Early Formation of the Ion‐Conducting Pore in Channelrhodopsin‐2

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)...

Section: Significancementioning

confidence: 97%

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

Suomivuori

Gámiz‐Hernández

Sundholm

et al. 2017

“…The static nature of the above experiments precluded the determination of the timing of these structural rearrangements. It was recently concluded from a combination of homology modeling and molecular dynamics (MD) simulations that helix B tilts outwardly by 3.9 Å and a water-filled pore between helices A-C and G is formed within less than 100 ns after isomerization of the retinal from the all-trans to 13-cis conformation (26). Thus, helical tilt changes and water influx might precede the onset of ion permeation by more than three orders of time (<100 ns vs. ∼200 μs).…”

Section: Significancementioning

confidence: 99%

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

Lórenz-Fonfrı́a

Bamann

Resler

et al. 2015

106

The discovery of channelrhodopsins introduced a new class of lightgated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time-resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(−) and 1,648(+) cm. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance.channelrhodopsin | optogenetics | channel gating | infrared spectroscopy | time-resolved spectroscopy I on channels are membrane proteins that mediate the passive movement of cations and anions across biological membranes, a process not only central for most living organisms but key to electrical excitability of cells. Upon activation, ion channels constitute a transient pathway to facilitate the permeation of ions across the cellular membrane. Ion channels can be switched, or gated, from a closed (nonconductive) to an open (conductive) state by external stimuli, such as ligands, voltage, or mechanical stress (1-3). Available structural information indicates that the ion-conducting pathway generally comprises a pore with wide regions solvated by water and constrictions sites formed by specific polar groups that confer ion selectivity (4). In the nonconductive state, the permeation of ions is prevented by energy barriers along the pore, either from physical occlusion or from the hydrophobic nature of residues lining the pore: the pore does not need to be physically occluded to be functionally closed (5). Habitual suspects for the gating mechanism, i.e., how the protein transits from the nonconductive to the conductive state, are diverse types of structural changes, such as orientation/rotation changes of transmembrane helices or of bulky (aromatic) side chains (6). Several computational studies have suggested a more subtle gating mechanism, where the thermodynamics and kinetics of the hydration of the ion-conducting pore might play a vital role (5,7,8). However, experimental support for this last proposal relies largely on studies on synthetic nanometric pores (9).Channelrhodopsins (ChRs) belong to the new class of lightgated ion channels, i.e., ion channels activated by light...

“…A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25).…”

mentioning

confidence: 99%

mentioning

confidence: 99%

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Becker‐Baldus

Bamann

Saxena

et al. 2015

217

Channelrhodopsin-2 from Chlamydomonas reinhardtii is a lightgated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15 N-labeled channelrhodopsin-2 carrying 14,15-13 C 2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P 500 1 K-like state, the slowly decaying late intermediate P 480 4 , and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.ince their discovery (1), channelrhodopsins (ChRs) have generated enormous interest because of the rapid development of their applications in optogenetics (2-7). Commonly, ChR2 from Chlamydomonas reinhardtii (8) and its variants are used thanks to their favorable expression levels. They are the only proteins known today functioning as light-gated ion channels (Fig. 1A). Like other microbial retinal proteins, they undergo a periodic photocycle. In ChRs, this photocycle is coupled to channel opening and closing as revealed in electrophysiological recordings (8). A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25). In the case of bacteriorhodopsin, for example, this state contains a mixture of two species terme...