Attractant and Repellent Signaling Conformers of Sensory Rhodopsin−Transducer Complexes

Sineshchekov, Oleg A.; Sasaki, Jun; Wang, Jihong; Spudich, John L.

doi:10.1021/bi100798w

Cited by 19 publications

(31 citation statements)

References 40 publications

(83 reference statements)

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“…At pH ≤ 3, irreversible absorption changes gave rise to a product with maximal absorption at ∼430 nm (Fig. 1D, red line), which is typical of partially unfolded microbial rhodopsins (13). Absorption changes in the alkaline region revealed two transitions.…”

Section: Resultsmentioning

confidence: 93%

Photochemical reaction cycle transitions during anion channelrhodopsin gating

Sineshchekov

Govorunova

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 93%

Photochemical reaction cycle transitions during anion channelrhodopsin gating

Sineshchekov

Govorunova

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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130

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“…A similar inversion of protein function was previously observed in haloarchaeal sensory rhodopsin I (SRI), in which a single point mutation either of the photoreceptor itself or of its cognate transducer converted SRI from an attractant to a repellent photoreceptor (16,17). The SRI inversion is attributable to a switch in its conformation from the C (retinylidene Schiff base accessible from the cytoplasm) to E (Schiff base accessible from the extracellular space) conformer (18)(19)(20). The stable open channel of GtACR1_E68R is likely to be particularly valuable for structural comparisons with the WT by enabling identification of residue determinants and structural changes distinguishing the closed and open conformations of the channel by structural methods such as molecular spectroscopy and crystallography.…”

Section: Discussionmentioning

confidence: 99%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Anion channelrhodopsins (ACRs) are a class of light-gated channels recently identified in cryptophyte algae that provide unprecedented fast and powerful hyperpolarizing tools for optogenetics. Analysis of photocurrents generated by Guillardia theta ACR 1 (GtACR1) and its mutants in response to laser flashes showed that GtACR1 gating comprises two separate mechanisms with opposite dependencies on the membrane voltage and pH and involving different amino acid residues. The first mechanism, characterized by slow opening and fast closing of the channel, is regulated by Glu-68. Neutralization of this residue (the E68Q mutation) specifically suppressed this first mechanism, but did not eliminate it completely at high pH. Our data indicate the involvement of another, yetunidentified pH-sensitive group X. Introducing a positive charge at the Glu-68 site (the E68R mutation) inverted the channel gating so that it was open in the dark and closed in the light, without altering its ion selectivity. The second mechanism, characterized by fast opening and slow closing of the channel, was not substantially affected by the E68Q mutation, but was controlled by Cys-102. The C102A mutation reduced the rate of channel closing by the second mechanism by ∼100-fold, whereas it had only a twofold effect on the rate of the first. The results show that anion conductance by ACRs has a fundamentally different structural basis than the relatively well studied conductance by cation channelrhodopsins (CCRs), not attributable to simply a modification of the CCR selectivity filter.ion transport | channel gating | channelrhodopsins | optogenetics R ecently we reported a class of rhodopsins from the cryptophyte alga Guillardia theta that act as anion-conducting channels when expressed in cultured animal cells and therefore can be used to suppress neuronal firing by light (1). These proteinsnamed anion channelrhodopsins (ACRs)-show distant sequence homology to cation channelrhodopsins (CCRs) from chlorophyte (green) algae (2), but completely lack permeability for protons and metal cations. This property, as well as large current amplitudes and fast kinetics, makes ACRs superior hyperpolarizing optogenetic tools, compared with proton and chloride pumps (3, 4), or engineered Cl − -conducting CCR variants (5, 6). Two G. theta (Gt) ACRs differ in their spectral sensitivity and channel kinetics (1). GtACR1, with its absorption peak at 515 nm (Fig. S1), has an advantage over a more blue-shifted GtACR2 with maximal efficiency at 470 nm, because it allows using light of longer wavelengths that is less scattered by biological tissue.Because a CCR could be altered to exhibit Cl − channel activity by mutation of a single amino acid residue (5), a fundamental question about ACRs is whether they differ from CCRs only by their selectivity filter. In our search for an answer, we analyzed photocurrents generated by GtACR1 in HEK293 cells under single-turnover conditions in which secondary photochemistry does not complicate the photocycle. We found that GtACR1 cond...

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“…36 Light-induced displacement/rotation of helix F in the photocycle of the SRI–HtrI attractant signaling is in the direction that is the opposite of that in BR and SRII, which agrees with other data showing that SRI undergoes a C → E conversion in the SRI–HtrI molecular complex. 24,26,37 …”

Section: Discussionmentioning

confidence: 99%

Cation-Specific Conformations in a Dual-Function Ion-Pumping Microbial Rhodopsin

et al. 2015

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A recently discovered rhodopsin ion pump (DeNaR, also known as KR2) in the marine bacterium Dokdonia eikasta uses light to pump protons or sodium ions from the cell depending on the ionic composition of the medium. In cells suspended in a KCl solution, DeNaR functions as a light-driven proton pump, whereas in a NaCl solution, DeNaR conducts light-driven sodium ion pumping, a novel activity within the rhodopsin family. These two distinct functions raise the questions of whether the conformations of the protein differ in the presence of K+ or Na+ and whether the helical movements that result in the canonical E → C conformational change in other microbial rhodopsins are conserved in DeNaR. Visible absorption maxima of DeNaR in its unphotolyzed (dark) state show an 8 nm difference between Na+ and K+ in decyl maltopyranoside micelles, indicating an influence of the cations on the retinylidene photoactive site. In addition, electronic paramagnetic resonance (EPR) spectra of the dark states reveal repositioning of helices F and G when K+ is replaced with Na+. Furthermore, the conformational changes assessed by EPR spin–spin dipolar coupling show that the light-induced transmembrane helix movements are very similar to those found in bacteriorhodopsin but are altered by the presence of Na+, resulting in a new feature, the clockwise rotation of helix F. The results establish the first observation of a cation switch controlling the conformations of a microbial rhodopsin and indicate specific interactions of Na+ with the half-channels of DeNaR to open an appropriate path for ion translocation.

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Attractant and Repellent Signaling Conformers of Sensory Rhodopsin−Transducer Complexes

Cited by 19 publications

References 40 publications

Photochemical reaction cycle transitions during anion channelrhodopsin gating

Photochemical reaction cycle transitions during anion channelrhodopsin gating

Gating mechanisms of a natural anion channelrhodopsin

Cation-Specific Conformations in a Dual-Function Ion-Pumping Microbial Rhodopsin

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