branching occurs involving (i) a direct M → D transition and (ii) formation of late photointermediates N and O. 3) The DC pair residue Asp196 (Asp156 in CrChR2) deprotonates in N → O transition. Interestingly, the D196N mutation increases 15-syn-retinal at the expense of 15-anti, which is the predominant isomer in the wild type, and abolishes the peak current in electrophysiological measurements. This suggests that the peak current is formed by 15-anti species, whereas 15-syn species contribute only to the stationary current.

Highlights

Channelrhodopsins (ChRs) are light-gated ion channels widely used for activating selected cells in large cellular networks
The concept of parallel 15-anti and 15-syn photocycles delivers an explanation for the observed isomeric light-dark adaptation of ChRs by assuming transitions between the different subspecies by C13ϭC14 and C15ϭN double isomerization: the initial dark state (IDA) of ChRs, which is formed after hours of dark adaptation, adopts a pure all-trans-retinal isomer according to recent NMR studies [16, 17]
To estimate the independent spectral components contributing to the signal change, we evaluated the results using a combination of singular value decomposition (SVD) and a rotation procedure [46, 47] as performed for CrChR2-C128T [15]

Summary

Introduction

Channelrhodopsins (ChRs) are light-gated ion channels widely used for activating selected cells in large cellular networks. This phenomenon is called inactivation or desensitization and was interpreted previously in CrChR2 as an equilibration process between two conducting states with different conductivities, which become populated by two distinct dark states (D and DЈ) under continuous illumination [1, 12,13,14] It was suggested [11, 15, 16] that the dark states represent protein subspecies with a 15-anti- and 15-syn-retinal conformation, respectively, whereby their conducting states differ with respect to ion selectivity, conductivity, and channel kinetics. The concept of parallel 15-anti and 15-syn photocycles delivers an explanation for the observed isomeric light-dark adaptation of ChRs by assuming transitions between the different subspecies by C13ϭC14 and C15ϭN double isomerization: the initial dark state (IDA) of ChRs, which is formed after hours of dark adaptation, adopts a pure all-trans-retinal isomer according to recent NMR studies [16, 17].

Results

Discussion

Conclusion