Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Grimm, Christiane; Vierock, Johannes; Hegemann, Peter; Wietek, Jonas

doi:10.3791/55497

Cited by 21 publications

(15 citation statements)

References 46 publications

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“…Electrophysiology in HEK-293 cells. HEK-293 cells (ECACC 85120602, Sigma-Aldrich, Munich, Germany) were cultured and electrophysiologic experiments were performed as described elsewhere 22,75 . In detail, cells were supplemented with 1 µM all-trans retinal, seeded at a density of 1 × 10 5 /ml on poly-D-lysine-coated coverslips, and transiently transfected using Fugene HD (Promega, Madison, WI).…”

Section: Methodsmentioning

confidence: 99%

MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins

et al. 2019

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Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. Here we describe a metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of optogenetic tools from metagenomic databases and enhance general understanding of ChR function.

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Section: Methodsmentioning

confidence: 99%

MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins

et al. 2019

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“…3a–d ). Recent studies have indicated that the highly red-shifted absorption of Chrimson is partly due to the protonation of the counterion residue Glu165 22 , 27 , which weakens the stabilization effect for the PSB, thus causing red-shifted absorption similar to that of the acidic form of BR (BR 605 ) 28 . Consistent with these studies, the purified Chrimson protein showed a large pH-dependent spectrum shift (Supplementary Figure 6a and b).…”

Section: Resultsmentioning

confidence: 99%

“…HEK cell preparation and whole-cell patch-clamp experiments were performed as previously described 22 , 28 . HEK293 (human embryonic kidney) cells (85120602, Sigma-Aldrich) were cultured in Dulbecco’s Modified Medium (DMEM) with stable glutamine (Biochrom, Berlin, Germany) supplemented with 10% (v/v) fetal bovine serum (FBS Superior; Biochrom, Berlin, Germany), 1 μM all-trans retinal and 100 µg/ml penicillin/streptomycin (Biochrom, Berlin, Germany).…”

Section: Methodsmentioning

confidence: 99%

Crystal structure of the red light-activated channelrhodopsin Chrimson

et al. 2018

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Channelrhodopsins are light-activated ion channels that mediate cation permeation across cell membranes upon light absorption. Red-light-activated channelrhodopsins are of particular interest, because red light penetrates deeper into biological tissues and also enables dual-color experiments in combination with blue-light-activated optogenetic tools. Here we report the crystal structure of the most red-shifted channelrhodopsin from the algae Chlamydomonas noctigama, Chrimson, at 2.6 Å resolution. Chrimson resembles prokaryotic proton pumps in the retinal binding pocket, while sharing similarity with other channelrhodopsins in the ion-conducting pore. Concomitant mutation analysis identified the structural features that are responsible for Chrimson’s red light sensitivity; namely, the protonation of the counterion for the retinal Schiff base, and the polar residue distribution and rigidity of the retinal binding pocket. Based on these mechanistic insights, we engineered ChrimsonSA, a mutant with a maximum activation wavelength red-shifted beyond 605 nm and accelerated closing kinetics.

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“…Electrophysiological recordings were performed on stably expressing ChR2-mVenus fusion construct HEK cell line (31) as previously described in detail (43). Briefly, HEK cells were cultured at 5% CO2 and 37 °C in Dulbecco's minimal essential medium supplemented with 10% fetal For a comparison with the photocycle under the "shortcut condition" the flash frequency was increased (trelax = 5 s, fflash = 0.2 Hz).…”

Section: Preparation Of Hek Cellsmentioning

confidence: 99%

A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

Kühne

Vierock

Tennigkeit

et al. 2018

Preprint

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Although Channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light-adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well-understood from a molecular perspective. Herein, we address these open questions using single turn-over electrophysiology, time-resolved step-scan FTIR and Raman spectroscopy of fully dark adapted ChR2. This yields a unifying parallel photocycle model explaining all data: in dark-adapted ChR2, the protonated Schiff base retinal chromophore (RSBH + ) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H + and Na + conductance in a late M-like state and an N-like open-channel state. In contrast, the 13cis,C=N-syn isomer represents a second closed-channel state identical to the long lived P480-state, which has been previously assigned to a late intermediate in a single photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn-cycle and we show that deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light-adaptation. Parallel anti-and syn-photocycles explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools. Significance statementUnderstanding the mechanisms of photoactivated biological processes facilitates the development of new molecular tools, engineered for specific optogenetic applications, allowing the control of neuronal activity with light. Here, we use a variety of experimental and theoretical techniques to examine the precise nature of the light-activated ion channel in one of the most important molecular species used in optogenetics, channelrhodopsin-2. Existing models for the photochemical and photophysical pathway after light absorption by the molecule fail to explain many aspects of its observed behavior including the inactivation of the photocurrent under continuous illumination. We resolve this by proposing a new branched photocycle explaining electrical and photochemical channel properties and establishing the structure of intermediates during channel turnover.

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Whole-cell Patch-clamp Recordings for Electrophysiological Determination of Ion Selectivity in Channelrhodopsins

Cited by 21 publications

References 46 publications

MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins

MerMAIDs: a family of metagenomically discovered marine anion-conducting and intensely desensitizing channelrhodopsins

Crystal structure of the red light-activated channelrhodopsin Chrimson

A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

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