Molecular mechanism and functionalizationof optogenetic toolfrom the protein perspective
2020/12/10
Literature Seminar
B4 Habazaki

1

Abstract

What is Optogenetics?
Optogenetics is the techniquethat allows precise and fast

control of the activity oftargeted brain cells!
https://mcgovern.mit.edu/2020/04/29/optogenetics-with-soul/

One protein in Rhodopsin family

Channelrhodopsin-2 (ChR2)= light-gated cation channel
Key protein of optogenetics

Heberle et al. J. Am. Chem. Soc. 2009, 131 (21), 7313-7319.

2

Abstract

A new ChR2 achieved minimally invasive optogenetic stimulation
Brain damage…

Buchen, Nature 2010, 465, 26-28.

Non-invasive!

https://www.clea-japan.com/products/animal/inbred

A significant disadvantage of optogenetics is permanent damage to the brain…

Now it is possible to activate any mouse brain region, non-invasively
using ultra light-sensitive Channelrhodopsin mutant, SOUL!
Feng et al. Neuron 2020, 107 (1), 38-51.

3

Contents◆Introduction• Artificial control of neuronal activity• New technique to control neuronal activity using light; Optogenetics◆Photoactivation mechanism of ChR2
• Photoreceptor protein; Rhodopsin• Structure and photocycle of ChR2
• Methods used to investigate the molecular mechanisms of ChR2
• Light absorption and photoisomerization of retinal• Rearrangement of hydrogen-bond network and channel opening◆Application of optogenetics◆Development of less invasive optogenetics tools• Highly light-sensitive ChR2 mutant ①; SFO family• Highly light-sensitive ChR2 mutant ②; TC mutant• New ultra light-sensitive ChR2; SOUL
• Less invasive optogenetic stimulation using SOUL

◆Summary & Perspective
4

Contents◆Introduction• Artificial control of neuronal activity• New technique to control neuronal activity using light; Optogenetics◆Photoactivation mechanism of ChR2
• Photoreceptor protein; Rhodopsin• Structure and photocycle of ChR2
• Methods used to investigate the molecular mechanisms of ChR2
• Light absorption and photoisomerization of retinal• Rearrangement of hydrogen-bond network and channel opening◆Application of optogenetics◆Development of less invasive optogenetics tools• Highly light-sensitive ChR2 mutant ①; SFO family• Highly light-sensitive ChR2 mutant ②; TC mutant• New ultra light-sensitive ChR2; SOUL
• Less invasive optogenetic stimulation using SOUL

◆Summary & Perspective
5

Introduction

Artificial control of neuronal activity
Question of neuroscientists :How does the activity of certain neurons in living animals relate to the function of the brain?
• Observing the relationship between neural activity and brain function• Manipulating neural activity and examining what happens in the brain

Electrical stimulation• The previous major method of artificial induction ofneuronal activity

Fast control of neuronal activity
Permanent damage to the brain
Unspecific excitation of a wide spread neuronal networkhttps://kids.frontiersin.org/article/10.3389/frym.2017.00051

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Introduction

Artificial control of neuronal activity
Question of neuroscientists :How does the activity of certain neurons in living animals relate to the function of the brain?
• Observing the relationship between neural activity and brain function• Manipulating neural activity and examining what happens in the brain

Caged reagent• Neurotransmitters whose activity is controlled by lightLess invasive than electrical stimulation
Excitation of specific location
Unspecific excitation of a wide spread neuronal network
Kramer et al. Nat Neurosci 2013, 16, 816-823.

7

Introduction

New technique to control neuronal activity using light; Optogenetics
One protein in Rhodopsin family

Channelrhodopsin-2 (ChR2)= light-gated cation channel
Key protein of optogenetics

Heberle et al. J. Am. Chem. Soc. 2009, 131 (21), 7313-7319.

https://sites.google.com/site/gs2013b4/benefits

Needs : Cell type-specific neural activity manipulation technique was desired.

Discovery : ChR2 was discovered as a light-sensitive protein which has channel function.
Optogenetics
Idea : Expressing ChR2 in neurons induces light-induced depolarization
Deisseroth et al. Nat Neurosci 2005, 8, 1263-1268.

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Introduction

New technique to control neuronal activity using light; Optogenetics• Fast control(millisecond-scale)!

• Excitation of specificlocation!
• Excitation of specificcell type!
• Reversible control!

Buchen, Nature 2010, 465, 26-28.

9

Contents◆Introduction• Artificial control of neuronal activity• New technique to control neuronal activity using light; Optogenetics◆Photoactivation mechanism of ChR2
• Photoreceptor protein; Rhodopsin• Structure and photocycle of ChR2
• Methods used to investigate the molecular mechanisms of ChR2
• Light absorption and photoisomerization of retinal• Rearrangement of hydrogen-bond network and channel opening◆Application of optogenetics◆Development of less invasive optogenetics tools• Highly light-sensitive ChR2 mutant ①; SFO family• Highly light-sensitive ChR2 mutant ②; TC mutant• New ultra light-sensitive ChR2; SOUL
• Less invasive optogenetic stimulation using SOUL

◆Summary & Perspective
10

Photoactivation mechanism of ChR2

Structure of ChR2
Photoreceptor protein; Rhodopsin• Apoprotein; Opsin• Chromophore; retinal

Opsin protein• Seven transmembrane α-helices (TM)Retinal Schiff Base
(RSBH+)
Retinal chromophore• Forming a Schiff base linkage with alysine residue in the middle of TM7• Isomerization from all-trans-retinal to
13-cis-retinal by the photoactivation

Ernst et al. Chem. Rev. 2014, 114 (1), 126-163.

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Photoactivation mechanism of ChR2

Structure of ChR2
cavityChR2 dimer

IC2

IC1

EC2

EC1

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

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Photoactivation mechanism of ChR2

Methods used to investigate the molecular mechanisms of ChR2
Analysis of molecular mechanism

Structural Analysis

Spectroscopy

X-ray crystallography
Cryo-EM, XFEL
Computer simulation

Time-resolved spectroscopy
Raman spectroscopy
EPR, NMRElectrophysiological Studies
Patch-Clamp Recordings

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Photoactivation mechanism of ChR2

Methods used to investigate the molecular mechanisms of ChR2
Time-resolved spectroscopy

UV/Vis spectroscopy• Isomeric state of retinal• Environment of retinal Schiff base(e.g. Protonation state (RSB or RSBH+))
Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

FTIR spectroscopy• Changes in interactions(e.g. Hydrogen bonding)• Large conformational changes in theprotein backbone

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Photoactivation mechanism of ChR2

Photocycle of ChR2
Important points in the opticalresponse of ChR2
① Photoisomerization of retinal= Trigger of photoactivation② Change in the protonation state of RSB= Important switch of channel gating

Retinal Schiff Base ③ Changes in the H-bonding networks(RSB, RSBH+)= Key factors of conformational change☆Channel gating does not rely on thesteric effect of retinal isomerization!

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

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Photoactivation mechanism of ChR2

Photocycle of ChR2
Channel closing phase

Channel opening phase③Conformational change①Retinal isomerization

all-trans→13-cis②RSBH+ deprotonation②D156 reprotonation

RSBH+→D256①Retinal reisomerization③Conformational change

Channel closing

Channel opening②RSB reprotonation

Conducting state

D156→RSB
Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

Heberle et al. J. Am. Chem. Soc. 2009, 131 (21), 7313-7319.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

Ernst et al. Chem. Rev. 2014, 114 (1), 126-163.

16

Photoactivation mechanism of ChR2

Light absorption and photoisomerization of retinal
Channel opening phase①Retinal isomerization

all-trans→13-cis②RSBH+ deprotonation

RSBH+→D256
Conducting state③Conformational change

Channel opening

Conducting state• The initial photochemical reaction in rhodopsins is known to be one of thefastest and most efficient chemical events in biology!
Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

Ernst et al. Chem. Rev. 2014, 114 (1), 126-163.

17

Photoactivation mechanism of ChR2

Light absorption and photoisomerization of retinal
Observation using X-ray free-electron lasers (XFELs) of Bacteriorhodopsin
The ultrafast structural changes to theretinal was probed!
Asp212

Water402
Asp82

Ultrafast charge transfer along retinal is adriving force for ultrafast response of thecounterion cluster.
→Possibly contributing to thestereoselectivity and efficiency.

ultrafast charge transfer

Standfuss et al. Science 2018, 361, 6398.

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Photoactivation mechanism of ChR2

Light absorption and photoisomerization of retinal

Standfuss et al. Science 2018, 361, 6398.

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Photoactivation mechanism of ChR2

Rearrangement of hydrogen-bond network and channel opening
Channel opening phase
Opening of Central gate
& Extracellular gate
①Retinal isomerization

all-trans→13-cis②RSBH+ deprotonation

RSBH+→D256
Conducting state③Conformational change

Channel opening

Conducting state
Ultrafast isomerization of retinal can not directly cause major structural changes.
→The high-energy H-bonding network causes structural changes with energetic relaxation!
Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366. 20

Photoactivation mechanism of ChR2

Rearrangement of hydrogen-bond network and channel opening
Proton transfer from RSBH+ to D253 and rearrangement of D253
Spectroscopic data :• D253 serves as the proton acceptor
of RSBH+.
• The strong interaction of the
protonated D253 with the E123
carboxylate is observed afterisomerization of retinal.

Salt bridge between
RSBH+ and E123/D253

Isomerization causes the deprotonation of
RSBH+ and the rearrangement of D253.

Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

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Photoactivation mechanism of ChR2

Rearrangement of hydrogen-bond network and channel opening
Rearrangement of H-bonding network and opening of two gates

H-bonding network
containing D253

Opening of Central and Extracellular gate
Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

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Photoactivation mechanism of ChR2

Rearrangement of hydrogen-bond network and channel opening
Channel opening phase
Opening of
Intracellular gate
①Retinal isomerization

all-trans→13-cis②RSBH+ deprotonation

RSBH+→D256
Conducting state③Conformational change

Channel opening

Conducting state
Ultrafast isomerization of retinal can not directly cause major structural changes.
→The high-energy H-bonding network causes structural changes with energetic relaxation!
Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366. 23

Photoactivation mechanism of ChR2

Rearrangement of hydrogen-bond network and channel opening
Rearrangement of H-bonding network and opening of the remaining gate
Structural & Spectroscopic data :• The global reorientation of TM 2 occurs during channel opening.

The core residue of
Intracellular gate

TM2
Residues of TM2

Residues of TM2
contained in the H-bonding
network including D253

Hegemann, Gerwert et al. Angew. Chem. Int. Ed. 2015, 54, 4953-4957.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

Bartl et al. J. Biol. Chem. 2008, 283, 35033-35041. 24

Photoactivation mechanism of ChR2

Rearrangement of hydrogen-bond network and channel opening
Rearrangement of H-bonding network and opening of the remaining gate

Opening of Intracellular gate

TM2

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

Residues of TM2
contained in the H-bonding
network including D253
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Photoactivation mechanism of ChR2

Short Summary
ClosedOpenedhνPhotoisomerization of retinal

Deprotonation of RSB
Changes in the H-bonding networks

Changes in the protein conformation
Opening of three gates
Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

26

Contents◆Introduction• Artificial control of neuronal activity• New technique to control neuronal activity using light; Optogenetics◆Photoactivation mechanism of ChR2
• Photoreceptor protein; Rhodopsin• Structure and photocycle of ChR2
• Methods used to investigate the molecular mechanisms of ChR2
• Light absorption and photoisomerization of retinal• Rearrangement of hydrogen-bond network and channel opening◆Application of optogenetics◆Development of less invasive optogenetics tools• Highly light-sensitive ChR2 mutant ①; SFO family• Highly light-sensitive ChR2 mutant ②; TC mutant• New ultra light-sensitive ChR2; SOUL
• Less invasive optogenetic stimulation using SOUL

◆Summary & Perspective
27

Application of optogenetics

Application of optogenetics
Regulation of parkinsonian motor behaviours by optogenetic control
D1→direct-pathway
D2→indirect-pathway• Direct-pathway activation completely
restored motor behaviour to pre-lesion

levels in in Parkinson’s disease-model
mice!
Kreitzer et al. Nature 2010, 466, 622-626.

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Contents◆Introduction• Artificial control of neuronal activity• New technique to control neuronal activity using light; Optogenetics◆Photoactivation mechanism of ChR2
• Photoreceptor protein; Rhodopsin• Structure and photocycle of ChR2
• Methods used to investigate the molecular mechanisms of ChR2
• Light absorption and photoisomerization of retinal• Rearrangement of hydrogen-bond network and channel opening◆Application of optogenetics◆Development of less invasive optogenetics tools• Highly light-sensitive ChR2 mutant ①; SFO family• Highly light-sensitive ChR2 mutant ②; TC mutant• New ultra light-sensitive ChR2; SOUL
• Less invasive optogenetic stimulation using SOUL

◆Summary & Perspective
29

Development of less invasive optogenetic tools

Development of less invasive optogenetic tools
Brain damage…

Optogenetics•

Permanent damage to the brain…

Development of less invasive optogenetic tools

is a very important challenge!
Buchen, Nature 2010, 465, 26-28.

One of the strategies of less invasive optogenetic tools :Engineering high light-sensitive ChR2 mutants!
… ChR2 mutants that can drive a certain amount of photocurrent even by very
weak light (only a little photon).
Blue light(photon)
ChR2
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Development of less invasive optogenetic tools

Highly light-sensitive ChR2 mutant ①; SFO family

IC2

Step-Function Opsin (SFO) family• Mutation : C128X or D156A(X = S, T, A)

IC1• Phenotype :Significantly stable open channel state→Accumulation of the conducting state

EC2

DC gateEC1Deisseroth, Hegemann et al. Nat Neurosci 2009, 12, 229-234.
Bamann et al. Biochemistry 2010, 49 (2), 267-278.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366.

31

Development of less invasive optogenetic tools

Highly light-sensitive ChR2 mutant ①; SFO family
Wild-type ChR2

Step-Function Opsin (SFO) family• Mutation : C128X or D156A• Phenotype :Significantly stable open channel state

SFO (C128S)→Accumulation of the conducting state
Deisseroth, Hegemann et al. Nat Neurosci 2009, 12, 229-234.

Stable Step-Function Opsin (SSFO)

SSFO
SFO

• Mutation : C128X and D156A• Phenotype :More stable open channel state!
Deisseroth, Hegemann, Yizhar et al. Nature 2011, 477, 171-178.
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Development of less invasive optogenetic tools

Highly light-sensitive ChR2 mutant ①; SFO family
SFO/SSFO act as photon integrators across time!

WT

Deisseroth, Hegemann, Yizhar et al. Nature 2011, 477, 171-178.

SFO/SSFO family

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Development of less invasive optogenetics tools

Highly light-sensitive ChR2 mutant ①; SFO family
Channel closing phase
Reprotonation and③Conformational change

channel closing②D156 reprotonation

DC gate①Retinal reisomerization

Channel closing

Conducting state②RSB reprotonation

D156→RSB

The mutations of SFO family slow down the steps related to channel closing.
→Accumulation of conducting states!
Heberle et al. PNAS 2013, 110 (14), E1273–E1281.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366. 34

Development of less invasive optogenetics tools

Highly light-sensitive ChR2 mutant ①; SFO family

From RSB to D156

Spectroscopic data :• D156 and C128 forms strong H-bondin the DC gate.
• D156 serves as the proton donor of
RSB.

① In the transition from 𝐏𝟐 𝟑𝟗𝟎 (RSB)
In the conducting states, theH-bonding network from RSB to D156
is formed through T127 & C128.
Heberle et al. PNAS 2013, 110 (14), E1273–E1281.

to 𝐏𝟑 𝟓𝟐𝟎 (RSBH+), the proton istransferred from D156 to RSB.
Heberle et al. Photochem. Photobiol. Sci. 2010, 9, 194-198.
Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366. 35

Development of less invasive optogenetics tools

Highly light-sensitive ChR2 mutant ①; SFO family

From RSB to D156

Spectroscopic data :• D156 is reprotonated when thechannel is closed.

Structural &Electrophysiological data :• A proton is taken up from inside thecell.

② In 𝐏𝟑 𝟓𝟐𝟎 , RSBH+ points toward thecytoplasmic side due to stabilizationby the deprotonated D156.
→Reprotonation of D156 is the triggerof channel closing!

Bamberg et al. PNAS 2009 , 106 (30), 12317-12322.
Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366. 36

Development of less invasive optogenetics tools

Highly light-sensitive ChR2 mutant ①; SFO family
Mechanism of D156A mutation

From RSB to D156
D156A mutation• Missing of proton donor→Accumulation of conducting 𝐏𝟐 𝟑𝟗𝟎

Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366. 37

Development of less invasive optogenetics tools

Highly light-sensitive ChR2 mutant ①; SFO family
Mechanism of C128X mutation

C128

From RSB to D156

T128

C128X mutation (X = S, T, A)• The mutation disturbs the structureof the retinal pocket.
→The deprotonated D156 is stabilized

in a different interaction network.
→Accumulation of conducting 𝐏𝟑 𝟓𝟐𝟎
Heberle et al. PNAS 2013, 110 (14), E1273-E1281.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366. 38

Development of less invasive optogenetic tools

Highly light-sensitive ChR2 mutant ②; TC mutant

IC2

TC mutant• Mutation : T159C

IC1• Phenotype :T159

Dramatic increase of photocurrent
amplitude

EC2 ring
Close to β-iononeof retinal

EC1

Oertner et al. PNAS 2011, 108 (18), 7595-7600.

Gordeliy, Bamberg, Büldt et al. Science 2017, 358, 6366. 39

Development of less invasive optogenetic tools

Highly light-sensitive ChR2 mutant ②; TC mutant
TC mutant• Mutation : T159C• Phenotype :Dramatic increase of photocurrent
amplitude
Oertner et al. PNAS 2011, 108 (18), 7595-7600.

WT

TC mutant40

Development of less invasive optogenetic tools

New ultra light-sensitive ChR2; SOUL
Idea
SOUL

SFO family(C128X, D156A)

TC mutant(T159C)

Highly light sensitive

Highly light sensitive(C128S, D156A,
T159C)

Ultra light sensitive!

Brain damage…
Non-invasive!

https://blogs.lt.vt.edu/stems/2014/05/01/optogenetics/

https://www.clea-japan.com/products/animal/inbred

Highlight :Achieving non-invasive activation of any region of the mouse brain!
Feng et al. Neuron 2020, 107 (1), 38-51.

41

Development of less invasive optogenetics tools

New ultra light-sensitive ChR2; SOUL
Idea
SSFO family(C128X+D156A)

TC mutant(T159C)

Highly light sensitive

Highly light sensitive

SFO/SSFO family

SOUL(C128S+D156A+T159C)

Ultra light sensitive!

SOUL

42

Development of less invasive optogenetic tools

New ultra light-sensitive ChR2; SOUL
In vitro and ex vivo characterization of SOULSSFO (C128S + D156A)

SOUL (C128S + D156A + T159C)• SOUL retains a prolonged open state like SSFO.
• SOUL-expressing neurons had a significantly higher operational light sensitivity

compared to SSFO.
Feng et al. Neuron 2020, 107 (1), 38-51.

43

Development of less invasive optogenetic tools

New ultra light-sensitive ChR2; SOUL
In vivo characterization of SOUL
Encoding SOUL into the deepest region
in the mouse brain
50 mW blue lightMarker protein
of firing

LH

SOUL can be activated via transcranial optical stimulation in mice
regardless of the depth!
Feng et al. Neuron 2020, 107 (1), 38-51.

44

Development of less invasive optogenetics tools

Less invasive optogenetic stimulation using SOULTranscranial optogenetic control of mouse behavior by SOUL
Previous finding :Photoactivation of excitatory neurons in LH

suppresses feeding in food-deprived mice.

LH
The result of this report :Food-deprived SOUL-expressing mice showedsignificantly reduced food consumption via
transcranial blue light.

SOUL can manipulate neuronal activity and behavior
through non-invasive transcranial delivery of lighteven in the deepest mouse brain region!
Feng et al. Neuron 2020, 107 (1), 38-51.

45

Development of less invasive optogenetics tools

Less invasive optogenetic stimulation using SOULTransdural optogenetic activation of macaque cortex by SOULTranscranial

Any depth

Transdural

Deep corticalneurons*BS = baseline
PB = post blue light

External optical stimulation of SOUL through native dura achieved minimally invasive
activation of neurons across all depths in superficial cortical regions!
→ Large scale optogenetic activation in primates!
Feng et al. Neuron 2020, 107 (1), 38-51.

Lawrence & Chang, J. Neurophysiol. 2020, 124 (5), 1312-1314.

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Contents◆Introduction• Artificial control of neuronal activity• New technique to control neuronal activity using light; Optogenetics◆Photoactivation mechanism of ChR2
• Photoreceptor protein; Rhodopsin• Structure and photocycle of ChR2
• Methods used to investigate the molecular mechanisms of ChR2
• Light absorption and photoisomerization of retinal• Rearrangement of hydrogen-bond network and channel opening◆Application of optogenetics◆Development of less invasive optogenetics tools• Highly light-sensitive ChR2 mutant ①; SFO family• Highly light-sensitive ChR2 mutant ②; TC mutant• New ultra light-sensitive ChR2; SOUL
• Less invasive optogenetic stimulation using SOUL

◆Summary & Perspective
47

Summary & Perspective

Summary & Perspective• Optogenetics is the technique that allows precise and fast control of theactivity of targeted brain cells using light-gated cation channel,
channelrhodopsin-2 (ChR2).
• The key factors of channel-gating mechanism is…
①Photoisomerization of retinal②Change in the protonation state of retinal Schiff base (RSB)③Conformational change induced by changes in the H-bonding networks• Non-invasive / minimally invasive optogenetic activation ofmouse / macaque monkey brain was achieved by ultra light-sensitive ChR2
mutant, SOUL.
• SOUL-based optogenetics may be explored for minimally invasive treatmentof neurological and psychiatric disorders.
48