Grutter et al. 10.1073/pnas.0509024102.

Supporting Information

Files in this Data Supplement:

Supporting Materials and Methods
Supporting Figure 4
Supporting Table 1
Supporting Figure 5
Supporting Figure 6
Supporting Figure 7
Supporting Movie 1
Supporting Movie 2
Supporting Figure 8




Supporting Figure 4

Fig. 4. Expression in HEK cells of the a7/Gly chimeras. (a) Molecular model of Torpedo nAChR (9). (b) Sequence alignment between nAChRs and GlyRs. Numerals refer to chick a7 sequence. (c) Shown is 4.5 nM a-125I-BgTx binding on intact cells in the presence (black bars) or absence (white bars) of 1 mM nicotine for a7(205)/Gly, a7(206)/Gly, and a7(210)/Gly chimeras fused after residues R205, R206, and Y210, respectively. (d) Immunofluorescence localization of a7/Gly chimeras. Transfected cells were assayed for cell-surface expression or for total expression (permeabilized in the presence of 0.1% Triton X-100) of HA-tagged chimeras.





Supporting Figure 5

Fig. 5. Amino acid sequence alignments of nAChR, GlyR, and GABAA receptor subunits. The sequences shown represent the Cys-loop and M2-M3-loop (2-3L). Numbers refer to mature a7 nAChR. Also indicated under sequence alignment is the secondary structure.





Supporting Figure 6

Fig. 6. Allosteric scheme for a three-state model. Shown are B (channel closed), A (open), and D (desensitized) states.





Supporting Figure 7

Fig. 7. Bar plot comparing rate constants k of a7/Gly, a7(Cys-L)/Gly, and microchimeras (C1 to C11). kopen values are indicated, except for C7, C8, C10, and C11 mutants. Because, for these mutants, kapp was not concentration-dependent, kclosed are indicated (dashed bars). The sequence of the Cys-loop is indicated, with residues from a7 nAChR in gray. Numbers refer to mature a7 nAChR.





Fig. 8. Snapshots of the gating mechanism of a7 nAChR (6). All frames of Movie 1 are superimposed on the same structure. The color code is the same as for Fig. 3d.





Supporting Movie 1

Movie 1. This movie shows the gating mechanism of a7 nAChR previously proposed (6), with the residues identified in the current work highlighted by the same color code as in Fig. 3d. At the beginning of the movie, the channel is closed (QuickTime, 2.8 MB).





Supporting Movie 2

Movie 2. The same as in Movie 1 at the level of one subunit (QuickTime, 1.3MB).





Table 1. Summary of whole-cell dose-response and kinetics data for chimeric receptors

Constructs

EC50, mM

n

H

k

open, s-1

k

closed, s-1

rEC50, mM

No. of cells

a

7/Gly

300 ± 140

1.1 ± 0.2

2.3 ± 0.6

0.20 ± 0.07

460 ± 80

5

a

7(Cys-L)/Gly

240 ± 80

2.3 ± 0.9**

16.4 ± 3.9**

1.77 ± 0.68**

470 ± 210

13

C1

46 ± 8*

2.8 ± 1.3*

28.1 ± 9.4**

4.84 ± 2.04**

190 ± 60*

6

C2

1,570 ± 900*

1.6 ± 0.4*

0.18 ± 0.10*

0.04 ± 0.01*

610 ± 250

4

C3

560 ± 375

1.4 ± 0.3

1.7 ± 0.4

0.06 ± 0.02

480 ± 40

3

C4

1,080 ± 380*

1.3 ± 0.3

0.18 ± 0.08*

0.05 ± 0.02

840 ± 45*

2

C5

80 ± 20

0.9 ± 0.4

4.4 ± 1.1

1.1 ± 0.4*

580 ± 200

4

C6

350 ± 110

1.2 ± 0.4

16.1 ± 2.9*

8.9 ± 2.7*

460 ± 350

4

C7

200 ± 16

1.3 ± 0.2

ND

0.66 ± 0.14*

ND

3

C8

3,160 ± 590*

1.2 ± 0.3

ND

1.25 ± 0.22*

ND

4

C9

470 ± 190

1.4 ± 0.4

26.7 ± 3.4**

10.5 ± 2.9**

360 ± 120

6

C10

63 ± 25*

1.9 ± 0.6

ND

0.76 ± 0.23**

ND

6

C11

1,760 ± 410*

1.7 ± 0.3*

ND

0.55 ± 0.04**

ND

6

a

7/(2-3L)Gly

140 ± 50

1.3 ± 0.6

10.3 ± 2.2**

0.75 ± 0.14**

930 ± 200*

5

a

1 GlyR

28 ± 4

1.1 ± 0.1

430 ± 90†

20 ± 11

90 ± 25

4

a

1 L142V/N144W GlyR

650 ± 130***

2.4 ± 0.9***

60 ± 3***

2.3 ± 0.6***

850 ± 210***

5

Values are means ± SD. *, P < 0.05 and **, P < 0.01 when compared with a7/Gly; ***, P < 0.05 when compared with WT GlyR. ND, Because kapp was not concentration-dependent (see Supporting Materials and Methods), kopen and rEC50 cannot be determined.

†

For WT GlyR, kopen values might be underestimated because the activation on-rates at high glycine concentrations approach the time resolution limit of the perfusion system (≈1,000 s-1).