Table 1 The Functional Effects of Alanine Mutations of Residues located at the M4-M1/M3 Interface of GLIC.

From: Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function

GLICa (WT EC50 = 9.33 ± 1.7 µM (pH50 = 5.03 ± 0.08) n = 38)

Single Alanine Mutations

Aromatic

Aliphatic

TMD α-helix

Mutant

EC50 (pH50) (µM)

n

TMD α-helix

Mutant

EC50 (pH50) (µM)

n

M1

F210A

10.5 ± 1.6 (4.98 ± 0.07)e

8

M1

I198A

NCb

10

W213A

NCb

17

I202A

25.7 ± 3.3 (4.59 ± 0.06)e

8

F216A

35.5 ± 3.1 (4.45 ± 0.04)e

8

L203A

1.6 ± 0.2 (5.79 ± 0.05)e

8

W217A

66.1 ± 3.0 (4.18 ± 0.02)e

8

M205A

6.3 ± 0.4 (5.20 ± 0.03)e

8

M3

Y254A

26.9 ± 1.8 (4.57 ± 0.03)e

8

L206A

6.2 ± 0.4 (5.21 ± 0.03)e

8

F265A

47.9 ± 1.1 (4.32 ± 0.01)e

8

L209A

4.9 ± 0.3 (5.31 ± 0.03)e

8

M4

F299A

9.1 ± 1.7 (5.04 ± 0.08)c

6

M3

I258A

6.5 ± 0.8 (5.19 ± 0.05)e

8

F303A

33.1 ± 6.2 (4.48 ± 0.08)c,e

8

M261A

5.4 ± 0.5 (5.27 ± 0.04)e

8

F314A

33.1 ± 1.5 (4.48 ± 0.02)d,e

10

I262A

NCb

10

F315A

20.4 ± 0.5 (4.69 0.01)d,e

8

V268A

2.6 ± 0.2 (5.59 ± 0.03)e

8

Charged

V275A

2.2 ± 0.1 (5.65 ± 0.02)e

8

M3

E272A

NCb

10

L279A

7.2 ± 0.9 (5.14 ± 0.05)e

8

K280A

15.1 ± 2.5 (4.82 ± 0.07)e

8

M4

I291A

16.6 ± 1.8 (4.78 ± 0.05)c,e

7

M4

R296A

22.4 ± 1.5 (4.65 ± 0.03)c,e

9

P300A

NCc,b

8

Hydrogen Bonding

V302A

15.5 ± 1.4 (4.81 ± 0.04)c,e

7

M3

Q276A

36.3 ± 3.2 (4.44 ± 0.04)e

8

L310A

33.9 ± 1.5 (4.47 ± 0.02)c,e

6

M4

T292A

13.8 ± 1.5 (4.86 ± 0.05)c,e

6

    

S295A

7.6 ± 1.6 (5.12 ± 0.09)c,f

6

    

N307A

38.0 ± 0.9 (4.42 ± 0.01)c,e

10

    

Double Alanine Mutations

Outer Leaflet Cluster

Inner Leaflet Cluster

 Mutant

EC50 (µM) (pH50)

n

Mutant

EC50 (µM) (pH50)

n

  Y254F

7.24 ± 0.8 (5.14 ± 0.05)

8

F210A + F303A

55.0 ± 11.5 (4.26 ± 0.09)

8

  Y254F + N307A

27.5 ± 6.4 (4.56 ± 0.10)

8

F216A + F299A

NCb

10

  Y254A + N307A

40.7 ± 9.4 (4.39 ± 0.10)

8

W217A + R296A

40.7 ± 9.5 (4.39 ± 0.10)

8

  Y254A + F314A

NCb

12

F265A + F299A

NCb

10

  Y254A + F315A

NCb

11

F265A + F303A

NCb

10

  F314A + F315A

NCb

11

Q276A + T292A

9.55 ± 1.8 (5.02 ± 0.08)

8

    

F299A + F303A

NCb

10

  1. aData collected 1–4 day(s) after cRNA injection (VHold ranging from −20 to −40 mV). Error Values represent the standard deviation.
  2. bNo current (NC) observed in oocytes after 1–4 day(s) of cRNA injection.
  3. cData taken from Henault et al.9, 18, 29.
  4. dData taken from Carswell et al.23, 24.
  5. e p < 0.001 relative to the pH50 of WT GLIC via one-way ANOVA followed by Dunnet’s post hoc test.
  6. f p < 0.01 relative to the pH50 of WT GLIC via one-way ANOVA followed by Dunnet’s post hoc test.
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