Mechanisms of photoswitch conjugation and lightactivation of an ionotropic glutamate receptorPau Gorostiza*†, Matthew Volgraf‡, Rika Numano*§, Stephanie Szobota*¶, Dirk Trauner‡�**, and Ehud Y. Isacoff*�**

*Department of Molecular and Cell Biology, ‡College of Chemistry, and ¶Biophysics Graduate Program, University of California, Berkeley, CA 94720; and�Lawrence Berkeley National Laboratory, Berkeley, CA 94720

Edited by Charles F. Stevens, The Salk Institute for Biological Studies, La Jolla, CA, and approved May 4, 2007 (received for review February 19, 2007)

The analysis of cell signaling requires the rapid and selectivemanipulation of protein function. We have synthesized photo-switches that covalently modify target proteins and reversiblypresent and withdraw a ligand from its binding site due tophotoisomerization of an azobenzene linker. We describe here theproperties of a glutamate photoswitch that controls an ion channelin cells. Affinity labeling and geometric constraints ensure that thephotoswitch controls only the targeted channel, and enablesspatial patterns of light to favor labeling in one location overanother. Photoswitching to the activating state places a tetheredglutamate at a high (millimolar) effective local concentration nearthe binding site. The fraction of active channels can be set in ananalog manner by altering the photostationary state with differentwavelengths. The bistable photoswitch can be turned on withmillisecond-long pulses at one wavelength, remain on in the darkfor minutes, and turned off with millisecond long pulses at theother wavelength, yielding sustained activation with minimalirradiation. The system provides rapid, reversible remote control ofprotein function that is selective without orthogonal chemistry.

azobenzene � ion channel � photoisomerization � optical switch �remote control

Much progress has been made recently in the real-timenoninvasive detection of protein function (1), but the

development of approaches for remote protein manipulationwithin the complex environment of the cell has lagged behind.A major advance has been the development of photolysablecages for soluble ligands (2). The caged ligand is allowed toslowly diffuse into tissue in its inert form, and a powerful lightflash cleaves a photolabile protecting group, releasing the activeligand rapidly (within microseconds) (3, 4). This provides faston-rates that are complemented with reasonably fast off-rates,which depend on native binding affinity, diffusion, sequestration,and breakdown. However, because native ligands often act onmultiple proteins, this approach has limited selectivity. Intro-ducing foreign receptors that bind nonnative ligands, on theother hand, enables cellular stimulation without the activation ofendogenous proteins (5, 6) and can even be used in a photolys-able form for rapid release (7).

Photoisomerizable moieties have also found use in the remoteand selective control of native protein function. In this case,reversible photochemically induced changes in the shape orelectronic character of functionally important amino acids havebeen used to control the function of proteins in response to light(8–10) or to alter the backbone structure of peptides (11),thereby controlling their interaction with other biological mac-romolecules (12). In an alternative strategy, photoisomerizationof a tethered ligand can be used to reversibly present, andwithdraw, a ligand from a binding site. To date, several ionchannel photoswitches have been reported wherein a ligand istethered to the channel via a linker containing a photoisomer-izable azobenzene moiety. The ligand in these photoswitches canoperate either as an active site (i.e., pore) blocker (10, 13), or asan allosteric ligand for an ionotropic receptor (14, 15).

To tune the properties of tethered ligand photoswitches,optimize their efficacy, and generalize their application to otherproteins, it is important to understand their basic physical andchemical properties. We set out here to characterize the opticalgating of the ionotropic glutamate receptor subtype 6 (iGluR6)with a photoisomerizable tethered agonist termed MAG(Maleimide–Azobenzene–Glutamate) (MAG-1 and MAG-2,Fig. 1b). We address several key issues that aim to characterizeboth the photochemical and functional properties of the modi-fied channel protein. Investigations into the photochemistry ofthe azobenzene photoswitch, including the half-life of thermalrelaxation and the wavelength dependence of activation anddeactivation, are discussed. We investigate the estimated localconcentration and efficacy of different length photoswitchesunder a variety of labeling conditions in an attempt to optimizethe degree of activation of the light-activated receptor. Lastly,the elucidation of other key properties, including affinity label-ing at concentrations that are approximately three orders ofmagnitude below the Kd, and thus will not activate receptorsappreciably, and selective targeting of MAG to individual cellswithin a culture using patterned illumination, provide a basis forthe development of light-activated ion channels as a powerfultool in neurobiology.

ResultsFurther Details. For further details, see supporting information(SI) Text, SI Appendix, and SI Figs. 8–11.

Modular Photoswitchable Tethered Ligands. The photoswitchabletethered ligand was designed to possess a maleimide for conju-gation to a cysteine residue on the exterior of the ligand-bindingdomain (LBD), a glutamate analog, and an azobenzene linkerenabling reversible state-dependent control over the reach of theglutamate analog (Fig. 1a) (15). The glutamate analog waschosen based on previously established structure–activity rela-tionships of the selective iGluR agonists (2S,4R)-4-allyl-glutamate (LY310214) and (2S,4R)-4-methyl-glutamate (SYM2081) (18–20), and on our iGluR6 agonist, termed the ‘‘tethermodel’’ (3; Fig. 1b) (15). The modularity of the design allows for

Author contributions: P.G., M.V., R.N., D.T., and E.Y.I. designed research; P.G., M.V., R.N.,and S.S. performed research; P.G. and M.V. contributed new reagents/analytic tools; P.G.,M.V., R.N., S.S., and E.Y.I. analyzed data; and P.G., M.V., D.T., and E.Y.I. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: MAG, maleimide–azobenzene–glutamate; LBD, ligand-binding domain;iGluR6, ionotropic glutamate receptor subtype 6.

†Present address: Institucio Catalana de Recerca i Estudis Avancats and Institut de Bioeng-inyeria de Catalunya, Parc Cientıfic de Barcelona, 08028 Barcelona, Spain.

§Present address: Laboratory Animal Research Center, Institute of Medical Science, Univer-sity of Tokyo, Tokyo 108-8639, Japan

**To whom correspondence may be addressed. E-mail: ehud@berkeley.edu ortrauner@berkeley.edu.

This article contains supporting information online at www.pnas.org/cgi/content/full/0701274104/DC1.

© 2007 by The National Academy of Sciences of the USA

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the introduction of additional glycine units in the tether withminimal synthetic investment. Initial studies were based uponmodels of docking MAG-1 in the iGluR6-SYM 2081 crystalstructure (19), though the exact tether length required foroptimal activation remained unknown. After the synthesis ofMAG-1, the elongated MAG-2 was synthesized by using chem-istry analogous to that described in ref. 15. MAGs of differentlengths allow for the study of tether length dependence onchannel activation and agonist binding using readily accessibleand minimally disruptive amino acid building blocks. The successof the MAG design, and the ease with which it can be modified,opens the possibility of replacement of the glutamate moiety forother iGluR agonists or antagonists, or application to othersimilarly functioning allosteric proteins with well defined ligandbinding modes.

Photostationary State Determination by NMR. In the thermallyrelaxed state, azobenzene exists entirely in the trans configura-tion. Upon illumination, a steady state mixture is generated, witha fraction of the azobenzene in the cis configuration and the restin trans. The balance between cis and trans (the photostationarystate) depends on the wavelength of irradiation. The cis popu-lation is maximally populated in the near UV, and trans popu-lation is maximally populated in the visible range of the lightspectrum (21). Usually UV/visible spectroscopy is used todetermine the fraction of azobenzene in the two states (22).Here we used a novel approach of NMR spectroscopy todistinguish between the two isomers (23). NMR was usedto determine the ratio of cis- to trans-MAG-1 conjugated to2-mercaptoethanol between 340 and 500 nm, at 20 nm incre-ments (Fig. 2a). Optimal wavelengths for cis and trans popula-tions were found to be 380 and 500 nm, respectively. At 380 nm,

92.7 � 0.3% of MAG-1 is in the cis state (n � 3, note that valuesthroughout are mean � SEM) and at 500 nm 83.3 � 0.3% ofMAG-1 is in the trans state (n � 3).

Spectral Sensitivity of Photoresponse Produces an Analog Output. Toquantify the relationship between the photostationary state ofMAG-1 in solution and after conjugation to iGluR6-L439C, thecurrent amplitude and switching kinetics were measured as afunction of wavelength. Experiments were carried out inHEK293 cells expressing CMV-iGluR6-L439C and recorded 1–2days after transfection by whole cell patch clamping. Recordingswere in voltage clamp mode with a holding potential of �60 mV,and desensitization was blocked with Con A (see SI Text). Weexamined activation by stepping wavelengths from maximalsteady state deactivation (500 nm) to a series of shorter wave-lengths. The step duration was selected to be 10 s, long enoughfor currents to reach steady state. We then examined deactiva-tion by starting at the wavelength of maximal steady stateactivation (380 nm) and stepping to longer wavelengths (Fig. 2b).The activation and deactivation components were each well fitwith a single exponential (Fig. 2c). The activation spectrum iscentered at 380 nm (Fig. 2d ‘‘On’’), falling off steeply at higherand lower wavelengths, whereas the deactivation spectrum isbroadly centered at �500 nm (Fig. 2d ‘‘Off’’). The NMR-baseddetermination of the photostationary states of MAG-1 in solu-tion, over the wavelength range of 320–500 nm, closely match theaction spectrum of channel activation when MAG-1 is conju-gated to the channel protein (Fig. 2a). Furthermore, the on andoff rates were fastest at wavelengths between 380 nm and 500 nm

a

b

Fig. 1. Modular photoswitchable tethered ligands. (a) The light-gatedglutamate receptor operates by reversibly binding of the photoswitchableagonist MAG (14), which is attached covalently to a cysteine introduced in theligand binding domain of the receptor. The ribbon structure of apo-iGluR2(Protein Data Bank ID code 1FTO) (16) is shown on the left, together with theball-and-stick structure of MAG in the extended (trans) and unbound confor-mation. Under 380-nm illumination, MAG can activate the receptor as isshown on the right with cis-MAG docked on the structure of iGluR6 in complexwith methylglutamate (Protein Data Bank ID code 1SD3) (17). Photoswitchingis reversible with 500-nm illumination. (b) MAG-1 (14) can be elongated byintroducing an additional glycine unit (MAG-2). Compound 3 is a nonphoto-switchable MAG-1 analog and an iGluR6 agonist termed the ‘‘tether model.’’

Fig. 2. Photostationary state determination by NMR and spectral sensitivityof photoresponses. (a) Fraction of MAG-1 in the cis form determined fromNMR spectroscopy. Maximal wavelengths for cis and trans populations are 380and 500 nm, respectively. Values are given as mean � SEM (n � 3). (b)Wavelength dependence of photoresponses of iGluR6-L439C channels ex-pressed in HEK293 cells and conjugated to MAG-1. Currents were measured bywhole-cell patch clamping in the voltage clamp mode at Vh � �60 mV, withdesensitization blocked by Con A. The current vs. time traces and correspond-ing wavelength step protocol used to record action spectra are indicated. Thefirst set of steps (activation spectrum) starts at the wavelength of maximaldeactivation (500 nm) and spans UV illuminations of increasing wavelength.The second set of steps (deactivation spectrum) starts at the wavelength ofmaximal activation (380 nm) and spans visible illuminations of increasingwavelength. (c) Each temporal trace can be fitted with a single exponentialfunction whose amplitude and time constant is used to build the actionspectra. (d) The activation spectrum (‘‘ON’’) is centered on 380 nm and falls offrather steeply at higher and lower wavelengths. The deactivation spectrum(‘‘OFF’’) is wider, with maximal amplitude between 460 and 560 nm. Values aremean � SEM (n � 5). (e) Wavelength dependence of photoswitch rate. Timeconstants �ON and �OFF from fits of traces in b are represented as switch rates(1/�). Values are mean � SEM (n � 5).

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[kon-380 nm � 2.8 � 0.1 s�1 (n � 5) and koff-500 nm � 2.6 � 0.2 s�1

(n � 5), see Fig. 2d].

Thermal Relaxation of MAG. Experimentally, it may be advanta-geous to control channel opening without continuous irradia-tion. In such situations, a single activating pulse of UV lightwould be used to initiate activation for extended periods of time(minutes). As such, the MAGs were designed with a 4,4�-azodianiline scaffold modified with amide linkages to the glu-tamate and maleimide moeities of the molecule. These amide-based azobenzene cores are known to possess half-lives ofminutes for the rate of thermal relaxation from the cis state tothe lower energy trans state in the dark (24).

We measured the rate of thermal relaxation from cis to transin the dark for free MAG-1 in solution. This was done byfollowing the accumulation of the trans form by measuringabsorbance at 360 nm, near the maximum absorbance of thetrans form at 380 nm. We obtained a half-life for the spontaneouscis to trans isomerization of free MAG-1 of 17.65 � 0.03 min (n �3) (Fig. 3a). This behavior of free MAG was compared with thespontaneous deactivation of iGluR6-L439C channels in the darkusing whole cell patch clamp in HEK293 cells. Because it was notpossible to maintain seals for long enough to observe fulldeactivation, we followed activation induced by a 5-s pulse ofillumination at 380 nm with an observation period of 2 min in thedark. We observed little decay in the current during these times,with the amplitude being 0.98 � 0.1 of its initial value after 2 minin the dark (n � 7) (Fig. 3b). These findings show that the slowthermal cis to trans isomerization of azobenzene is preserved inMAG attached to the functioning receptor. Indeed, it appearsthat binding of the glutamate end of MAG in the binding pocketeven stabilizes the cis state of the azobenzene. The significanceof persistent channel activity in the dark after a brief pulse ofillumination is that long-lasting currents can be maintained in theabsence of irradiation, thus reducing photo-bleaching of theazobenzene, photo-damage to the protein, and photo-toxicity tocells. Even if some stabilization of the cis state occurs, 500-nmlight is still able to rapidly turn the current off.

MAG Conjugation to iGluR6-L439C Occurs by Affinity Labeling. In ourfirst study (15), a model of MAG-1 in the cis state was docked ontothe crystal structure of iGluR6 in complex with SYM 2081. When

the glutamate moiety was fit in the agonist binding site, themaleimide end of MAG-1 was able to reach amino acid 439, wherean introduced cysteine permits conjugation and yields a light-gatedchannel (Fig. 1a). This provided a vivid picture of the photoacti-vated state, and raised the question of whether occupancy of thebinding site by MAG-1 would enhance the conjugation efficiency ofthe maleimide to the cysteine at position 439 by affinity labeling.Affinity labeling has been observed in a variety of systems (25),including in the conjugation of tethered blockers to the Shaker K�

channel (26), which served as a basis for the development of thephotoswitchable SPARK channel (13).

To investigate the nature of MAG conjugation, we designedtwo experiments that would test the effect of interfering withaffinity labeling. In a first experiment, we asked whether wecould hinder labeling by using visible light to favor the trans stateof MAG-1 conformation, which is expected to extend themaleimide away from cysteine 439 when the glutamate end of themolecule is docked in the binding pocket (Fig. 4a). We evaluatedthe efficiency of MAG-1 conjugation from the amplitude ofphoto-responses using calcium imaging to detect the activationof the calcium permeant iGluR6 channels, as described previ-ously (15). Incubation with 100 nM MAG-1 under 380-nm light(favoring the cis state) produced larger subsequent photo-responses (36.1 � 3.6%, n � 22, of the 300 �M glutamateresponse) than did incubation under 500-nm light, favoring thetrans state (10.7 � 0.9%, n � 22, of the 300 �M glutamateresponse) (Fig. 4c). This �3-fold difference is consistent withstate-dependent affinity labeling, which is expected to better

Fig. 3. Slow thermal relaxation of MAG allows for persistent activation indark. (a) Rate of thermal relaxation in the dark of free MAG-1 from cis to trans,measured by absorbance at 360 nm. Traces are exponential and display ahalf-life of 17.65 � 0.03 min (n � 3). (b) Minimal spontaneous deactivation ofiGluR6-L439C channels conjugated to MAG-1 over a 2-min period after acti-vation with a 5-s pulse at 380 nm. Photocurrent at end of 2 min in dark is aslarge as photocurrent evoked by new pulse at 380 nm after light-induceddeactivation at 500 nm.

Fig. 4. MAG-1 conjugation to iGluR6-L439C occurs by affinity labeling.MAG-1 conjugation at 100 nM can be interfered by two means: (a) favoringthe trans conformation with 500-nm illumination, which puts the maleimidegroup away from cysteine 439 when the glutamate is bound to the LBD, and(b) occupying the binding site with a competing concentration of glutamate,thus preventing docking of MAG-1 in a conformation that favors conjugationof the maleimide to the introduced cysteine. (c) Photoresponses obtained bycalcium imaging (Fura2 fluorescence ratio at 350 vs. 380 nm excitation) inHEK293 cells transfected with CMV-iGluR6(L439C) and conjugated to MAG-1under the conditions shown in a. Weak responses are obtained after MAG-1conjugation at 100 nM under visible illumination (trans-MAG, maleimidegroup facing away from cysteine 439), but a substantial increase in photore-sponses (by a factor 3.5 � 0.3, n � 22) is observed after conjugation under UV(cis-MAG; maleimide group facing cysteine 439). (d) Weak responses areobtained after MAG-1 conjugation at 100 nM in the presence of 300 �Mglutamate (ligand-binding pocket occupied), but they are increased a factor3.2 � 0.2 (n � 10) after MAG-1 conjugation at 100 nM in absence of glutamate(binding pocket free to dock glutamate end of MAG-1 and place maleimidenear cysteine 439).

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position the maleimide near the engineered cysteine whencis-MAG-1 is bound.

In a second experiment, we attempted to interfere withaffinity labeling of MAG-1 with a competing concentration offree glutamate (300 �M) during the incubation period (Fig. 4b).Incubation was carried under 380-nm light as shown above.Incubation of iGluR6-L439C with 100 nM MAG-1 in theabsence of glutamate for 15 min produced significantly largersubsequent photo-responses (53.1 � 7.7% of the 300 �Mglutamate response, n � 10) than did incubation in the presenceof 300 �M glutamate (17.9 � 3.3% of the 300 �M glutamateresponse, n � 10) (Fig. 4d). Again, this �3-fold difference isconsistent with the disruption of affinity labeling by MAG-1 byglutamate competition for the ligand binding site.

Spatially Controlled MAG Conjugation. The above experimentsdemonstrate that, at low concentrations, MAG conjugationoperates by affinity labeling. Furthermore, the ability to controlphotoswitch conjugation with light opens the possibility ofselective labeling only in regions of a sample illuminated atshorter wavelengths. We tested this idea using a simple lightpattern (Fig. 5a). We washed 100 nM MAG-1 into a dishcontaining HEK293 cells expressing iGluR6-L439C and illumi-nated a small region of cells through the objective of the invertedmicroscope with 374-nm light, while illuminating a neighboringregion of cells with 500-nm light delivered by a fiber optic cablefrom above. After a 10-min incubation, MAG was washed fromthe chamber. We then patched cells in the two regions andcompared the amplitude of photocurrent of each cell normalizedby the amplitude of the current evoked by 300 �M glutamate.The normalized photocurrents were �4.5-fold larger (P � 0.001)in cells located within the 374-nm spot [normalized values �0.227 � 0.015 within 374 nm light spot (n � 3), versus 0.050 �0.016 within 500 nm light spot (n � 4)] (Fig. 5b). This findingdemonstrates that, in addition to selecting cells for opticalcontrol by targeting iGluR6-L493C expression with cell-specificpromoters, further subselection can be achieved by using light topreferentially label regions illuminated by UV light. One can

imagine generating complex patterns of optical responsiveness ina tissue using patterned illumination.

Concentration Dependence of MAG Conjugation. Previous work haddemonstrated that the photo-currents of iGluR6-L439C conju-gated to MAG-1 were smaller than the saturating glutamateresponse. We asked whether the partial activation by iGluR6-L439C-MAG-1 is due to incomplete MAG conjugation. Chan-nels were labeled with MAG-1 for 1 h under 380-nm illumina-tion, using concentrations of 0.1, 10, and 200 �M, with the finalconcentration being the solubility limit of MAG-1. Using whole-cell patch clamp in HEK293 cells, the average photo-current wasfound to increase with increasing MAG-1 concentration. Rela-tive to currents evoked by 300 �M glutamate, the opticalactivation of iGluR6-L439C-MAG-1 at 380 nm was 21 � 3%(n � 7), 54 � 9% (n � 5), and 71 � 6% (n � 9) at 0.1, 10, and200 �M, respectively.

Under optimal excitation at 380 nm to maximize the activatingstate, MAG-1 will have a photostationary state with 93% of themolecules in the cis state (Fig. 2a). Assuming complete conju-gation, a tetrameric channel possessing four LBDs will then befully activated 75% (0.934) of the time by MAG-1. Thus, ourresults suggested that, at higher concentrations, we are close tocomplete labeling under the assumption that MAG-1 functionswith similar efficacy to glutamate.

MAG Functions as a Full Agonist. We asked whether MAG-1operates as a full agonist, or if it is a strong, but partial agonist.As shown earlier, when partial agonists bind they allow partialclosure of the LBD and thus only partial channel activation (27).Thus, If MAG-1 is a partial agonist then in the presence of 300�M glutamate, photoswitching to cis-MAG-1 should competewith glutamate and reduce the observed current (i.e., act as anantagonist).

We found that iGluR6-L439C labeled with 100 �M MAG-1for 15 min (i.e., expected to yield substantial, but likely incom-plete conjugation, see above) did not show a sign of partialagonism. Rather than decrease currents, photo-activation(isomerization to the cis state at 380 nm) of iGluR6-L439C-MAG-1 in the presence of glutamate slightly increased thecurrent (SI Fig. 11). This observation argues that MAG-1functions with similar efficacy to glutamate, i.e., is a full agonist.

High Effective Local Concentration of MAGs. We have shown thatagonist 3, a MAG analogue lacking a maleimide and full-lengthazobenzene, has an EC50 of 180 �M (15) at iGluR6. Althoughcompound 3 possesses relatively weak affinity, the local concen-tration of the glutamate end of cis-MAG-1 when conjugated toiGluR6-L439C is expected to be very high based on its shorttether.

To test this idea, we estimated the effective concentration ofthe glutamate end of MAG-1 using the competitive antagonistDNQX (26). DNQX inhibits iGluR activation by occupying theglutamate binding site and stabilizing an open conformation ofthe LBD (Fig. 6a) (16). We examined the ability of DNQX tocompetitively inhibit the responses of iGluR6-L439C to light-activation with MAG-1, and to perfusion with compound 3 orglutamate. DNQX inhibited the response to MAG-1 at 380 nmillumination in a concentration dependent manner and wascompletely reversible upon washout (Fig. 6b). The inhibitioncurve had a 50% inhibition (IC50) of the cis state light responseat 220 � 65 �M DNQX (n � 8) (Fig. 6c). However, even at theDNQX solubility limit of 4 mM, the block of the photo-currentwas incomplete.

To calculate the local concentration of the glutamate end ofMAG-1 we examined DNQX competition versus compound 3,the closest soluble MAG analogue. We measured the concen-tration dependence of DNQX inhibition using two known

Fig. 5. Spatial patterning of MAG conjugation with patterned illumination.(a) Illustration of coverslip (12 mm in diameter) on which HEK293 cells areadhered showing illumination pattern used during a 10-min MAG exposure.Simultaneous illumination at two wavelengths was with 374-nm laser lightthrough the �40 objective, yielding a small (530 �m in diameter) spot (violetarea) and with 500-nm light through a fiber light guide on the remaining areaof the coverslip (cyan area). After a 10-min exposure to MAG and the desig-nated light pattern, MAG was washed away before subsequent patch clamprecording. (b) Bar graph showing amplitude (mean � SEM) of inward currentsevoked by illumination at 380 nm normalized to amplitude of current evokedby 300 �M glutamate. Photocurrents are 4.5-fold larger in cells illuminatedat the shorter wavelength during MAG exposure, indicating that affinitylabeling could be biased spatially with patterns of light.

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concentrations of the tether model (3 mM and 10 mM, the latterbeing the solubility limit) to extrapolate effective MAG concen-trations from their DNQX IC50. At 3 mM and 10 mM concen-trations of compound 3 we obtained DNQX IC50 values of 39 �15 �M (n � 5) and 202 � 26 �M (n � 5), respectively (Fig. 6c).Thus, inhibition by DNQX reveals that in the cis state theglutamate moiety of MAG-1 has an effective concentration of12.5 mM (Fig. 6d). Such a high effective concentration (50-foldgreater than the EC50 of compound 3) suggests that the photo-switched tethered ligand functions as designed on the channel,generating a very high effective local concentration in the cisstate.

The antagonist competition experiment revealed the existenceof a basal current of �20% at 500 nm that was blocked byDNQX. We quantified the IC50 value of block of this basalcurrent by DNQX and found it to be 7 � 2 �M (n � 8) (Fig. 6c).This value indicates an effective glutamate concentration of 0.5mM, which is �30-fold lower than the value measured at 380 nm(Fig. 3d). This finding supports the model that light activates thechannel by changing the local concentration of the ligand uponcis–trans photoisomerization.

Tether Length Dependence on Channel Activation. We next investi-gated the dependence of light-gating on tether length using anelongated tethered ligand, MAG-2 (Fig. 7a). We found that foriGluR6-L439C conjugated to MAG-2 at 10 �M for 1h the

amplitude of the photoresponse at 380 nm was 24 � 2% (n � 12)that of the current evoked by 300 �M glutamate, about half thatmeasured for iGluR6-L439C-MAG-1 (Fig. 7b). Competitionstudies on iGluR6-L439C-MAG-2 using DNQX yielded an IC50under 380 illumination of 80 � 20 �M (n � 7), indicating aneffective concentration that was three-fold lower than thatobserved for MAG-1. Consistent with the lower effective con-centration of MAG-2 in the cis state, the basal activation waslower than for MAG-1 (Fig. 7c) and high concentrations ofDNQX were able to completely block the photo-current at 380nm for iGluR6-L439C-MAG-2 (Fig. 7d).

DiscussionWe have characterized the optical control of a glutamate recep-tor by a photoswitchable tethered glutamate that is covalentlyattached to the exterior of the receptor’s LBD. Properties ofMAG-1 when free in solution, such as the wavelength depen-dence of isomerization, are found to match those of the fullyconjugated, light-activated receptor. NMR experiments eluci-dating the photostationary state ratios of cis- to trans-MAG-1provide a greater understanding of the activation of this light-actuated system. For example, at 380 nm, despite the fact thatMAG-1 is a full agonist, the photocurrent is submaximal. This islikely due to the fact that even at the optimal wavelength of 380nm �7% of MAG-1 remains in the trans state. By the sametoken, the basal current at 500 nm can be attributed in part tothe �17% of azobenzene that is in the cis state at that wave-length. Further optimization could be achieved by buildingphotoswitches with different properties. For instance, fast-relaxing urea-based azobenzene cores (24) could reduce basalactivation by quickly isomerizing from the higher energy cis stateto the more stable trans conformation in the dark. Furthermore,azobenzene photoswitches with more complete conversion tothe cis state should improve the photo-efficacy of the tetheredligand.

Using a soluble mimic of the tethered ligand, the effectivelocal concentration was estimated to be very high (10 mM),consistent with the short tethers to which the ligand is attached.

Fig. 6. Effective local concentration of MAG-1 is in the millimolar range. (a)The competitive antagonist DNQX inhibits iGluR activation by occupying theglutamate binding site without allowing LBD closure. (b) Patch clamp currenttraces of iGluR6–L439C conjugated to MAG-1 show responses to perfusion of300 �M glutamate and to illumination. The corresponding wavelength–timetraces are shown below. Perfusion of DNQX partially inhibits photoresponsesto 380 nm illumination and reveals a basal activation under 500-nm illumina-tion. Inhibition by DNQX is reversible on washout after each DNQX perfusion.(c) Quantification of DNQX inhibition of photoresponses and comparison toits effect on free tether model compound 3. Current under 380-nm light (filledcircles) is inhibited by DNQX to 36% of total photoresponse (IC50 � 220 �M �65 �M DNQX, n � 8) and the current under 500-nm (open circles) is completelyblocked, which reveals a basal activation 20% of total photoresponse, IC50 �7 � 2 �M DNQX (n � 8). For comparison, DNQX blocks responses to 10 mMtether model 3 (filled squares, IC50 � 202 � 26 �M DNQX, n � 5) and 3 mMtether model 3 (open squares, IC50 � 39 � 15 �M DNQX (n � 5). (d) Determi-nation of the effective concentration of MAG-1 as a function of the DNQX IC50

values. The IC50 values for DNQX/tether model 3 are used to calibrate the localconcentration axis assuming a linear relationship (straight line), and yield 12.5mM and 0.5 mM for MAG-1 under UV and visible, respectively. Values aremean � SEM.

Fig. 7. Tether length dependence on channel activation. (a) DNQX titrationsof iGluR6-L439C conjugated to MAG-1 (UV, filled circles; visible, open circles)and MAG-2 (UV, filled triangles; visible, open triangles). (b) Amplitude ofMAG-1 and MAG-2 photoresponses after conjugation at 10 �M for 1 h,compared with 300 �M glutamate responses. (c) Basal activation of iGluR6L439C conjugated to MAG-1 and MAG-2, as obtained from low-DNQX pla-teaux under visible illumination in a. Residual photoresponse for MAG-1 andMAG-2 obtained from high-DNQX plateau under UV illumination in a. Valuesare mean � SEM.

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The increased concentration of the ligand in the cis state is afunction of the increased proximity of the glutamate moiety toits binding site, illustrating the geometric constraints imposed bythe azobenzene. Furthermore, MAG functions as a full agonist,indicating that the tether does not prevent full closure of theligand binding domain on the glutamate end of MAG. This issomething that cannot be taken for granted because the pathwayto the binding pocket is narrow, presenting potential stericclashes. For applications in neuroscience, the high effectiveconcentration allows for selective block of activation by synap-tically released glutamate with 25 �M DNQX (29) while main-taining the near maximal photoresponses at 380 nm (Fig. 7c).The length of the tether, here studied using an additional glycinebuilding block, is essential to the function of the photoswitch.Although the increased length of the tether decreased the basalcurrent under 500 nm illumination, the ease with which it isdisplaced by competing DNQX and the low efficacy of thephotoresponse reveals the importance of optimizing tetherlength to best suit an individual protein under photocontrol.

The high local concentration of the glutamate moiety whenthe maleimide end is attached makes the reverse geometrypossible too, so that binding of the glutamate end places themaleimide in a high effective concentration near the introducedcysteine, resulting in affinity labeling. This enables conjugationto the target receptor selectively, and to do so at concentrationsthat are far below the EC50 for activation, thus avoiding signif-icant activation and possible cytotoxicity. Moreover, we can usethe state-dependence of affinity labeling to pattern conjugationwith patterned illumination. Regions illuminated at �380 nm arepreferentially labeled, a valuable way of constraining where in atissue light gating will take place.

Our findings represent advances in the remote control ofprotein function and new insights into the mechanism of light-gating glutamate receptors. We find that, although MAG-1maintains similar action spectra when it is conjugated to theprotein compared with free in solution, binding of the glutamatemoiety within the binding pocket may stabilize the cis state of theazobenzene and slow thermal relaxation. As a result of the slowrelaxation of cis-MAG-1, the channels display a memory for ashort light pulse at 380 nm, which activates the receptors andkeeps them open in the dark for an extended period. This can be

useful in experimental setups where light cannot be deliveredcontinuously, and it makes it possible to reduce illumination timeand thus to minimize photo-toxicity to cells and photo-damageto other elements in a system, including to the azobenzene itself.

Light switching provides the ability to temporally and spatiallycontrol protein function in cells, and makes it possible to setswitching kinetics and the degree of activation. Future work willexploit these properties in complex cellular environments andcould be a powerful tool for the production of artificial bio-chemical circuits and artificial cells or reengineered cells forsynthetic biology. The properties of the MAG photoswitchessuggest that structure-based design could, in principle, providerapid, reversible, and remote control of any protein for whichtethered ligands can operate as active site blockers or as allo-steric agonists or antagonists.

MethodsSynthesis of iGluR6 Tethered Agonist MAG-2. MAG-2 was synthe-sized by using chemistry similar to that described for MAG-1(15). See SI Text.

Introduction of Cysteine into Glutamate-Binding Domain. Cysteinepoint mutations were introduced to the iGluR6 DNA, contain-ing Q at the position 621 RNA editing site (30) using theQuikChange site-directed mutagenesis kit (Stratagene, La Jolla,CA). The following PCR profile was used: one cycle (95°C for30 s); 20 cycles (95°C for 30 s, 55°C for 1 min, 68°C for 12 min).The forward and reverse oligonucleotide sequences designed forthe L439C mutant were 5�-GATTGTTACCACCATTTGC-GAAGAACCGTATGTTCTG-3� and 5�-CAGAACATACG-GTTCTTCGCAAAATGGTGGTAACAATC-3�, respectively.

We thank R. Kramer for discussion, M. Banghart for method of determiningphotostationary states by NMR, and S. Heinemann (Salk Institute, La Jolla,CA) for the iGluR6 clone. This work was supported by postdoctoralfellowships from Human Frontier Science Program (HFSP) and Nanotech-nology Program of Generalitat de Catalunya (to P.G.) and Japan Society forthe Promotion of Science (to R.N.), predoctoral fellowships from AmericanChemical Society Medical Chemistry Division (to M.V.) and NationalScience Foundation (to S.Z.), and grants from Lawrence Berkeley NationalLaboratory, HFSP, National Institutes of Health, and the Alfred P. SloanFoundation.

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