Supplemental Data for:

A triad of residues is functionally transferrable between 5-HT3 serotonin receptors and nicotinic acetylcholine receptors

Richard Mosesso, Dennis A. Dougherty

Division of Chemistry and Chemical Engineering

California Institute of Technology, Pasadena, CA 91125 USA

To whom correspondence should be addressed: Prof. Dennis A. Dougherty, Department of Chemistry and Chemical Engineering, California Institute of Technology, Crellin 359, 1200 E. California Blvd, Pasadena, CA 91125, Telephone: (626) 395-6089; FAX: (626) 564-9297; Email: dadoc@caltech.edu

*Running title: Transferrable Residues in Serotonin and Nicotinic Receptors

Table S1

Response to 5-HT of 5-HT3ARs incorporating GABAAR-type mutations.

Mutation(s)

EC50 (µM)

nH

|Imax| (μA)

Fold

N

ΔΔG†

-

1.3 ± 0.03

2.4 ± 0.11

1.0 - 78

1.0

21

-

T152E

13 ± 0.37

1.8 ± 0.08

0.25 - 3.6

10

15

-

E209R

NR

NR

NR

NR

12

NR

K211S

1.3 ± 0.05

2.1 ± 0.14

5.0 - 47

1.1

15

-

T152E E209R

NR

NR

NR

NR

8

NR

T152E K211S

NE

NE

NE

NE

0

NE

E209R K211S

NR

NR

NR

NR

12

NR

T152E E209R K211S

NR

NR

NR

NR

4

NR

†kcal•mol-1; NR = no response; NE = not established; ± indicates SEM.

Figure S1. Two-electrode voltage clamp traces for wild-type and mutant 5-HT3ARs. Four doses are shown for each variant: 1) a low dose of 5-HT evoking no response, 2) an intermediate dose below the EC50, 3) an intermediate dose near or above the EC50, 4) a dose evoking a maximal response. Mutations typically did not have dramatic effects on receptor activation/deactivation parameters, although all mutants display less apparent desensitization than wild-type at their respective maximal responses.

Figure S2. Dose-response curves and TEVC traces for wild-type and triple mutant T152K E209D K211T (ABC) 5-HT3ARs, showing data beyond [5-HT] which evoke maximal responses. At comparable levels of rundown, currents from wild-type 5-HT3ARs are limited by desensitization, while currents from ABC 5-HT3ARs suffer from open channel block (evident in the spike in current immediately following washout of 5-HT). Macroscopic activation and deactivation kinetics are slower in ACB 5-HT3ARs compared to wild-type. Error bars represent SEM.

Figure S3. Two-electrode voltage clamp traces for wild-type and mutant muscle-type nAChRs. Four doses are shown for each variant: 1) a low dose of ACh evoking no response, 2) an intermediate dose below the EC50, 3) an intermediate dose near or above the EC50, 4) a dose evoking a maximal response. Mutations typically did not have dramatic effects on receptor activation/deactivation parameters, although some displayed altered desensitization at high concentrations of ACh.

Figure S4. Dose-response curves for muscle-type nAChR mutants. A-G) Dose-response curves comparing wild-type to K145T, D200E, T202K, D200E T202K, T202K K145T, D200E K145T, and K145T D200E T202K, respectively. All variants display an increase in EC50 relative to wild-type, but meaningful non-additivity is observed for double and triple mutants. Error bars are SEM.

Figure S5. Mutant cycles based on nicotine Kd. Every pair of mutations couples meaningfully, demonstrating cooperativity between these residues in binding nicotine. EC50 values (M) are provided in parentheses.

Figure S6. Noncompetitive inhibition by 5-HT of ACh-evoked currents in the muscle-type nAChR. Minimal changes occur in ACh EC50 while maximal currents are sharply reduced at high [5-HT]. Each dose-response curve was performed on different oocytes from the same batch of oocytes injected with the same amount of mRNA from the same preparation of mRNA. The first four (lowest-concentration ACh) data points on the 1000µM 5-HT curve were never actually measured, but inferred for the purpose of fitting the Hill equation. Error bars represent SEM.

Figure S7. Possible interactions between residues aligning to those discussed in this study in various crystal structures. Interaction distances between heavy atoms >4.0Å were excluded from these images. Yellow dashed lines indicate interactions involving main chain amides; green dashed lines indicate side chain interactions or water-mediated side chain interactions. Residue numbers are assigned as in the crystal structure. Carbon atoms of “triad” residues are highlighted in yellow.

Figure S8. Dose-response curves examining coupling of positive charge-ablating K211 mutations to nearby mutations which introduce a net positive charge. A, Mutant cycles with N101K. B, Mutant cycles with E102Q. C, Mutant cycles with E209Q. D, Mutant cycles with T152K. Error bars represent SEM.

S-10

Table S2

Mutation(s)Slope

R

2

K

d

(µM)Fold

DD

G (kcal•mol-1)

wild-type0.94

±

0.070.9712

±

3.41.0-

T152K1.0

±

0.170.9318

±

4.71.5-

E209D1.3

±

0.180.96160

±

84.0013-

K211T1.07

±

0.160.9431

±

8.22.5-

T152K K211T1.1

±

0.04>0.995.7

±

0.850.461.1

E209D K211T0.78

±

0.150.9091

±

497.40.60

T152K E209D K211T0.88

±

0.060.982.6

±

0.780.211.2*

*measured in the background of K211T

Schild fit parameters for mouse muscle-type nAChR variants.

Table S3

Mutation(s)[Nicotine] (µM)EC

50

(µM)n

H

|Imax| (µA)FoldN

wt01.3±0.032.4±0.111.0 - 781.021

wt2.51.5±0.024.0±0.130.68 - 7.41.214

wt7.52.3±0.065.0±0.610.99 - 4.81.88

wt253.7±0.063.0±0.120.50 - 4.42.914

wt757.6±0.222.5±0.161.1 - 106.015

wt25015±0.383.1±0.201.7 - 4.4127

wt35024±0.772.6±0.200.20 - 3.1196

wt1000143±5.93.3±0.380.41 - 2.81106

T152K049±1.01.9±0.060.14 - 111.015

T152K7.571±1.72.1±0.081.9 - 7.01.56

T152K1595±2.52.1±0.093.0 - 171.98

T152K2592±2.32.4±0.110.59 - 5.91.97

T152K50219±132.4±0.280.81 - 5.64.58

T152K75284±9.12.4±0.160.60 - 115.810

E209D 013±0.212.2±0.070.53 - 201.015

E209D 5016±0.292.6±0.100.65 - 111.38

E209D 7529±1.01.8±0.102.1 - 2.92.22

E209D 25031±1.12.7±0.210.41 - 3.22.47

E209D 35058±1.42.4±0.121.4 - 4.24.53

K211T01.5±0.042.5±0.161.9 - 251.015

K211T252.7±0.052.8±0.146.0 - 171.87

K211T505.6±0.122.3±0.101.9 - 4.73.88

K211T755.1±0.072.7±0.093.6 - 123.55

K211T25014±0.302.6±0.130.30 - 4.19.48

K211T35021±0.722.5±0.181.7 - 6.1145

T152K K211T02.5±0.052.1±0.081.3 - 561.014

T152K K211T2.53.7±0.112.7±0.180.84 - 4.81.58

T152K K211T7.55.6±0.112.0±0.079.7 - 382.24

T152K K211T2514±0.322.9±0.150.15 - 585.67

T152K K211T7545±1.91.8±0.1222 - 46184

T152K K211T250198±3.02.7±0.108.8 - 61797

E209D K211T06.9±0.252.1±0.110.18 - 121.016

E209D K211T258.9±0.501.7±0.135.6 - 191.36

E209D K211T5013±0.411.9±0.091.5 - 5.71.98

E209D K211T7513±0.412.0±0.104.5 - 161.86

E209D K211T25018±0.642.3±0.160.74 - 3.12.67

E209D K211T35031±1.82.4±0.290.58 - 2.524.55

T152K E209D K211T 011±0.392.4±0.180.15 - 191.012

T152K E209D K211T 0.2512±0.172.2±0.194.4 - 131.26

T152K E209D K211T 2.520±0.411.9±0.141.4 - 5.31.97

T152K E209D K211T 7.529±1.21.9±0.121.6 - 162.77

T152K E209D K211T 2593±3.82.9±0.180.80 - 2.98.97

T152K E209D K211T 75267±152.8±0.310.33 - 2.67256

Response to 5-HT of 5-HT

3A

Rs in the presence of varying concentrations of (-)-nicotine.