A Chimera Na+-Pump Rhodopsin as an Effective Optogenetic Silencer

Hoque, Mohammad Razuanul; Ishizuka, Toru; Inoue, Kazuhide; Abe-Yoshizumi, Rei; Ishii, Hiroyuki; Mishima, Takaaki; Kandori, Hideki; Yawo, Hiromu

doi:10.1371/journal.pone.0166820

Cited by 31 publications

(41 citation statements)

References 50 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Based on this initial screen of membrane targeting and photocurrent amplitude, we decided to further investigate the pumping characteristics of FdNaR, NyNaR and SrNaR. It has been reported that KR2 constructs carrying an ER export signal and eYFP exhibited larger photocurrents in cortical neurons and cultured cells [ 17 , 25 ]. We therefore replaced the C-terminally fused GFP coding sequence in our rhodopsin expression constructs with an ER export signal and eYFP (namely KR2-3.0, FdNaR-3.0 and NyNaR-3.0 (Fig A in S3 Fig ).…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Functional characterization of sodium-pumping rhodopsins with different pumping properties

et al. 2017

Self Cite

View full text Add to dashboard Cite

Sodium pumping rhodopsins (NaRs) are a unique member of the microbial-type I rhodopsin family which actively transport Na+ and H+ depending on ionic condition. In this study, we surveyed 12 different NaRs from various sources of eubacteria for their electrophysiological as well as spectroscopic properties. In mammalian cells several of these NaRs exhibited a Na+ based pump photocurrent and four interesting candidates were chosen for further characterization. Voltage dependent photocurrent amplitudes revealed a membrane potential-sensitive turnover rate, indicating the presence of an electrically-charged intermediate(s) in the photocycle reaction. The NaR from Salinarimonas rosea DSM21201 exhibited a red-shifted absorption spectrum, and slower kinetics compared to the first described sodium pump, KR2. Although the ratio of Na+ to H+ ion transport varied among the NaRs we tested, the NaRs from Flagellimonas sp_DIK and Nonlabens sp_YIK_SED-11 showed significantly higher Na+ selectivity when compared to KR2.All four further investigated NaRs showed a functional expression in dissociated hippocampal neuron culture and hyperpolarizing activity upon light-stimulation. Additionally, all four NaRs allowed optical inhibition of electrically-evoked neuronal spiking. Although efficiency of silencing was 3–5 times lower than silencing with the enhanced version of the proton pump AR3 from Halorubrum sodomense, our data outlines a new approach for hyperpolarization of excitable cells without affecting the intracellular and extracellular proton environment.

show abstract

Section: Resultsmentioning

confidence: 99%

“…Fortunately we were able to identify three candidates (FdNaR, NyNaR and SrNaR) for further investigation. In addition, improvement was made for KR2, when the fluorescence tag and a signaling motif was replaced in the original construct [ 25 ]. But this was not the case with FdNaR and NyNaR ( Fig 1B ).…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of sodium-pumping rhodopsins with different pumping properties

et al. 2017

Self Cite

View full text Add to dashboard Cite

show abstract

“…We also suggest putative structures for the transient photocycle intermediates, which we verify based on calculations of absorption spectra. Understanding the molecular mechanism of light-driven Na + transport by KR2 is essential for the rational design of novel light-driven ion pumps that hyperpolarize the membrane potential and could be used as inhibitory optogenetic tools (6,7,13,14). …”

mentioning

confidence: 99%

“…Furthermore, mechanistic studies of the Na + pumping in KR2 have important neurophysiological applications, as the protein can be used as an inhibitory optogenetic tool, which does not affect the pH balance of the organism (6,7,13,14). Despite several experimental studies (11,(15)(16)(17)(18), as well as the recently resolved X-ray structure (7,8), the exact molecular mechanism by which KR2 pumps Na + remains elusive.…”

mentioning

confidence: 99%

Energetics and dynamics of a light-driven sodium-pumping rhodopsin

Suomivuori

Gámiz‐Hernández

Sundholm

et al. 2017

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

View full text Add to dashboard Cite

The conversion of light energy into ion gradients across biological membranes is one of the most fundamental reactions in primary biological energy transduction. Recently, the structure of the first light-activated Na + pump, Krokinobacter eikastus rhodopsin 2 (KR2), was resolved at atomic resolution [Kato HE, et al. (2015) Nature 521: [48][49][50][51][52][53]. To elucidate its molecular mechanism for Na + pumping, we perform here extensive classical and quantum molecular dynamics (MD) simulations of transient photocycle states. Our simulations show how the dynamics of key residues regulate water and ion access between the bulk and the buried light-triggered retinal site. We identify putative Na + binding sites and show how protonation and conformational changes gate the ion through these sites toward the extracellular side. We further show by correlated ab initio quantum chemical calculations that the obtained putative photocycle intermediates are in close agreement with experimental transient optical spectroscopic data. The combined results of the ion translocation and gating mechanisms in KR2 may provide a basis for the rational design of novel light-driven ion pumps with optogenetic applications.bacterial ion pumps | bioenergetics | QM/MM | optogenetics | retinal P rimary biological energy conversion is based on the efficient capture and conversion of light and chemical energy into ion gradients across biological membranes (1, 2). The established gradients are used to thermodynamically drive energy-requiring processes, such as active transport and synthesis of adenosine triphosphate (ATP) (3, 4). The rhodopsin family of proteins catalyzes such reactions by harnessing the energy from retinal photoisomerization and deprotonation reactions, followed by conformational changes that further trigger the pumping of ions across the membrane (5). All previously known ion-pumping rhodopsins function either as inward chloride or outward proton pumps, but the structure of the first Na + pumping rhodopsin, Krokinobacter eikastus rhodopsin 2 (KR2) (6), was recently resolved (7,8). In the absence of Na + ions, KR2 functions as an outward proton pump, similarly to bacteriorhodopsin (bR), but under physiological conditions, KR2 pumps Na + out of the cell (6, 9).KR2 shares the heptahelical transmembrane (7TM) structure common to all microbial rhodopsins (Fig. 1A) (5,7,8). In contrast to bR, however, where a highly conserved Asp-Thr-Asp (DTD) motif is used to pump protons, KR2 employs a unique NDQ motif comprising residues Asn112, Asp116, and Gln123 (6). In bR, Asp85 and Asp96 function as proton acceptors and donors for the protonated Schiff base (PSB), respectively, whereas in KR2, Asn112 and Gln123 occupy these positions, and Asp116 replaces Thr89. In analogy to bR, it has been suggested that Gln123 aids in capturing ions from the solvent and that Asn112 might be part of a Na + binding site (6,10).Similarly to other microbial rhodopsins, four photocycle intermediates have been spectroscopically identified in KR2 (Fig. 1B) (6)...

show abstract

“…11,[51][52][53] Therefore, understanding the molecular level structure and dynamics of water will help us to better understand the mechanism of Na + pumps, and rhodopsins in general, which eventually could be used for the rational design of novel light-driven ion pumps and development of inhibitory optogenetic tools. 3,4,54,55 Given the above background, in this work we have carried out an extensive atomistic molecular dynamics (MD) simulation to understand the localisation, local structure, hydration and dynamics of water inside KR2 channel in the resting (dark) state. Our study identifies several spatially disjoint specific water binding sites and their nature in terms of available space, local interaction and water dynamics.…”

Section: Introductionmentioning

confidence: 99%

Structural and dynamical heterogeneity of water trapped inside Na⁺-pumping KR2 rhodopsin in the dark state

Santra

Seal

Bhattacharjee

et al. 2020

Preprint

View full text Add to dashboard Cite

Photoisomerisation in retinal leads to a channel opening in the rhodopsins that triggers translocation of an ion/proton. Crystal structures of rhodopsins contain several structurally conserved water molecules. It has been suggested that water plays an active role in facilitating the ion pumping/translocation process by acting as a lubricant in these systems. In this work, we investigate the localisation, local structure and dynamics of water molecules along the channel for the resting/dark state of KR2 rhodopsin. Employing 1.5 μs long atomistic molecular dynamics (MD) simulations of this trans-membrane protein system, we demonstrate the presence of five distinct water containing pockets/cavities separated by gateways controlled by the protein side-chains. We present evidence of significant structural and dynamical heterogeneity in the water molecules present in these cavities. The exchange time-scale of these buried water with bulk ranges from tens of nanoseconds to > 1.5 μs. The translational and rotational dynamics of buried water are found to be strongly dependent on protein cavity size and local interactions with possible functional significance.

show abstract

A Chimera Na+-Pump Rhodopsin as an Effective Optogenetic Silencer

Cited by 31 publications

References 50 publications

Functional characterization of sodium-pumping rhodopsins with different pumping properties

Functional characterization of sodium-pumping rhodopsins with different pumping properties

Energetics and dynamics of a light-driven sodium-pumping rhodopsin

Structural and dynamical heterogeneity of water trapped inside Na⁺-pumping KR2 rhodopsin in the dark state

Contact Info

Product

Resources

About

A Chimera Na+-Pump Rhodopsin as an Effective Optogenetic Silencer

Cited by 31 publications

References 50 publications

Functional characterization of sodium-pumping rhodopsins with different pumping properties

Functional characterization of sodium-pumping rhodopsins with different pumping properties

Energetics and dynamics of a light-driven sodium-pumping rhodopsin

Structural and dynamical heterogeneity of water trapped inside Na+-pumping KR2 rhodopsin in the dark state

Contact Info

Product

Resources

About

Structural and dynamical heterogeneity of water trapped inside Na⁺-pumping KR2 rhodopsin in the dark state