An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli

Doi, Satoko; Tsukamoto, Takashi; Yoshizawa, Susumu; Sudo, Yuki

doi:10.1038/srep41879

Cited by 17 publications

(32 citation statements)

References 27 publications

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“…By taking advantage of the expression system, we performed mutational analysis on the conserved “basic” amino acid residues among ACRs, and found that the positive charge of R84 located at the extracellular side plays an inhibitory role in its anion transport activity ( Fig. 1C ) [ 25 ], although the precise position and the interaction remain unknown. The prediction of protein structure by molecular simulation and informatics is still developing.…”

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confidence: 99%

Mutational analysis of the conserved carboxylates of anion channelrhodopsin-2 (ACR2) expressed in <i>Escherichia coli</i> and their roles in anion transport

et al. 2018

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Anion channelrhodopsin-2 (ACR2), a light-gated channel recently identified from the cryptophyte alga Guillardia theta, exhibits anion channel activity with exclusive selectivity. In addition to its novel function, ACR2 has become a focus of interest as a powerful tool for optogenetics. Here we combined experimental and computational approaches to investigate the roles of conserved carboxylates on the anion transport activity of ACR2 in Escherichia coli membrane. First, we replaced six conserved carboxylates with a neutral residue (i.e. E9Q, E56Q, E64Q, E159Q, E219Q and D230N), and measured anion transport activity using E. coli expression system. E159Q and D230N exhibited significantly lower anion transport activity compared with wild-type ACR2 (1/12~1/3.4), which suggests that E159 and D230 play important roles in the anion transport. Second, to explain its molecular aspects, we constructed a homology model of ACR2 based on the crystal structure of a cation channelrhodopsin (ChR). The model structure showed a cavity formed by four transmembrane helices (TM1, TM2, TM3 and TM7) similar to ChRs, as a putative anion conducting pathway. Although E159 is not located in the putative pathway, the model structure showed hydrogen bonds between E159 and R129 with a water molecule. D230 is located in the pathway near the protonated Schiff base (PSB) of the chromophore retinal, which suggests that there is an interaction between D230 and the PSB. Thus, we demonstrated the functional importance and the hypothetical roles of two conserved carboxylates, E159 and D230, in the anion transport activity of ACR2 in E. coli membrane.

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confidence: 99%

Mutational analysis of the conserved carboxylates of anion channelrhodopsin-2 (ACR2) expressed in <i>Escherichia coli</i> and their roles in anion transport

et al. 2018

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“…Although the secondary Cl − binding site has not been identified yet, we estimate that it is located near or along the Cl − conducting pathway in the protein. Previously, the inhibitory role of the Arg residue on the extracellular surface of Gt ACR2, which is a candidate consisting of the Cl − conducting pathway, for its anion channel function has been reported 8 . One of the authors’ discussion points regarding the inhibition mechanism is that the positively charged Arg84 interacts with the negatively charged Cl − , which prevents the Cl − from being transported through the protein 8 .…”

Section: Discussionmentioning

confidence: 99%

“…Previously, the inhibitory role of the Arg residue on the extracellular surface of Gt ACR2, which is a candidate consisting of the Cl − conducting pathway, for its anion channel function has been reported 8 . One of the authors’ discussion points regarding the inhibition mechanism is that the positively charged Arg84 interacts with the negatively charged Cl − , which prevents the Cl − from being transported through the protein 8 . Together with the fact that Psu ACR1 conserves the corresponding residue as Arg84, we estimate that the inhibitory role of Arg84 is related to the secondary Cl − binding and therefore the Arg84 in Psu ACR1 is one candidate for the secondary Cl − binding site.…”

Section: Discussionmentioning

confidence: 99%

“…ACRs from a marine cryptophyte alga Guillardia theta (abbreviated as Gt ACR1 and 2) are the first natural ACRs reported in 2015 3 . Several in vivo and in vitro investigations have revealed their optogenetic availability 3 , their channel gating mechanism during the photocycle 5 – 7 , the roles of positively charged residues for anion conductance 8 , and their structure and structural changes around the chromophore 9 , 10 . On the other hand, another homologous ACR from a marine cryptophyte alga Proteomonas sulcata (abbreviated as Psu ACR1 or Ps ACR1) has also been investigated regarding its electrophysiological 4 , 11 and spectroscopic properties 12 .…”

Section: Introductionmentioning

confidence: 99%

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Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl− concentrations

Tsukamoto

Kikuchi

Suzuki

et al. 2018

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Natural anion channelrhodopsins (ACRs) have recently received increased attention because of their effectiveness in optogenetic manipulation for neuronal silencing. In this study, we focused on Proteomonas sulcata ACR1 (PsuACR1), which has rapid channel closing kinetics and a rapid recovery to the initial state of its anion channel function that is useful for rapid optogenetic control. To reveal the anion concentration dependency of the channel function, we investigated the photochemical properties of PsuACR1 using spectroscopic techniques. Recombinant PsuACR1 exhibited a Cl− dependent spectral red-shift from 531 nm at 0.1 mM to 535 nm at 1000 mM, suggesting that it binds Cl− in the initial state with a Kd of 5.5 mM. Flash-photolysis experiments revealed that the photocycle was significantly changed at high Cl− concentrations, which led not only to suppression of the accumulation of the M-intermediate involved in the Cl− non-conducting state but also to a drastic change in the equilibrium state of the other photo-intermediates. Because of this, the Cl− conducting state is protracted by one order of magnitude, which implies an impairment of the rapid channel closing of PsuACR1 in the presence of high concentrations of Cl−.

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“…Natural ACRs showed a larger (several nA) photocurrent and a stronger inhibition of the generation of action potential by light than those of ChR-based anion channels (Govorunova et al 2015). We recently demonstrated that the ion transport activity of GtACR2 is strongly (10-fold) enhanced by a mutation of the conserved Arg residue (R84E) located on a loop (the B-C loop) between TM helix 2 and TM helix 3, when it is expressed in Escherichia coli cells as a recombinant protein (Doi et al 2017). In the future, further modification(s) will be applied to natural ACRs to obtain efficient neural silencers.…”

Section: Conversion Of Cation Channelsmentioning

confidence: 99%

Conversion of microbial rhodopsins: insights into functionally essential elements and rational protein engineering

et al. 2017

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Technological progress has enabled the successful application of functional conversion to a variety of biological molecules, such as nucleotides and proteins. Such studies have revealed the functionally essential elements of these engineered molecules, which are difficult to characterize at the level of an individual molecule. The functional conversion of biological molecules has also provided a strategy for their rational and atomistic design. The engineered molecules can be used in studies to improve our understanding of their biological functions and to develop protein-based tools. In this review, we introduce the functional conversion of membrane-embedded photoreceptive retinylidene proteins (also called rhodopsins) and discuss these proteins mainly on the basis of results obtained from our own studies. This information provides insights into the molecular mechanism of light-induced protein functions and their use in optogenetics, a technology which involves the use of light to control biological activities.

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An inhibitory role of Arg-84 in anion channelrhodopsin-2 expressed in Escherichia coli

Cited by 17 publications

References 27 publications

Mutational analysis of the conserved carboxylates of anion channelrhodopsin-2 (ACR2) expressed in <i>Escherichia coli</i> and their roles in anion transport

Mutational analysis of the conserved carboxylates of anion channelrhodopsin-2 (ACR2) expressed in <i>Escherichia coli</i> and their roles in anion transport

Implications for the impairment of the rapid channel closing of Proteomonas sulcata anion channelrhodopsin 1 at high Cl− concentrations

Conversion of microbial rhodopsins: insights into functionally essential elements and rational protein engineering

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