Identification of a Natural Green Light Absorbing Chloride Conducting Channelrhodopsin from Proteomonas sulcata

Abstract

Chloride conducting channelrhodopsins (ChloCs) are new members of the optogenetic toolbox that enable neuronal inhibition in target cells. Originally, ChloCs have been engineered from cation conducting channelrhodopsins (ChRs), and later identified in a cryptophyte alga genome. We noticed that the sequence of a previously described Proteomonas sulcata ChR (PsChR1) was highly homologous to the naturally occurring and previously reported ChloCs GtACR1/2, but was not recognized as an anion conducting channel. Based on electrophysiological measurements obtained under various ionic conditions, we concluded that the PsChR1 photocurrent at physiological conditions is strongly inward rectifying and predominantly carried by chloride. The maximum activation was noted at excitation with light of 540 nm. An initial spectroscopic characterization of purified protein revealed that the photocycle and the transport mechanism of PsChR1 differ significantly from cation conducting ChRs. Hence, we concluded that PsChR1 is an anion conducting ChR, now renamed PsACR1, with a red-shifted absorption suited for multicolor optogenetic experiments in combination with blue light absorbing cation conducting ChRs.

The light‐driven sodium ion pump: A new player in rhodopsin research

“…In addition, as shown in recent successful examples [20,33,[69][70][71][72][73][74][75][76][77][78], the wealth of structural data available and advances in genomic technologies will lead to further engineering and discovery of functionally novel rhodopsins, and it is expected that the scope and impact of rhodopsin research will continue to expand.…”

“…Recently, the structural information on channelrhodopsin (ChR), a light-gated cation channel, has prompted the engineering of a light-gated anion channel [69][70][71][72], and soon after that, light-gated anion channels were discovered in a natural source [73,74]. Structurebased molecular engineering accomplished a 100 nm blue shift in the absorption spectrum of a proton pumping rhodopsin [75], and de novo transcriptome sequencing of green alga has led to the discovery of a novel ChR with a very red-shifted spectral peak (590 nm) [76].…”

The form and function of channelrhodopsin

“…Replacement of E3 [hydrogen-bonded to Ser 63 and Asn 258 forming the central gate (86, 87); ChR2 residue numbering] had the largest impact. Replacement of inner-gate His 134 /E2 or access-channel E4/E5 also reduced H conductance (62, 64, 88, 89). Further support for this proton-wire hypothesis is derived from cryptophyte anion-conducting ChRs (90), which show reduced H + conductance with fewer helix-2 glutamates (90); interestingly, these glutamates have little influence on kinetics, and only mutation of E1 decelerates closing (88, 91).…”

Microbial Rhodopsins: Diversity, Mechanisms, and Optogenetic Applications

“…A resonance Raman study of Gt ACR1 showed that the retinal chromophore exists in an all- trans configuration with a protonated Schiff base very similar to that of BR (149). The most striking difference between the photocycle of all three so far tested ACRs and other type 1 rhodopsins is an extremely slow appearance and decay of a blue-shifted M-like intermediate with a deprotonated retinylidene Schiff base (133, 136). In CCRs M formation occurs within microseconds to tens of microseconds and precedes channel opening (91, 95–96).…”

“…The spectral sensitivities of Gt ACR1 and Gt ACR2 photocurrents peak at 515 and 470 nm, respectively. Another cryptophyte alga, Proteomonas sulcata , contained a channelrhodopsin initially named Ps ChR1 (86), but renamed Psu ACR1 (also known as Ps ACR1) when shown to conduct exclusively anions (132–133). Screening sequences obtained by an ongoing transcriptome sequencing projects (134–135) expanded the list of functional ACRs to include 20 proteins derived from various marine cryptophyte species.…”