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.

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

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)

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

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

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).

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 ).

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

Photoactivate of nAChR α3β4E61C receptor α2β4E61C receptor recepter With MAACh in Xenopus oocyte 6.8+1.3% (mean+s.e.m.) n=10) 16.1+2.1% (mean+s.e.m., n = 15) the saturating cholinergic current

Mutant property α3β4E61C receptor α2β4E61C receptor in Xenopus oocyte WT Mutant(E61C) Mutant(E61C) With MAACh (in the dark)

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

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)

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

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

There are other Cysteine Discussion 1970-80s 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).

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.

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):6517-6524

Neurotransmitter recepter

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.

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.