Photochemical reaction cycle transitions during anion channelrhodopsin gating

Abstract

A recently discovered family of natural anion channelrhodopsins (ACRs) have the highest conductance among channelrhodopsins and exhibit exclusive anion selectivity, which make them efficient inhibitory tools for optogenetics. We report analysis of flashinduced absorption changes in purified wild-type and mutant ACRs, and of photocurrents they generate in HEK293 cells. Contrary to cation channelrhodopsins (CCRs), the ion conducting state of ACRs develops in an L-like intermediate that precedes the deprotonation of the retinylidene Schiff base (i.e., formation of an M intermediate). Channel closing involves two mechanisms leading to depletion of the conducting L-like state: (i) Fast closing is caused by a reversible L⇔M conversion. Glu-68 in Guillardia theta ACR1 plays an important role in this transition, likely serving as a counterion and proton acceptor at least at high and neutral pH. Incomplete suppression of M formation in the GtACR1_E68Q mutant indicates the existence of an alternative proton acceptor. (ii) Slow closing of the channel parallels irreversible depletion of the M-like and, hence, L-like state. Mutation of Cys-102 that strongly affected slow channel closing slowed the photocycle to the same extent. The L and M intermediates were in equilibrium in C102A as in the WT. In the position of Glu-123 in channelrhodopsin-2, ACRs contain a noncarboxylate residue, the mutation of which to Glu produced early Schiff base proton transfer and strongly inhibited channel activity. The data reveal fundamental differences between natural ACR and CCR conductance mechanisms and their underlying photochemistry, further confirming that these proteins form distinct families of rhodopsin channels.photochemical conversions | Schiff base | channel gating | channelrhodopsins | optogenetics T he genomes of cryptophyte algae harbor nucleotide sequences that encode anion-conducting channelrhodopsins (ACRs) (1, 2). Although these proteins show distant sequence homology to cation-conducting channelrhodopsins (CCRs) from green (chlorophyte) algae, they completely lack permeability for protons and metal cations and, thus, represent a distinct structural and functional class among microbial rhodopsins. Using ACRs permits optogenetic inhibition of neuronal firing at much lower light intensities than other currently used silencers (1).Analysis of photocurrents generated by ACR1 from Guillardia theta (GtACR1) in human embryonic kidney (HEK) cells under single-turnover conditions revealed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH, and involving different amino acid residues (3). The first mechanism, characterized by fast closing of the channel, is regulated by Glu-68, a homolog of Glu-90 in channelrhodopsin-2 from the green alga Chlamydomonas reinhardtii (CrChR2). Replacement of Glu-90 with Arg in CrChR2 made the channel permeable for Cl − (4), whereas the same substitution of Glu-68 in GtACR1 did not influence its selectivity for anions, but inverted its gating, ren...

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K

“…Previous studies have shown that the Glu68 → Gln substitution (E68Q) has significant effects on the channel gating mechanism 6 but only a weak effect on the visible absorption. 5,10 In agreement, at 80 K, UV–visible absorption of E68Q is very similar to that of WT (Figure S6A). However, the difference spectrum compared to that of WT (Figure 7 and Figure S6B) displays an increased negative intensity and a downshift in the wavelength of the major negative band (from 510 to 503 nm).…”

“…This result may indicate the K and L intermediates produced at 80 K exist in equilibrium, similar to the conclusion from room-temperature visible absorption measurements. 5 In addition, successive first-push decay difference spectra recorded at approximately 1 min intervals after the exciting light is turned off (Figure S3) reveal very little change compared to the initial “2–1” difference spectrum. This result eliminates the possibility that an L-like photoproduct corresponding to the 1545 cm −1 band is a transient species at 80 K that accumulates during illumination.…”

“…The final buffer used for storage of the membranes at 5 °C consisted of 5 mM K 2 HPO 4 and 100 mM NaCl (pH 7.3). All mutants were generated using a similar procedure as described above (see also refs 5 and 10 ).…”

“…Recent studies of the light-induced photocurrent and photochemical reaction cycle of an ACR from Guillardia theta (designated Gt ACR1) reveal several distinct differences. 5,6 Most importantly, in 6 ns laser flash photolysis measurements, the Schiff base-deprotonated M intermediate occurs much later in ACRs than in any other microbial rhodopsins, including CCRs. The formation of a blue-shifted L-like intermediate (which may be in rapid equilibrium with pre-M red-shifted intermediate(s)) in the Gt ACR1 photocycle is correlated with the anion conducting “open” state of the channel while decay of the L intermediate (and formation of an M intermediate) with channel closing.…”

“…The formation of a blue-shifted L-like intermediate (which may be in rapid equilibrium with pre-M red-shifted intermediate(s)) in the Gt ACR1 photocycle is correlated with the anion conducting “open” state of the channel while decay of the L intermediate (and formation of an M intermediate) with channel closing. 5,6 This is an unusual feature of ACRs because the “activated state” of most microbial rhodopsins, such as signaling states of sensory rhodopsins, is associated with M formation and not the earlier K or L intermediates. For example, in the case of CCRs, the open channel state occurs after formation of an M-like intermediate often designated P 2 3807 and subsequent red-shifted (N/O) intermediates.…”