Photocurrent attenuation by a single polar-to-nonpolar point mutation of channelrhodopsin-2

Sugiyama, Yuka; Wang, Hongxia; Hikima, Takuya; Sato, Minami; Kuroda, Jun; Takahashi, Tetsuo; Ishizuka, Toru; Yawo, Hiromu

doi:10.1039/b815762f

Cited by 53 publications

(75 citation statements)

References 42 publications

(35 reference statements)

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…However, these negatively charged residues represent a striking feature of helix 2 in our alignment as well (supplemental Fig. S2), and at least some of those were shown to affect the photocurrent (42,43).…”

Section: Discussionmentioning

confidence: 99%

Bioinformatic and Mutational Analysis of Channelrhodopsin-2 Protein Cation-conducting Pathway

Plazzo

Franceschi

Broi

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: ChR2 is a light-gated ion channel allowing fast non-invasive control of cell membrane potential. Results: We combined bioinformatic modeling and electrophysiology to infer structure/function details on ChR2. Conclusion: We show a complete structural model of the channel, describe the ion-conducting pathway and identify key residues involved in ionic permeability and in photoactivation. Significance: These results expand our knowledge on the structural determinants of ChR2.

show abstract

Section: Discussionmentioning

confidence: 99%

Bioinformatic and Mutational Analysis of Channelrhodopsin-2 Protein Cation-conducting Pathway

Plazzo

Franceschi

Broi

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…They are known to be involved in H ϩ selectivity and H ϩ release. Substitution of Glu-90 by Gln or Asp or His-134 by Arg or Asn inhibited H ϩ conductance and promoted Na ϩ selectivity (27,32,33), suggesting that both residues are responsible for H ϩ transfer and release into the medium or part of the internal selectivity filter. Ritter et al (34) and Radu and co-workers (35,36) monitoring FTIR differences between the dark state and the intermediate P500 reported that ChR2 has two indicative band patterns around 1700 cm Ϫ1 , implying the existence of two protonated carboxyl residues at least.…”

Section: Alignment-throughout This Study the Residue Numbering Ofmentioning

confidence: 99%

“…Sugiyama et al (32) substituted the glutamates of helix B individually by alanine and found strong inhibition of photoreceptor currents for Ala-82, Ala-83, Ala-97, and Ala-101 but not for Glu-90. The same finding has been reported by Ruffert et al (27), proposing a model for helix B that is very similar to our alignment A, because they propose Glu-90 to be located in the middle of helix B.…”

Section: Alignment-throughout This Study the Residue Numbering Ofmentioning

confidence: 99%

Structural Model of Channelrhodopsin

Watanabe

Welke

Schneider

et al. 2012

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background:The channelrhodopsins are widely used for optogenetic application, whereas a structural model was not available before now. Results: Our modeled structure identifies remarkable structural motifs and elucidates important electrophysiological properties of Channelrhodopsin-2. Conclusion: Channel function relies on the unusual properties of helices 1 and 2. Significance: The atom level structure promotes further understanding of function and may guide the engineering of channelrhodopsins for novel optogenetic applications.

show abstract

“…S4. Glu-60 was also included in this analysis, although its predicted location is close to the cytoplasmic surface, because this position corresponds to Glu-82 of Chlamydomonas reinhardtii channelrhodopsin 2 (CrChR2), in which its replacement with Ala strongly inhibited photocurrents (10). All but one tested GtACR1 mutant exhibited biphasic current rise and decay, as did wild type (WT), although the rates and contributions of the phases to the total amplitude quantitatively varied (Fig.…”

Section: Significancementioning

confidence: 99%

Gating mechanisms of a natural anion channelrhodopsin

Sineshchekov

Govorunova

et al. 2015

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

102

View full text Add to dashboard Cite

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...

show abstract

Photocurrent attenuation by a single polar-to-nonpolar point mutation of channelrhodopsin-2

Cited by 53 publications

References 42 publications

Bioinformatic and Mutational Analysis of Channelrhodopsin-2 Protein Cation-conducting Pathway

Bioinformatic and Mutational Analysis of Channelrhodopsin-2 Protein Cation-conducting Pathway

Structural Model of Channelrhodopsin

Gating mechanisms of a natural anion channelrhodopsin

Contact Info

Product

Resources

About