Ligand-Directed Chemistry of AMPA Receptors Confers Live-Cell

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Ligand-directed chemistry of AMPA receptors confers live-cell fluorescent biosensors Shigeki Kiyonaka, Seiji Sakamoto, Sho Wakayama, Yuma Morikawa, Muneo Tsujikawa, and Itaru Hamachi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b01042 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 14, 2018

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Graphical Abstract

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1 2 3

Ligand-directed chemistry of AMPA receptors confers live-cell fluorescent

4

biosensors

5 6 7

Shigeki Kiyonaka*†, Seiji Sakamoto†, Sho Wakayama†, Yuma Morikawa†,

8

Muneo Tsujikawa† and Itaru Hamachi*†, ‡

9 10 11 12 13 14

†

Engineering, Kyoto University, Nishikyo-ku, Kyoto 615-8510, Japan

15 16 17

Department of Synthetic Chemistry and Biological Chemistry, Graduate School of

‡

CREST(Core Research for Evolutional Science and Technology, JST), Chiyodaku, Tokyo, 102-0075, JAPAN

18 19

*Correspondence : [email protected]

20

[email protected]

21

1 ACS Paragon Plus Environment

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1

ABSTRACT

2

AMPA-type glutamate receptors (AMPARs) mediate fast excitatory synaptic

3

transmission in the central nervous system. Disregulation of AMPAR function is

4

associated with many kinds of neurological, neurodegenerative and psychiatric

5

disorders. As a result, molecules capable of controlling AMPAR functions are potential

6

therapeutic agents. Fluorescent semisynthetic biosensors have attracted considerable

7

interest for the discovery of ligands selectively acting on target proteins. Given the large

8

protein complex formation of AMPARs in live cells, biosensors using full-length

9

AMPARs retaining original functionality are ideal for drug screening. Here, we

10

demonstrate that fluorophore-labeled AMPARs prepared by ligand-directed acyl

11

imidazole chemistry can act as turn-on fluorescent biosensors for AMPAR ligands in

12

living cells. These biosensors selectively detect orthosteric ligands of AMPARs among

13

the glutamate receptor family. Notably, the dissociation constants of agonists and

14

antagonists for AMPARs were determined in live cells, which revealed that the

15

ligand-binding properties of AMPARs to agonists are largely different in living cells,

16

compared with non-cellular conditions. We also show that these sensors can be applied

17

to detecting allosteric modulators or subunit-selective ligands of AMPARs. Thus, our

18

protein-based biosensors can be useful for discovering pharmaceutical agents to treat

19

AMPAR-related neurological disorders.

20



1

INTRODUCTION

2

Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS).

3

Fast excitatory neurotransmission in the CNS is achieved mainly via ion-channel-type

4

glutamate receptors (iGluRs) including three subgroups (AMPA, NMDA and kainate

5

receptors).1 Among them, recent studies revealed that AMPA receptors (AMPARs) play

6

critical roles in nearly all aspects of brain function such as learning, memory and

7

cognition.2 Disregulation of AMPARs is associated with many kinds of neurological,

8

neurodegenerative and psychiatric disorders, and thus molecules capable of controlling

9

AMPAR function are regarded as potential therapeutic agents.3,4

10

To discover the small molecules that selectively inhibit AMPAR functions, a

11

cell-based assay using a voltage- or Ca2+-sensitive dye was reported recently, in which

12

changes in glutamate-induced cation influx in live cells expressing AMPARs were

13

measured.5,6 Although powerful for high-throughput screening, this assay evaluated the

14

ion-channel function of AMPARs indirectly, which would result in the erroneous

15

inclusion of many false-positive compounds. For examining the direct binding of the

16

compounds to the receptors, competitive ligand binding assays have conventionally

17

been utilized, in which the selective binding of radioisotope-labeled ligands to soluble

18

proteins or membrane fractions including AMPARs were evaluated.7,8 However, it is

19

difficult to apply this method for cell-based or high-throughput assays. More seriously,

20

the complex formation of AMPARs would be disrupted in the membrane fractions or its

21

solubilized state.8 An assay for the direct binding of small compounds to AMPARs

22

under live cell conditions would be ideal.

23

Protein-based fluorescent biosensors have attracted considerable interest as

24

chemical tools for evaluating ligand binding to target proteins.9 Semisynthetic

25

biosensors are prepared using site-specific chemical modifications of target proteins

26

with environmentally sensitive fluorescent dyes, in which the ligand-binding events are

27

transduced into a fluorescence change in the labeled fluorophore. Fluorescent


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1

biosensors for many kinds of analytes have been reported,10–19 and some of these have

2

been applied to high-throughput screening.13,15 For glutamate sensing, Hirose’s group

3

has reported fluorescent sensors consisting of the ligand-binding domain (S1S2)20 of

4

GluA2, an AMPAR subunit.16,17 This was targeted on live cells via avidin-biotin

5

interactions to visualize extracellular glutamate concentrations in neuronal cultures.

6

Johnsson’s group reported an in situ biosensor in live cells, termed Snifits18 consisting

7

of a SNAP-tag, CLIP-tag and S1S2 of a kainate receptor subunit (GluK1), which

8

enabled ratiometric imaging of the extracellular glutamate concentration.19 These

9

approaches are useful for the fluorescent visualization of glutamate under live cell

10

conditions. However, S1S2 is just an artificially prepared ligand-binding region of

11

iGluRs including AMPARs, whose sizes (about 30 kDa) are largely different from those

12

of the tetrameric complexes of AMPARs (400–500 kDa). Thus, the pharmacological

13

properties of S1S2 would be different from those of full-length AMPARs. Semisynthetic

14

biosensors using full-length AMPARs that maitain their original function in live cells

15

are preferable for drug screening.

16

We have recently reported novel chemical labeling reagents, CAM2 for the

17

fluorescent labeling of native AMPARs with negligible disturbances to receptor

18

functions in living cells.21 This is based on an affinity-driven protein labeling method,

19

termed ligand-directed acyl imidazole (LDAI) chemistry (Figure 1a).22,23 On the basis

20

of the Hirose’s S1S2-based glutamate biosensors16,17 and our previous results with other

21

proteins22, we expected that the fluorescence of AMPARs labeled with CAM2 reagents

22

would be altered upon glutamate binding. Indeed, we found that Alexa488-labeled

23

AMPARs using CAM2(Ax488) are able to act as turn-on fluorescent biosensors for

24

AMPAR ligands in live cells. This biosensor selectively detects orthosteric ligands

25

including agonists and antagonists. Notably, quantitative analyses of ligand affinity with

26

AMPARs revealed that ligand-binding properties determined in live cell environments

27

are largely different compared with those obtained in non-cellular conditions using



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1

S1S2 and membrane fractions containing full-length AMPARs, highlighting the benefit

2

of our sensor construction using the entire AMPAR scaffold in living cells.

3 4 5

RESULTS AND DISCUSSION

6

Fluorescent response of Alexa488-labeled AMPARs to glutamate on the surface of

7

live cells

8

We evaluated the fluorescent response of fluorophore-labeled AMPARs on the surface

9

of live cells upon the addition of an agonist, L-glutamate. Chemical labeling of

10

AMPARs using CAM2(Ax488-5S) (Figure 1b), whose original name was

11

CAM2(Ax488), was conducted according to our previous report using CAM2(Fl)21. As

12

shown in Figure 1c, western blotting indicated the selective labeling of AMPARs. This

13

band was not observed in the presence of NBQX, a competitive inhibitor, suggesting

14

that the selective labeling was facilitated by affinity-based effects.

15

Confocal live cell imaging clearly revealed that the fluorescence was

16

predominantly observed at the plasma membrane in the GluA2 transfected cells (Figure

17

S1), and that this fluorescence was enhanced by the addition of 1 mM L-glutamate

18

(Figure 1d). To quantify the fluorescence changes, we used epifluorescence imaging

19

with a perfusion system, in which extracellular buffer solution was quickly (15 µL/sec)

20

and reversibly exchanged (Figure 2a). As shown in Figure 2b, the fluorescence of

21

Alexa488-AMPARs increased immediately after the addition of L-glutamate, and

22

returned to its original intensity after exchanging the buffer solution. This fluorescence

23

change did not occur after the addition of other amino acids (Figure 2c). A small but

24

prominent fluorescence change was observed after the addition of D-glutamate, which is

25

consistent with previous reports showing a low affinity of D-glutamate for GluA2.24

26

Importantly,

27

L-glutamate-induced

these

fluorescence

changes

occurred

reversibly,

and

the

fluorescence responses were observed repeatedly (Figures 2d,e).


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1

Thus, GluA2 labeled with CAM2(Ax488-5S) on the surface of live cells acted as a

2

fluorescent biosensor while keeping its original substrate selectivity.

3 4

Details about the ligand-induced fluorescent responses of Alexa488-labeled

5

AMPARs

6

It was expected that the glutamate-induced increase in fluorescence occurred due to

7

changes in the microenvironment of the labeled Alexa488 in the AMPAR scaffold. We

8

examined the effects of the orientation of the labeled fluorophore on the fluorescence

9

responses. Another CAM2 series were prepared, in which the substituted position of

10

Alexa488 and the linker length between the reactive acyl imidazole and Alexa488 were

11

varied (Figure 3a). Confocal imaging indicated that these CAM2 reagents successfully

12

labeled AMPARs in live cells (Figure S2). As shown in Figure 3b and Figure S3a,

13

elongation of the ethylene glycol linker increased the fluorescence changes (F/F0)

14

(Ax488-5S vs Ax488-5M), and a liker length of 16 Å was sufficient for this change.

15

With respect to the substituted position of Alexa488, the F/F0 change of the 5-isomer

16

was much larger than that of the 6-isomer (Ax488-5S vs Ax488-6S), and this tendency

17

was similarly observed in other CAM2s bearing different linkers (Figure 3b and

18

Figure S3b). These indicate that the orientation of the fluorophore affects the

19

glutamate-induced fluorescence responses of Alexa488-AMPARs.

20

AMPARs are composed of amino-terminal, ligand-binding, transmembrane and

21

carboxyl-terminal domains (Figure 1a).1,3 Recent structural analyses have revealed that

22

glutamate binding induces large structural changes, not only in the ligand-binding

23

domain but also in other domains.25–28 To examine the influence of other domains on the

24

glutamate-induced fluorescence changes, we prepared recombinant S1S2 of GluA225

25

(Figure S4a). As with the case of the chemical labeling of the full-length AMPARs in

26

live cells, CAM2(Ax488-5S) successfully labeled the S1S2 protein in test tubes (Figure

27

S4b). The obtained Alexa488-labeled S1S2 showed prominent fluorescence increases



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1

after the addition of L-glutamate (Figure S4c). This indicates that the ligand-binding

2

domain is sufficient by itself for the glutamate-induced fluorescent responses, as in

3

Hirose’s report.16,17

4

According to recent structure-function relationship studies, glutamate binding

5

induces the closure of the ligand binding domain to activate AMPARs.25,27 In contrast,

6

antagonists stabilize the open conformation of the domain, which results in the

7

inhibition of the receptor. To examine the impacts of domain closure on the

8

glutamate-induced

9

Alexa488-AMPAR or -S1S2. To our surprise, an F/F0 increase was clearly observed

10

after the addition of DNQX, a well-known antagonist for AMPARs1 (Figure S5a)

11

which stabilizes the open conformation of the ligand-binding domain25. Similar

12

fluorescence increases were observed in the case of S1S2 (Figure S5b), which indicates

13

that this fluorescence change is not caused by domain closure.

fluorescence

changes,

we

added

AMPAR

antagonists

to

14

We next examined the effect of the chemical structure of the labeled fluorophore

15

upon the ligand-induced fluorescent changes. AMPARs were labeled with the CAM2

16

series bearing Oregon green (OG), Alexa568 or Cy3.5 (Figure 3a) on the live cells, and

17

fluorescent responses upon addition of glutamate or NBQX were examined. In the case

18

of OG-AMPARs whose fluorophore size is similar to that of Alexa488, prominent F/F0

19

increase was observed (Figure 3c and Figure S6). In contrast, when AMPARs were

20

labeled with the large fluorophore (Alexa568 or Cy3.5), marginal fluorescent changes

21

were only occurred. A plausible explanation for the fluorescence changes is as follows:

22

fluorescence of the labeled Alexa488 or OG is weakened by the partial incorporation of

23

the fluorophore into the ligand binding domain. Addition of the competitive ligands

24

ejects the fluorophore, which results in an increase in the fluorescence. For Alexa568 or

25

Cy3.5, the fluorophore size would be too large to enter the ligand binding domain. This

26

mechanism is supported by the fact that the simple application of non-competitive


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1

ligands, such as allosteric modulators and channel-pore blockers, failed to increase the

2

fluorescence, as described below.

3 4

Determination of Kd values for various AMPAR ligands in live cells

5

Quantitative analyses of the affinity of AMPAR ligands have been examined using

6

competitive binding assays of radioisotope-labeled ligands.7,8,29–31 In these assays,

7

membrane fractions or solubilized receptors prepared from cells expressing AMPARs

8

were utilized, and Ki values (i.e. apparent dissociation constants) for various AMPAR

9

ligands have been determined. However, these values were largely different from the

10

affinity (Kb values (i.e. apparent dissociation constants for antagonists) or EC50 (for

11

agonists)) determined by a function-based assay (electrophysiology or Ca2+ influx

12

imaging) in live cells. The discrepancy was large for agonists, such as glutamate or

13

AMPA32,33, whereas it was almost negligible for antagonists, such as NBQX or DNQX34

14

(Table 1). Such discrepancies can be partially ascribed to difference in the buffer

15

compositions and/or the assay methods.8,35,36 Although the assay conditions, such as

16

non-cellular as opposed to live cell, may have large impacts, this aspect has not been

17

clearly discussed, due to a lack of reliable methods for ligand binding assays in live cell.

18

We sought to quantitatively examine the affinity of agonists for AMPARs in live

19

cells using our biosensors. As described above (Figure 2e), glutamate-induced

20

fluorescence increases in Alexa488-AMPARs were observed repeatedly and constantly.

21

Then, we monitored the fluorescence responses to altering the extracellular glutamate

22

concentration. As shown in Figures 4a and 4c, concentration-dependent fluorescence

23

changes were observed. Using this, the dissociation constant of glutamate (Kd = 252 ±

24

14 µM) was determined, which was almost identical with the EC50 values (EC50 = 296–

25

634 µM)32,33 previously determined by function-based assays in live cell conditions

26

(Table 1). Interestingly, this value was largely different from the Ki values (Ki = 0.17–

27

0.48 µM)29–31 obtained by radioisotope assays using the membrane fraction. Similar to



1

glutamate, the Kd value of AMPA in live cells (Kd = 18.3 ± 2.3 µM) was almost the

2

same as the previously reported EC50 values (EC50 = 11–66 µM), but about 103 times

3

larger than those for the radioisotope assay-based Ki (Ki = 0.012–0.025 µM) (Table 1

4

and Figures S7a and S7c). We also compared the dissociation constants of antagonists.

5

For DNQX, the Kd value determined by our method was 1.40 ± 0.39 µM, which was

6

almost the same as the Kb value (1.66 µM) from the function-based assay and the Ki

7

value (0.45 µM) from the radioisotope assay (Table 1, Figure 4d and Figure S8). In the

8

case of NBQX, the Kd value (0.26 ± 0.03 µM) from our method was almost identical

9

with the Kb value (0.31 µM) and Ki value (0.39 µM) (Table 1 and Figure S9). Taken

10

together, the Kd values determined by our biosensors showed good agreement with the

11

EC50 and Kb values determined using function-based assays for both agonists and

12

antagonists in live cells. In contrast, the Ki values found by the radioisotope assay

13

method using the membrane fractions differed significantly, particularly for agonists.

14

There is a concern that the ligand affinity may be affected by the covalent

15

labeling with the fluorophore near the ligand-binding site in our method. We previously

16

confirmed that the ion channel activity of AMPARs was not affected by CAM2 labeling

17

in a prior report.21 Here, we compared the Kd value of glutamate for Alexa488-S1S2

18

using fluorescence titration (Figures 4b and 4c) with the reported Ki values for S1S2

19

using the radioisotope method. As summarized in Table 1, the Kd value of glutamate (Kd

20

= 1.67 ± 0.01 µM) for Alexa488-S1S2 was slightly larger, but nearly comparable to the

21

reported Ki values (Ki = 0.45 µM)25 for S1S2. A similar tendency was seen with AMPA

22

(Table 1 and Figures S7b and S7c). In addition, neither the orientation nor the linker

23

length of the fluorophore significantly affects the Kd value of glutamate in S1S2 (Figure

24

S10), indicating that the localization of the fluorophore near the ligand binding domain

25

would negligibly impact on its ligand binding properties. Thus, the difference between

26

the Kd values for the full-length AMPAR in live cells found by our methods and the Ki

27

values found by the radioisotope method using the membrane fractions was not due to


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1

the chemical modification. We further examined the differences in buffer conditions.35 It

2

was shown using Alexa488-S1S2 that the Kd value for glutamate in HBS buffer was

3

7-fold higher than that obtained in a radioisotope-ligand binding buffer (Table S1), but

4

it is not substantial.

5

Given that the ligand affinity values obtained by our method in live cells are quite

6

similar to those found in live cells using function-based assays, while discrepancies are

7

seen in the values from radioisotope assays using the membrane fraction, it is likely that

8

the ligand-binding properties of AMPARs are different in cellular and non-cellular

9

conditions, especially for agonists. Indeed, the agonist affinities of the S1S2 domain

10

determined using the fluorescence or radioisotope methods in test tubes were similar to

11

those from the full-length AMPAR determined by the non-cellular membrane fraction

12

radioisotope assays. We also confirmed the affinity of glutamate for Alexa488-S1S2 on

13

the live cell surface. Targeting Alexa488-S1S2 to the cell surface was conducted using

14

avidin-biotin interaction (for details see Figure S11a). The Kd value was almost

15

comparable to that obtained in the test tube (Figure S11), but significantly smaller than

16

that for the tetrameric Alexa488-AMPARs in live cells (Table S1). These data indicate

17

that tethering the ligand binding domain in live cells is not a main cause for changing

18

the affinity to glutamate. Taking into considering our results, the plausible reason for the

19

lower Ki values relative to the EC50 values found in the previous study can be ascribed

20

to the non-cellular conditions, in which the native structure of tetrameric AMPARs are

21

disturbed. This appears consistent with many of previous reports that suggested the

22

existence of high and low affinity sites for 3H-AMPA in the membrane fraction and

23

convergence into the high affinity site after detergent treatment8,35.

24 25

Fluorescence response of various kinds of glutamate receptor ligands

26

Fluorescent on-cell biosensors for AMPAR ligands in hand, we next examined the

27

applicability of our method for evaluating various glutamate receptor ligands. As shown



1

in Figures 5a and 5b, prominent increases in the fluorescent ratio were observed after

2

the addition of competitive agonists or antagonists for AMPARs. In contrast, negligible

3

fluorescence responses were observed with competitive ligands for other glutamate

4

receptor subtypes, such as NMDA receptors (NMDARs) and GPCR-type metabotropic

5

glutamate receptors (mGluRs), indicating the subtype selectivity of our sensor. Notably,

6

neither allosteric modulators nor channel-pore blockers for AMPARs induced

7

fluorescent changes. These results indicate that our biosensor selectively detects

8

competitive ligands for AMPARs.

9

Allosteric modulators are also attractive targets for pharmacology related to

10

AMPARs.37 Unfortunately, we found that the simple application of allosteric modulators

11

failed to increase the fluorescence in our sensors, as described above (Figures 5a and

12

5b). However, the fluorescence of Alexa488-AMPARs induced by agonists may be

13

altered by allosteric modulators, which allows for an assay method for allosteric

14

modulators. As a proof of principle, we focused on cyclothiazide (CTZ), a

15

representative allosteric modulator for AMPARs that increases synaptic currents by

16

inhibiting desensitization of AMPARs, and decreases the affinity for AMPA.38,39 Our

17

sensors revealed increases in the Kd values of AMPA, from 18.3 ± 2.3 µM to 99 ± 26

18

µM, in the presence of CTZ (Figure S13). We then examined the CTZ-induced

19

fluorescence changes in the presence of 20 µM AMPA. As shown in Figure 5c, CTZ

20

decreased the fluorescence in a concentration-dependent manner, and the EC50 value for

21

CTZ was determined to be 13.3 ± 5.2 µM. This indicates that our system can be used for

22

detecting allosteric modulators for AMPARs in the presence of adequate concentrations

23

of agonists.

24

AMPARs have four subunits (GluA1–GluA4), whose distribution and

25

physiological roles are different in the brain.1 Thus, subunit-selective ligands are highly

26

desirable for treating neurological disorders with minimal side effects.40 In our system,

27

we can construct subunit selective biosensors for AMPAR ligands by changing the


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1

expression plasmids. We labeled the GluA4 subunit using CAM2(Ax488-5S) in

2

HEK293T cells transfected with expression plasmids encoding GluA4 (Figure S14). As

3

with GluA2, Alexa488-GluA4 showed a prominent increase in the fluorescence after

4

glutamate was added (Figure 5d). Notably, the addition of Cl-HIBO41, a

5

GluA2-selective agonist, increased the fluorescence in cells expressing GluA2 but not

6

GluA4, indicating the successful detection of GluA2-selective ligands. It is expected

7

that our method could be expanded for discovering subunit selective agonists or

8

antagonists for AMPARs.

9 10 11

CONCLUSION

12

We constructed on-cell fluorescent biosensors for AMPAR ligands using ligand-directed

13

acyl imidazole (LDAI) chemistry. Our study clarified that the dissociation constants of

14

AMPAR agonists are largely dependent on the assay condition (living cell or

15

non-cellular), as well as the AMPAR scaffold (full-length or ligand-binding domain).

16

This highlights the importance of constructing fluorescent biosensors in live cell

17

conditions. Our sensor allowed for the quantitative detection of competitive ligands,

18

allosteric modulators and subunit-selective ligands for AMPARs on live cells, which

19

represents a new approach for discovering pharmaceutical agents for treating

20

AMPAR-related neurological disorders.

21

We envision that our method can be expanded to construct on-cell biosensors for

22

other receptor ligands by changing the labeling reagents and receptor pairs, if the

23

fluorescent intensity of the labeled fluorophore changes after addition of ligands. To

24

sidestep this limitation, we recently reported the construction of on-cell biosensors by

25

coupling LDAI chemistry with a Bimolecular Fluorescence Quenching and Recovery

26

(BFQR) system.15,42 We anticipate that ligand-directed chemistry for cell-surface



1

receptors will be a powerful approach for the construction of biosensors under natural

2

habitat.

3


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1

METHODS

2

Synthesis

3

All synthetic procedures and compound characterizations are described in the

4

Supporting Notes. CAM2(OG) and CAM2(Cy3.5) were prepared as described

5

previously.22

6 7

General methods for biochemical and biological experiments

8

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting were carried

9

out using a Bio-Rad Mini-Protean III electrophoresis apparatus. Chemiluminescent

10

signals generated with Chemi-Lumi One (nacalai tesque) or ECL Prime (GE

11

Healthcare) were detected with an LAS4000 imaging system (Fuji Film).

12 13

Chemical labeling of AMPARs in HEK293T cells

14

HEK293T cells (ATCC) were cultured in Dulbecco’s modified Eagle’s medium

15

(DMEM)-Glutamax (Invitrogen) supplemented with 10% fetal bovine serum

16

(Invitrogen), penicillin (100 units ml–1), streptomycin (100 µg ml–1), and amphotericin

17

B (250 ng ml–1), and incubated in a 5% CO2 humidified chamber at 37 °C. Cells were

18

transfected with a plasmid encoding flip form of GluA2 (GluA2flip(Q)) or GluA4

19

(GluA4flip(Q)) tagged with HA-tag on the N-terminus21 or the control vector using the

20

lipofectamine 2000 (Invitrogen) and subjected to labeling experiments after 36 h of the

21

transfection.

22

serum free DMEM-Glutamax (25 mM HEPES), and treated with CAM2 reagents in the

23

absence or presence of NBQX in the serum free medium for indicated periods at 17 °C

24

to suppress internalization of AMPARs43.

For chemical labeling, the cells expressing GluA2 were washed with

25 26

Confocal imaging of labeled AMPARs in HEK293T cells

27

For live imaging experiments, HEK293T cells were co-transfected with GluA2 and



1

mCherry as a transfection marker. After chemical labeling as described above, the cells

2

were washed 3 times with ice-cold HBS (20 mM HEPES, 107 mM NaCl, 6 mM KCl, 2

3

mM CaCl2, and 1.2 mM MgSO4 , 11.5 mM glucose at pH 7.4). Cell imaging was

4

performed with a confocal microscopy (FV1000, IX81, Olympus) equipped with a 60×,

5

numerical aperture (NA) = 1.35 oil objective. Fluorescence images were acquired using

6

a 488 nm line of an argon laser for excitation of Alexa488 and a HeNe Green laser for

7

excitation of mCherry.

8 9

Western blotting analyses of labeled AMPARs in HEK293T cells

10

For western blot analysis, after chemical labeling, cells were washed 3 times with

11

ice-cold HBS, lysed with radio immunoprecipitation assay (RIPA) buffer containing 1%

12

protease inhibitor cocktail set III (Calbiochem), and mixed with a quarter volume of 5×

13

SDS-PAGE loading buffer containing 250 mM DTT. The samples were applied to

14

SDS-PAGE and electrotransferred onto immune-blot polyvinylidene fluoride (PVDF)

15

membranes (Biorad), followed by blocking with 5% nonfat dry milk in Tris-buffered

16

saline (TBS) containing 0.05% Tween (Sigma-Aldrich). The Alexa488-labeled

17

AMPARs were detected by chemiluminescence analysis using rabbit anti-Alexa488

18

antibody (Invitrogen, A11094, x1,000) and anti-rabbit IgG-HRP conjugate (CST, 7074S,

19

x5,000).

20

anti-HA antibody (Convance, MMS-101P, x2,000) and anti-mouse IgG-HRP conjugate

21

(Santa Cruz, sc-2005 x5,000). The signals were developed with Chemi-Lumi One

22

(Nacalai tesque) or ECL Prime Western Blotting Detection Reagent (GE Healthcare)

23

and detected with Imagequant LAS4000 (GE Healthcare).

The immunodetection of GluA2 or GluA4 was performed with a mouse

24 25

Fluorescent response analysis of labeled GluA2 in HEK293T cells

26

HEK293T cells transfected with GluA2 were plated on glass coverslips. Thirty-four h

27

after transfection, the cells were washed with serum free DMEM-Glutamax (25 mM


Page 16 of 32

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1

HEPES) and treated with 2 µM CAM reagents in serum free DMEM-Glutamax (25

2

mM HEPES) at 17 °C for 4 h. The coverslips were placed on the stage of a fluorescent

3

microscopy (IX83 Olympus) equipped with a 20×, numerical aperture (NA) = 0.75

4

objective and continually perfused with HBS (20 mM HEPES, 107 mM NaCl, 6 mM

5

KCl, 2 mM CaCl2, 1.2 mM MgSO4 and 11.5 mM glucose at pH 7.4). Fluorescence

6

images of the cells were captured by a high-sensitivity electron-multiplying

7

charge-coupled device (EM-CCD) camera (ImagEM-X2, Hamamatsu Photonics)

8

operated with HCImage software (Hamamatsu photonics). Fluorescence images were

9

acquired using blue light for excitation of Ax488 at 5 s intervals. AMPAR ligands or

10

amino acids were applied during periods indicated by bars. For determination of

11

dissociation constants, the change in fluorescence intensity was fitted by an equation (1)

12

using Kaleida Graph (Synergy Software) where L and h represent the concentrations of

13

ligand and Hill’s coefficient, respectively.

14

∆F/F0 = (∆Fmax/F0)/[1 + (L/logKd)h] (1)

15

∆F denotes the difference in the fluorescence intensity of Alexa488-AMPAR between in

16

the absence (F0) and presence of ligand at each concentration (F). When all the

17

Alexa488-AMPAR forms the complex with ligand, ∆F is equal to ∆Fmax.

18 19 20

Chemical labeling of ligand binding domain of GluA2 (S1S2)

21

Production, refolding and purification of a ligand binding domain of GluA2 (S1S2)

22

were carried out as previously described44 (The expression plasmid was kindly gifted

23

from Prof. Gouaux). One µM S1S2 was incubated with 3 µM CAM2(Ax488-5S) with

24

or without 50 µM NBQX inhibitor in the 20 mM HEPES buffer (100 mM NaCl, pH

25

7.2) at 17 °C. After 4 hr incubation, each sample was mixed with an equal volume of 2×

26

SDS-PAGE loading buffer (125 mM Tris-HCl, 100 mM DTT, 4% SDS, 20% glycerol,

27

and 0.01% bromophenol blue (BPB), pH 6.8). The samples were subjected to



Page 18 of 32

1

SDS-PAGE, and Alexa488-labeled S1S2 was detected and analyzed by an in-gel

2

fluorescence imaging system (LAS4000). After fluorescence imaging, the gel was

3

stained by Imperial Protein Stain, a coomassie R-250 dye-based reagent (Themo

4

Scientific).

5 6

Fluorescent measurement of labeled S1S2

7

Fluorescent labeling of the S1S2 protein was conducted as described above with slight

8

midifications. In brief, 3 µM of S1S2 was incubated with 6 µM of CAM2(Ax488-5S for

9

48 h at 17 °C in 20 mM HEPES (pH 7.2) buffer containing 100 mM NaCl. The labeled

10

S1S2 was purified by removal of unlabeled dye and ligand moiety with dialysis using a

11

10 K Da cut-off membrane. Fluorescent measurement of labeled S1S2J was performed

12

on

13

Alexa488-S1S2J in HEPES buffer (20 mM HEPES, 100 mM NaCl (pH 7.4)), radio

14

isotope-labeled ligand binding buffer (50 mM Tris HCl, 100 mM KSCN, 2.5 mM CaCl2

15

(pH7.4)) or HBS (20 mM HEPES, 107 mM NaCl, 6 mM KCl, 2 mM CaCl2, 1.2 mM

16

MgSO4 and 11.5 mM glucose at pH 7.4) was titrated with each concentration of ligands

17

at 20 °C. Excitation wavelength was 480 nm.

18

constants, the change in fluorescence intensity was fitted by a single-site binding

19

equation (2) using Kaleida Graph (Synergy Software) where P0 and L0 represent the

20

initial concentrations of Alexa488-S1S2 and ligand, respectively.

21

∆F/F0 = {(∆Fmax/F0)/2P0}[(P0+L0+Kd)-{(P0+L0+ Kd)2 -4P0L0}1/2]

22

∆F denotes the difference in the fluorescence intensity of Alexa488-S1S2 between in

23

the absence (F0) and presence of ligand at each concentration (F). When all the

24

Alexa488-S1S2 forms the complex with ligand, ∆F is equal to ∆Fmax.

a

Perkin

Elmer

LS55

fluorescent/phosphorescent

spectrophotometer.

For determination of dissociation

(2)

25 26

Targeting Alex488-S1S2 on the cell surface

27

Recombinant

S1S2

protein

was labeled with

both CAM2(Ax488-5S)


and

Page 19 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1

Biotin-PEG4-NHS (Thermo Scientific). Three µM of S1S2 was incubated both with 6

2

µM of CAM2(Ax488-5S) and 30 µM of Biotin-PEG4-NHS for 24 h at 17 °C in 20 mM

3

HEPES buffer (pH 7.2) containing 100 mM NaCl. The biotynylated Alex488-S1S2 was

4

purified by removal of unreacted Biotin-PEG4-NHS, unlabeled dye, and ligand moiety

5

with dialysis using a 10 K Da cut-off membrane. The biotynylated Alex488-S1S2 was

6

immobilized on the surface of HEK293T cells according to the method previously

7

reported by Hirose and coworkers with slight modifications.16 Briefly, HEK293T cells

8

were incubated with 1 mM of Biotin-PEG4-NHS for 30 min at room temperature in

9

HBS (20 mM HEPES, 107 mM NaCl, 6 mM KCl, 2 mM CaCl2, 1.2 mM MgSO4 and

10

11.5 mM glucose at pH 7.4).

After washing with DMEM medium, cells were seeded

11

on a micro glass plates and incubated with 1 µM of streptavidin at room temperature for

12

10 min in HBS. The cells were washed with HBS and incubated with 1 µM of

13

biotynylated Alex488-S1S2 for 30 min. The cells were washed with HBS for removing

14

free Alex488-S1S2 and used for subsequent measurements.

15



1

ASSOCIATED CONTENT

2

A pdf file of Supporting Information, which contains Supporting Notes, Table S1

3

and Figures S1–S14, is available free of charge on the ACS Publications website at

4

http://pubs.acs.org.

5 6 7

ACKNOWLEDGEMENTS

8

We thank E. Gouaux (Oregon Health and Science University) for the S1S2 construct.

9

This work was funded by a Research Fellowship from the Japan Society for the

10

Promotion of Science (JSPS) for Young Scientists to S.W. (26-3123), SUNBOR Grant

11

from Suntory Foundation for Life Sciences to S.K., the Takeda Science Foundation to

12

S.K., Daiichi Sankyo Foundation of Life Science to S.K., and the Japan Science and

13

Technology Agency (JST) Core Research for Evolutional Science and Technology

14

(CREST) of Molecular Technologies to I.H.. This work was also supported by a

15

Grant-in-Aid for Scientific Research (B) to S.K. (JSPS KAKENHI Grant No.

16

JP16K03290), and on Innovative Areas “Chemistry for Multimolecular Crowding

17

Biosystems” to I.H. (JSPS KAKENHI Grant No. 17H06348).

18


Page 20 of 32

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Table 1 Previously reported Ki, EC50 and Kb values of agonists and antagonists and those Kd values determined in this study for full-length AMPARs (GluA2) or its S1S2. Previously reported (µM) Binding assay Ki values

This study (µM)

Function-based assay EC50 or Kb values 4)

Binding assay Kd values 5)

AMPAR

S1S2

GluA2

GluA2

S1S2

Non-cellular 1,2)

Test tube 1,3)

Live cell

Live cell

Test tube 1)

Glu

0.17–0.48

0.45

296–634

252 ± 14

1.67 ± 0.01

AMPA

0.012–0.025

0.025

11–66 6)

18.3 ± 2.3

0.023 ± 0.003

DNQX

0.45

0.55

1.66 6)

1.40 ± 0.39

1.86 ± 0.17

NBQX

0.39

–

0.31 6)

0.26 ± 0.03

1.23± 0.31

Agonist

Antagonist 1)

These values were evaluated using radioisotope-labeled ligand binding buffer. Ki values were determined using membrane fractions of cells expressed with GluA2. 3) When IC50 values were measured, we coverted them to Ki values with the equation: Ki = IC50 / (1 + [L]/KDL), where [L] and KDL are the concentration of radioligand and its Kd value, respectively. 4) EC50 values for agonists and apparent dissociation constants (Kb values) for antagonists are shown. 5) Protein labeling was performed using CAM2(Ax488-5S). 6) These values were for GluA3. Ligand binding properties for GluA3 are similar to that for GluA2 (ref. 1). 2)



Page 28 of 32

Figure 1 Chemical labeling of AMPARs with CAM2(Ax488-5S) under live cell condition. a) Schematic illustration of AMPAR labeling by LDAI chemistry. Lg, ligand moiety; FL; fluorophore; Nu, nucleophilic amino acid residue. b) Chemical structure of CAM2(Ax488-5S). c) Western blotting of AMPARs labeled with CAM2(Ax488-5S) in HEK293T cells. HEK293T cells transfected with GluA2 or vector control were treated with 2 µM CAM2(Ax488-5S) in the presence or absence of 50 µM NBQX in serum free DMEM. d) Confocal live imaging of Alexa488-labeled AMPARs before and after addition of 1 mM L-glutamate (Glu) in HEK293T cells transfected with GluA2. Scale bar, 10 µm.


Page 29 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2 Glutamate-induced fluorescence response of AMPARs labeled with CAM2(Ax488-5S) in live cells. Chemical labeling was conducted as described in Figure 1c. a) Schematic illustration of the perfusion system for quick and reversible exchange of extracellular solution for epifluorescence imaging. b) Glutamate-induced fluorescence responses for Alexa488-labeled AMPARs in live cells. An averaged time course of the fluorescent changes (F/F0) is shown. The bar indicates a period of 1 mM L-glutamate (Glu) addition. (n = 8). c) Maximum of F/F0 value after addition of various kinds of amino acids. [Amino acid] = 1 mM. (n = 7–9). Data points represent mean ± SEM. d) Reversibility of the glutamate-induced fluorescence responses. This data indicates that application of other amino acids does not affect glutamate-induced fluorescence responses. (n = 9). e) Repetition of glutamate-induced fluorescent responses with subtle loss of the fluorescent changes. The bar indicates a period of 1 mM L-glutamate addition. (n = 20).



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Figure 3 Effects of the orientation or the chemical structure of labeled fluorophores for AMPARs upon the ligand-induced fluorescence responses. a) Chemical structure of CAM2 derivatives. b) Effects of the linker length and substitution position (5-isomer or 6-isomer) on glutamate-induced fluorescent changes for Alexa488-labeled AMPARs. Maximum of F/F0 values after addition of 1 mM L-glutamate are shown. (n = 14–24). Averaged time courses of the fluorescent changes (F/F0) are shown in Figure S3. Chemical labeling was conducted by similar procedures as described in Figure 1c. c) Effects of the fluorophore structure on glutamate- or NBQX-induced fluorescent changes. Maximum of F/F0 values after addition of 1 mM L-glutamate or 10 µM NBQX are shown. (n = 8–10). Averaged time courses of the fluorescent changes (F/F0) are shown in Figure S6. Data points represent mean ± SEM.


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Figure 4 Determination of Kd values of various AMPAR ligands for full-length AMPARs in live cells or S1S2 in test tubes using with CAM2(Ax488-5S). a) Fluorescence imaging of glutamate-induced responses for Alexa488-AMPARs in live cells. An averaged time course of the fluorescent changes (F/F0) is shown. The bar indicates a period of adding each concentration of L-glutamate. Chemical labeling was conducted as described in Figure 1c. (n = 7). b) Fluorescent spectra of Alexa488-labeled S1S2 after titrating L-glutamate in radioisotope-labeled ligand binding buffer in cuvettes. [Alexa488-S1S2] = 0.1 µM. Excitation wavelength is 480 nm. c, d) Concentration-dependency of fluorescence responses for glutamate (in c) and DNQX (in d) for AMPAR in live cells (closed circle) or S1S2J in cuvettes (open circle). Raw data for DNQX were shown in Figure S8.



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Figure 5 Fluorescent responses of Alexa488-AMPARs after adding various ligands for glutamate receptors in live cells. a) Fluorescent imaging of Alexa488-AMPARs after adding a competitive antagonist (ZK200775) or an allosteric modulator (GYKI53655) in live cells. An averaged time course of the fluorescent changes (F/F0) is shown. The bar indicates a period of adding 1 mM glutamate, 30 µM ZK200775 or 30 µM GYKI53655 (n = 10). Chemical labeling was conducted as described in Figure 1c. b) Maximum of F/F0 values after addition of various ligands for glutamate receptors (n = 10). Chemical structures of these ligands are shown in Figure S12. c) Concentration-dependency of CTZ-induced fluorescence responses for Alexa488-AMPARs in the presence of 20 µM AMPA in live cells. Left, averaged time course. Right, a concentration dependent curve for CTZ. d) Fluorescent imaging of Alexa488-AMPARs after adding 1 mM L-glutamate or 0.1 mM Cl-HIBO, an GluA2-subtype selective agonist in live cells. An averaged time course of the fluorescent changes (F/F0) is shown. The bar indicates a period of adding 1 mM L-glutamate or 0.1 mM Cl-HIBO (n = 6–10).


Ligand-Directed Chemistry of AMPA Receptors Confers Live-Cell

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