Response of acetylcholine receptors to rapid photochemically produced increases in agonist concentration

Lester, Henry A.; Chang, Hai Won

doi:10.1038/266373a0

Cited by 53 publications

(44 citation statements)

References 13 publications

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“…6. On the other hand, the reasonably high 2P brightness of the cis isomer provides an advantage for 2P control of trans-activated MAGs and related azobenzene PTLs (2,20,25).…”

Section: Discussionmentioning

confidence: 99%

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Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

Carroll

Berlin

Levitz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

101

116

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Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, , to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0 460 . 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca 2+ -imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca 2+ imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.optogenetics | pharmacology | multiphoton | photoswitch | azobenzene M odern neurobiology relies heavily on optical microscopy to observe, and, increasingly, to manipulate (1, 2), biological processes in live tissue. Among these methods, 2-photon-excited fluorescence microscopy (2PM) with near-infrared (NIR) light has emerged as an important technique for extending optical microscopy to highly scattering tissue (3, 4). Remarkably, barely 25 years after the first 2P-excited fluorescence image was published (5), 2PM is now performed in awake, behaving animals (4, 6-8). Naturally, optical manipulations in the brain can benefit from the many advantages of 2PM (9). In particular, the inherent spatial confinement of 2P absorption is critical for optical manipulation of individual cells (10) in the intact mammalian brain, where it is difficult to control the spatial extent of gene expression or confine soluble reagents. However, 2P-excited optogenetics is not yet as widespread in adoption as 2PM, being widely perceived to require sophisticated optical techniques (11-13) or reagent concentrations that compromise pharmacological specificity (2, 14).The rapid time to adoption of 2PM owes at least some credit to the availability of spectroscopic data on the 2P-excited efficacy, or brightness, of synthetic and genetically encoded fluorophores (15-17). Brightness, defined for fluorophores as the product of absorption cross-section and fluorescence quantum yield, gives the experimenter an objective metric to assess fluorescent reporters and identify appropriate optical parameters, such as the optimal excitation wavelength and range of light intensities (18). By ...

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“…6. On the other hand, the reasonably high 2P brightness of the cis isomer provides an advantage for 2P control of trans-activated MAGs and related azobenzene PTLs (2,20,25).…”

Section: Discussionmentioning

confidence: 99%

“…PTLs comprise a modular class of genetically-targeted photochemical tools first conceived in the 1970s by Eranger and Lester for acetylcholine receptors (19,20). PTLs have since been developed for a variety of other receptors and channels (2).…”

mentioning

confidence: 99%

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

Carroll

Berlin

Levitz

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

101

116

View full text Add to dashboard Cite

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“…At the low agonist concentrations used, the current is expected to increase with a time constant equal to the average channel open time. This phenomenon was first noted by Lester & Chang (1977) in electroplaque and was not extensively studied here.…”

Section: Methodsmentioning

confidence: 66%

“…The first relaxation after a flash seemed similar to the relaxation found in flash experiments with Electrophorus. This phenomenon was interpreted by Lester & Chang (1977) to represent the equilibration of agonist with its receptor, the time constant of relaxation equalling the reciprocal of the sum of the closing and opening rates, 1/(ac+6).…”

Section: Discussionmentioning

confidence: 99%

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Activation and desensitization of acetylcholine receptors in fish muscle with a photoisomerizable agonist.

Weinstock¹

1983

The Journal of Physiology

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SUMMARY1. Voltage-clamped muscle fibres of the fish XenoMy8tus nigri8 were bathed in a solution containing 3,3-bi8[a-(trimethylammonium)methyl]azobenzene (ci8-Bis-Q; 100 nM). A flash of light photoisomerized some of the ci8-Bis-Q (non-agonist form) to tran8-Bis-Q (agonist form). This resulted in agonist-induced current.2. Current noise of channels activated by both trans-Bis-Q and ACh was analysed to find the channel conductances and open times associated with these two agonists. Channels activated by tran8-Bis-Q and ACh had similar conductances (20-30 pS) and open times (3-4 msec) despite the photolabile azobenzene group of Bis-Q.3. Light flashes subsequent to the first flash caused further increases in the ratio of tran8-Bis-Q to ci8-Bis-Q and accompanying increases in agonist-induced current. Eventually, agonist-induced currents levelled off as a photo-equilibrium state was reached with a constant tran8 cis ratio.4. After the photo-equilibrium current level was reached, light flashes caused temporary increases in agonist-induced current which decayed back to equilibrium in seconds. This result is interpreted according to a model in which trans-Bis-Q molecules are bound to a subpopulation of desensitized receptors, preventing recovery to native receptor. A flash of light then converts some tran8-Bis-Q molecules bound to the desensitized receptor to the cis isomer. The newly formed cis-Bis-Q molecule may then unbind, allowing the desensitized receptor to recover. 5. When light was flashed on muscle exposed to 300-600 nM-cis-Bis-Q, large (100-200 nA) agonist-induced currents were produced. These currents decayed exponentially over several seconds as the fibre desensitized. This result confirms that a first-order process underlies the onset of desensitization.

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Switchable Proteins and Channels

Volgraf¹,

Banghart²,

Trauner³

2011

Molecular Switches

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Response of acetylcholine receptors to rapid photochemically produced increases in agonist concentration

Cited by 53 publications

References 13 publications

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics

Activation and desensitization of acetylcholine receptors in fish muscle with a photoisomerizable agonist.

Switchable Proteins and Channels

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