1. synthetic photoisomerizable azobenzene-regulated K+ channel
Control neuron proteins activity
M. Banghart, K. Borges, E. Isacoff, D. Trauner, R. H. Kramer,Nat. Neurosci. 2004, 7, 1381 – 1386.

2. Nicotinic acetylcholine receptor(nAChR)
Structure
channel is composed of 5 subunit
Binding sites about many kind of Agonist and antagonist is reported
function
control cationic ions flow(Ca+,K+,Na+) by pore`s close and open.
Inactivate state pore close
Active sate pore open

3. Variety of neuronal nAChR subtype
Research Objective
neuronal nAChR
interest for treating various nervous-system disorders
ex, Altjeomer`s disease,ADHD,depression
But physiological role is unrevealed
･lack of subtype selective reagent in pharmacology
･many types in neuronal system
-> approach by opt chemical genetics
･selective control by genetics and photo-irradiation
Variety of neuronal nAChR subtype
In the brain
Taly, A., Corringer, P-J., Guedin, D., Lestage, P. & Changeux, J-PNat. Rev. Drug Discov. 8, 733–750 (2009)

4. Photochromic molecule part
Research Objective
photo activation
opt chemical genetics
Photo switchable Ligand
Interaction part
Photochromic molecule part
photo inhibition
Genetic manipulation
Control
nAChR function

5. Design of Photoswichable tehered ligands
agonist
(make AChR activate)
Antagonist
(inhibit AChR role )

6. Attachment site screening
cis-MAACh
trans-MAACh
α-unit
β-unit
AC-5 azide
Bindig site
Brejc, K. et al..Nature 411,269–276 (2001).

7. Attachment site screening
α-unit
β-unit
Experimental screening result
(α4β4 by two-electrocode voltage-clamp recording)
Xenopus oocyte
E61C, R113C, S117C mutant
control photoactivate property(ON 380nm /OFF dark ).

8. Attachment site screening
Carbamylcholine Chloride site
E61Cmutant is selected by fitting simulation
Brejc, K. et al..Nature 411,269–276 (2001).

9. Photoactivate of nAChR
α3β4E61C receptor
α2β4E61C receptor
recepter With MAACh
in Xenopus oocyte
% (mean+s.e.m.) n=10)
% (mean+s.e.m., n = 15)
the saturating cholinergic current

10. Mutant property α3β4E61C receptor α2β4E61C receptor in Xenopus oocyteWT
Mutant(E61C)
Mutant(E61C)
With MAACh
(in the dark)

11. Photoinibiton of nAChR in Xenopus neuron
α3β4E61C receptor
α2β4E61C receptor
With MAHoCh
in Xenopus oocyte
380 nm ill
500 nm ill

12. Photoinibiton of nAChR in Xenopus neuron
α3β4E61C receptor
α2β4E61C receptor
500 nm ill
380 nm ill
The percentage of current ranged
from 80+5% at 30 mM ACh to 68+4%
at a saturating 10 mM ACh (mean+s.e.m., n =5)
The percentage of current ranged
From 75+10% at 100 mM ACh to 81+7%
at a saturating 3 mM ACh(mean+s.e.m., n=5)

13. Thermal relaxation effect
photo activation reagand
(MAACh-labelled a4b2E61C receptor )
In dark
500 nm ill
380 nm ill
t1/2(MAACh in solution) = 28 (s)
The thermal decay of the photoactivatable current
could only be fitted to a biexponential decay curve

14. Thermal relaxation effect
photo inhibition reagent
(MAHoCh-labelled a4b2E61C receptor )
380 nm ill
In dark
500 nm ill

15. There are other Cysteine
Discussion s
Erlanger`s research
This work
muscle nAChRs
Only one target
Homo-pentamer
There are other Cysteine
(Cys-loops)
Subtype selection
Hetero-pentamer
muscle nAChRs
subuunit
Remove by genetics
Azobenzene unit
λ1=330nm
Azobenzene unit
λ1=380nm
similation
In slico
Inactivate scheme
Bartels, E., Wassermann, N. H. & Erlanger, B. F. Proc. Natl Acad. Sci. USA 68, 1820–1823 (1971).
Lester, H. A., Krouse, M. E., Nass, M. M., Wassermann, N. H. &Erlanger, B. F. A J. Gen.Physiol. 75, 207–232 (1980).

16. summery
They provided the optochemical control of nAChRs with photoswitchable tethered agonists and antagonists.
Using structure-based design, heteromeric a3b4 and a4b2 nAChRs that can be activated or inhibited with deep-violet light, but respond normally to acetylcholine in the dark.
they showed second time scale control about common neuronal nACRh receptors.
This study reports the reversible photoactivation and inhibition of
common neuronal a4b2 and a3b4 nAChRs using light following the genetically targeted conjugation of PTLs.

17. Future direction
it is now possible to express heterologously mutant receptors in Xenopus oocytes, mammalian cells, neurons and even transgenic animals
Other research in Neuron cell
In vitro light irradiation
In future physiological investigations, LinAChRs will be expressed in dissociated neurons, intact neuronal tissues and live animals.
Banghart, Trauner, D. & Kramer, R. H Nat. Neurosci. 7,1381–1386 (2004).
Haruyuki Kamiya The Journal of Neuroscience, 9 May 2012,32(19):

18. Neurotransmitter recepter

25. effect of heteromeric nAChR by various PTL
Photoactivation
Photoinhibiton
1:α3β4WT+ MAACh
2: α3β4 + MAACh,
3: α3β4 + MAHoCh
4: α3β4E61C + AcAACh
5: α3β4E61C + MAHoCh
6: α3β4E61C + MAACh
7: α4β2WT + MAACh
8: α4β2 + MAACh
9: α4β2 + MAHoCh
10: α4β2E61C + AcAACh,
11: α4β2E61C + MAHoCh
12: α4β2E61C + MAACh.

28. Table S1. Characterization of cysteine-mutant and photoswitchable ligand-conjugated nicotinic acetylcholine receptors. Acetylcholine EC50 values and Hill coefficients of the dose-response curves for the wild-type, cysteine-mutant and PTL-conjugated cysteine mutant α3β4 and α4β2 nAChRs. All data are mean±SEM, n=5.