MOL Manuscript #108886

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Supplemental Materials

Article title: Molecular basis of altered hERG1 channel gating induced by ginsenoside Rg3

Authors: Alison Gardner, Wei Wu, Steven Thomson, Eva-Maria Zangerl-Plessl, Anna Stary-

Weinzinger and Michael C. Sanguinetti

Journal: Molecular Pharmacology

Supplemental Fig. 1. Scanning mutagenesis of select residues located in the S5-pore region of

hERG1. The shift in V0.5 for activation (black bars) and fold-change in deact (grey bars) induced

by 3 µM ginsenoside Rg3 are plotted for wild-type (WT) and select mutant hERG1 channels as

indicated on x-axis (n indicated below V0.5 bars).

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Supplemental Fig. 2. Plot of the fold change in deact (logarithmic scale) as a function of deact (at

a Vret of −70 mV) under control conditions for all mutant channels examined in this study. The 4

mutant channels with a control deact > 1.4 s (see Suppl. Table 2) are not included. There was no

correlation between the control deact and the fold change in this parameter induced by Rg3

(linear regression adjusted R2 = 0.099).

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Supplemental Fig. 3. Plot of the fold change in Itail-peak induced by ginsenoside Rg3 as a function

of deact (at a Vret of −70 mV) under control conditions for all the mutant channels examined in

this study. Itail was fitted to an exponential function, extrapolated to the time point where Vt was

pulsed to −70 mV to estimate the initial value of Itail-peak. Most of the faster deactivating mutants

had a greater than average fold change in Itail-peak. Specifically, 9 of the 11 mutant channels with a

deact < 130 ms had a fold change in Itail-peak > 1.3 (indicated by vertical dashed line). WT channel

is indicated by red circle. Y420A/L452A and the two other outlying mutant channels are labeled.

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Supplemental Fig. 4. Surface representations of the top view (upper) and side view (lower) of

the VSD with the ginsenoside Rg3 shown as sticks in the activated state (A) and the activated1

state (B).

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Supplemental Fig. 5. Root mean square deviation (RMSD) plots of ginsenoside Rg3 over

simulation time. Rmsd of the two 50 ns runs in the activated model (black and blue) of the

activated state (A) and the activated1 model (B).

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Supplemental Fig. 6. Root mean square fluctuations (RMSF) of two 50 ns runs (black and blue)

for all residues of the hERG1 voltage sensor. Both states reveal relatively similar RMSF profiles,

with the loop regions being most flexible.

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Supplemental Fig. 7. Comparison of the VSDs from cryo-EM structures of hERG1 and rat

EAG1. Cartoon representation of the VS module of hERG1 (blue) and EAG1 (light orange).

The missing loops in the hERG1 structure are indicated by red dots. The overall structure of the

VSD is similar between EAG1 and hERG1, with an overall RMSD value of all atoms of 1.25 Å.

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Supplemental Table 1. Voltage dependence of activation for WT and mutant hERG1

channels. Normalized g/gmax-Vt plots were fitted with a Boltzmann function to determine the

half-point of activation (V0.5) and slope factor (k) for the relationships. Data represent the average

± S.D. determined before (control) and after treatment of oocytes with 3 µM ginsenoside Rg3

unless indicated otherwise (n = number of oocytes).

V0.5 (mV) k

Mutation Control S.D. 3µM Rg3 S.D. Control S.D. 3µM Rg3 S.D. n

None (WT) -31.8 1.3 -41.1 2.0 8.21 0.40 9.03 0.74 5

L416A -28.5 1.4 -41.7 1.7 6.81 0.14 6.94 0.35 3

L417A -5.7 6.9 -43.1 7.8 12.2 1.12 5.03 0.42 5

V418A -44.4 2.9 -68.6 2.6 5.98 1.20 6.86 0.21 3

I419A -14.2 2.2 -25.3 5.0 7.98 0.22 9.04 0.38 4

Y420A -7.0 5.7 -9.0 6.6 12.3 1.02 15.1 1.32 9

Y420A; 30 µM -7.0 5.0 -15.3 4.5 12.3 0.90 11.1 0.85 7

Y420A/L452A 5.2 6.7 7.6 6.0 10.9 1.95 10.6 1.16 14

Y420A/L452A; 30 µM 6.8 5.4 5.4 4.8 10.9 1.56 8.46 0.51 9

Y420A/I521A -38.2 4.2 -40.1 4.2 8.38 1.25 8.56 1.10 6

Y420A/I521A; 30 µM -38.2 2.9 -34.4 2.1 8.38 0.88 7.8 0.88 3

T421A 10.9 3.5 1.3 3.5 7.47 0.38 9.71 0.38 3

V423A -32.2 0.7 -38.8 0.9 7.45 0.29 8.36 0.16 3

F424A -29.7 2.2 -38.4 2.0 9.14 3.35 12.1 6.53 5

T425A -30.0 0.3 -39.2 1.0 6.21 0.12 7.61 0.71 3

Y427A -36.1 1.4 -49.3 2.0 6.19 0.58 6.34 0.62 4

S428A -31.6 2.6 -44.8 1.7 6.52 0.17 6.66 0.05 3

L432A -33.2 0.3 -45.0 1.0 6.74 0.81 6.80 0.57 3

L433A -24.4 2.9 -36.9 2.4 7.70 0.38 7.77 0.97 3

K434A -30.8 1.0 -45.9 0.6 7.32 0.58 7.37 0.56 4

4E2C/6A1 -34.0 0.9 -47.0 0.9 6.60 0.29 6.53 0.27 5

3E1P/4R2 -24.1 2.6 -38.7 1.9 7.10 0.21 7.14 0.23 3

T436A -28.4 2.9 -45.0 4.3 7.80 0.66 8.62 1.44 3

T443A -29.3 1.6 -45.8 2.3 7.20 0.24 7.55 1.06 3

Y447A -27.8 0.3 -39.7 1.0 7.00 0.29 7.29 0.12 3

Q450A -24.0 3.8 -37.0 4.2 8.62 1.13 8.73 0.71 3

L452A -8.0 4.2 -14.2 3.8 8.95 0.10 9.95 0.24 4

L452A; 30 µM -8.0 3.6 -13.2 2.3 8.95 0.09 9.22 0.28 3

V454A -20.4 1.0 -37.9 0.5 7.69 0.73 8.23 1.16 3

V455A -41.1 1.4 -49.8 1.4 7.56 0.73 7.79 1.47 3

D456A 0.4 3.1 -12.7 2.1 7.22 0.05 8.59 0.12 3

D460A -4.6 1.9 -29.9 2.6 9.61 0.69 11.9 2.10 3

F463A -17.4 0.3 -20.8 2.4 8.77 1.35 8.03 0.87 7

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F463A; 30 µM -17.4 0.2 -12.7 2.6 8.77 1.02 7.33 0.56 4

D466A -32 2.0 -49.1 2.7 9.70 0.85 6.47 0.60 5

F508A -23.3 0.9 -36.8 0.7 5.94 0.31 6.45 0.09 3

D509A 7.8 2.4 -8.7 2.0 10.3 0.73 8.77 1.05 6

L510A 11.7 6.5 -6.8 5.8 11.4 0.85 10.5 1.39 5

L511A -22.5 1.1 -39.3 2.2 7.25 0.38 7.16 0.31 5

I512A -25.5 1.1 -46.3 0.9 6.28 0.11 6.80 0.34 5

F513A -18.7 2.4 -29.4 2.8 7.52 0.54 7.79 1.00 4

G514A -32.2 2.0 -43.5 2.2 7.48 0.29 7.60 0.76 6

S515A -34.7 1.8 -44.5 1.8 7.11 0.27 7.45 0.54 9

G516A -35.8 2.0 -52.4 2.2 7.53 0.14 7.44 0.22 4

S517A -12.5 2.2 -26.4 1.8 8.38 1.50 7.78 1.07 5

E518A -4.5 2.7 -16.4 2.5 9.02 1.10 8.04 0.67 5

E519A -23.5 3.0 -37.1 3.3 6.18 0.80 6.59 0.73 11

L520A -23.4 2.4 -32.8 3.4 6.69 0.32 7.54 0.61 6

I521A -45.6 2.5 -43.6 3.4 6.67 1.27 7.97 1.90 8

I521A; 10 µM -39.6 2.9 -27.4 2.0 10.2 4.47 8.80 2.10 5

I521A; 30 µM -39.6 2.3 -29.2 4.2 10.2 3.46 9.60 2.30 3

G522A -42.7 1.3 -47.3 2.4 6.54 0.71 7.59 0.50 7

L523A -59.8 1.2 -72.3 0.9 8.55 0.59 11.2 1.16 3

L524A -21.4 2.0 -33.2 2.9 6.99 0.67 6.29 0.60 5

K525A -70.1 2.0 -69.0 3.1 9.13 1.12 9.22 1.81 5

K525A; 10 µM -75.7 11.9 -75.9 14.1 9.44 0.85 8.20 5.37 8

T526A -33.2 2.2 -40.9 2.4 6.73 0.12 7.15 0.16 4

A527V -52.2 1.8 -62.0 2.7 7.36 0.31 7.07 0.31 5

R528A -25.6 1.4 -43.6 2.4 10.1 0.58 9.32 1.18 4

L553A -30.7 1.7 -43.2 0.5 8.23 1.54 8.37 0.88 3

F557L -21.3 1.3 -33.4 1.8 10.6 0.58 9.97 0.65 5

I567A -14.4 1.0 -32.4 2.3 8.37 0.80 8.92 1.18 3

Y569A -19.0 3.4 -26.5 4.9 6.69 0.20 6.90 0.16 5

A570V -35.4 1.8 -44.4 0.6 7.00 0.10 7.52 0.28 4

N573A -34.1 1.2 -46.0 1.7 7.51 0.29 7.56 0.31 3

M574A -33.7 2.4 -45.5 3.1 7.73 0.48 8.11 0.81 3

E575A -29.5 2.0 -45.8 1.0 7.51 0.38 7.45 0.32 4

Q576C -29.9 0.9 -45.1 0.7 6.42 0.05 7.67 0.54 3

H578A -26.4 0.9 -42.6 2.0 6.32 0.38 6.65 0.31 5

D580A -31.9 1.9 -45.1 3.1 6.49 0.21 7.05 0.90 3

S581C -31.6 2.6 -47.2 2.6 6.58 0.48 6.38 0.48 4

R582C -28.3 0.7 -42.7 1.6 6.30 0.23 6.57 0.35 3

I583C -27.4 2.4 -40.5 2.4 7.37 0.24 7.46 0.55 3

H587A -29.4 1.2 -44.9 1.4 6.48 0.68 6.74 0.43 3

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D591A -29.4 0.9 -43.6 1.9 6.13 0.14 6.62 0.23 3

Q592C -30.0 0.2 -43.6 1.7 5.57 0.29 5.63 0.95 3

I593M -25.5 0.7 -39.2 2.2 6.84 0.54 7.61 1.42 6

N598C -40.0 3.8 -49.3 2.2 8.17 0.31 7.25 0.25 5

S600C -29.2 1.4 -43.6 1.0 7.2 0.76 7.60 1.22 4

L602C -29.4 2.9 -45.4 2.3 7.22 0.42 6.30 0.94 3

S606C -29.5 2.1 -45.5 1.9 6.05 0.35 5.97 0.48 3

I607C -24.5 0.3 -40.7 1.0 7.02 0.10 6.80 0.14 3

K608A -34.1 3.4 -50.5 2.6 6.65 0.84 6.85 1.34 4

D609A -26.7 1.0 -42.2 1.2 7.34 0.17 7.11 0.16 3

K610A -32.3 0.7 -46.8 1.7 6.57 0.16 6.67 0.21 3

Y611A -37.8 1.9 -53.3 2.4 6.94 0.33 6.75 0.19 3

F619A -43.3 2.2 -57.1 2.4 7.58 0.54 7.52 0.70 4

L622C -21.5 1.4 -36.9 2.6 6.19 0.14 6.09 0.12 4

G628C/S631C -27.0 2.5 -37.3 1.1 9.23 1.05 9.63 0.45 5

S631A -23.4 3.8 -36.4 3.4 6.79 0.76 7.12 0.66 4

S631C -30.4 1.6 -44.8 3.3 7.6 0.66 7.14 0.81 3

N658A -40.4 2.2 -54.0 1.8 5.49 0.66 6.40 0.22 4

V659A -50.9 2.2 -62.3 2.0 12.1 0.80 12.5 1.90 4 1 E435A/E437A/E438A/E444A/C445A/C449A; 2 E435R/E437R/E438R/P440R

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Supplemental Table 2. Effects of Rg3 on the maximum value of Itail-peak and time constant

of current deactivation (deact) for WT and mutant hERG1 channels. Data represent the

average ± S.D. determined before (control) and after treatment of oocytes with 3 µM ginsenoside

Rg3 unless indicated otherwise (n = number of oocytes).

Mutation

Maximum Itail-peak deact (in ms) at −70 mV

fold-change S.D. Control S.D. 3µM Rg3 S.D. n

None (WT) 1.10 0.04 685 94 3441 1158 5

L416A 1.06 0.10 998 94 9462 4694 3

L417A 1.27 0.16 43 13 696 336 7

V418A 1.27 0.05 327 102 3785 1112 3

I419A 1.08 0.06 369 52 7447 3450 4

Y420A 1.02 0.03 432 99 1886 306 9

Y420A; 30 µM 0.95 0.03 432 87 1694 275 7

Y420A/L452A 1.35 0.07 73 30 83 15 14

Y420A/L452A; 30 µM 1.31 0.09 73 24 90 36 9

Y420A/I521A 1.10 0.05 523 100 1395 171 6

Y420A/I521A; 30 µM 0.98 0.07 523 71 747 59 3

T421A 1.52 0.07 70 9 158 23 3

V423A 1.11 0.03 1132 118 4340 1353 3

F424A 1.05 0.09 607 282 2712 1440 5

T425A 1.03 0.07 175 23 1967 362 3

Y427A 0.97 0.12 247 20 3958 3460 4

S428A 1.08 0.03 804 253 5968 2120 3

L432A 1.06 0.03 555 215 3812 1131 3

L433A 1.14 0.03 443 88 2859 941 3

K434A 1.11 0.02 847 158 4339 1268 4

4E2C/6A1 0.99 0.07 734 74 2674 411 5

3E1P/4R2 1.05 0.02 208 42 2787 693 3

T436A 1.18 0.07 978 68 2713 573 3

T443A 1.08 0.05 1049 187 4850 1526 3

Y447A 1.14 0.03 730 50 3122 262 3

Q450A 1.16 0.03 958 355 8638 1093 3

L452A 1.23 0.10 478 36 2222 188 4

L452A; 30 µM 1.16 0.07 478 31 3086 346 3

V454A 1.17 0.03 511 57 6902 2262 3

V455A 1.08 0.03 875 61 2835 480 3

D456A 1.79 0.23 57 2 191 59 3

D460A 1.60 0.07 67 12 288 78 3

F463A 1.65 0.19 62 13 150 26 7

F463A; 30 µM 1.24 0.20 62 10 174 8 4

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D466A 1.39 0.11 59 4 249 45 5

F508A 1.11 0.03 230 45 2246 61 3

D509A 1.78 0.15 37 7 63 12 6

L510A 1.69 0.18 31 7 48 9 5

L511A 1.27 0.09 156 9 2024 114 5

I512A 1.10 0.16 1627 221 15550 5288 5

F513A 1.19 0.04 257 74 1144 190 4

G514A 1.09 0.05 2123 385 4840 3037 6

S515A 1.09 0.06 1249 288 7361 3540 9

G516A 1.10 0.02 1977 172 2471 536 4

S517A 1.08 0.07 302 65 2410 387 5

E518A 1.07 0.11 496 94 3638 313 5

E519A 1.07 0.07 248 90 2380 886 11

L520A 1.01 0.02 455 81 2912 637 6

I521A 1.07 0.11 734 266 2222 707 8

I521A; 10 µM 0.98 0.04 683 110 1703 255 5

I521A; 30 µM 0.91 0.10 683 85 1717 204 3

G522A 1.06 0.03 511 135 4858 1029 7

L523A 1.01 0.03 278 50 965 83 3

L524A 1.15 0.11 1450 563 5291 946 5

K525A 1.03 0.11 189* 18 521* 134 5

K525A; 10 µM 0.88 0.06 287* 11 445* 110 8

T526A 1.15 0.02 314 22 2209 300 4

A527V 1.13 0.09 1268 96 5821 5264 5

R528A 1.18 0.00 2611 172 4421 410 4

L553A 0.98 0.09 618 69 6218 904 3

F557L 1.06 0.16 572 264 5895 3517 5

I567A 1.11 0.02 497 64 8990 4228 3

Y569A 0.79 0.16 104 25 1170 436 5

A570V 1.13 0.04 1014 90 4968 1176 4

N573A 1.08 0.02 730 28 2264 752 3

M574A 1.19 0.02 614 45 3024 1375 3

E575A 1.18 0.12 904 68 3535 1952 4

Q576C 1.12 0.05 764 71 4094 1891 3

H578A 1.06 0.04 1317 127 6617 3935 5

D580A 0.99 0.09 691 92 4071 580 3

S581C 1.01 0.04 1140 118 ND 4

R582C 0.95 0.10 956 31 5839 475 3

I583C 1.11 0.07 749 71 2325 168 3

H587A 1.17 0.03 927 83 3270 580 3

D591A 1.16 0.03 658 35 4944 1032 3

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Q592C 1.17 0.03 888 182 4276 618 3

I593M 1.02 0.05 573 44 6679 980 6

N598C 0.97 0.07 677 293 2427 651 5

S600C 1.11 0.06 938 86 3462 1388 4

L602C 1.06 0.05 934 249 4709 4727 3

S606C 0.87 0.10 671 43 3871 1005 3

I607C 0.93 0.05 512 40 2451 223 3

K608A 0.93 0.06 550 104 2913 808 4

D609A 1.19 0.01 1019 50 4074 466 3

K610A 1.08 0.05 820 118 3016 615 3

Y611A 0.92 0.19 2625 59 13438 3532 3

F619A 1.95 0.14 1352 232 ND 4

L622C 1.02 0.02 748 120 4024 188 4

G628C/S631C 1.06 0.01 431 103 1374 246 5

S631A 1.21 0.08 527 146 7757 3906 4

S631C 1.09 0.29 1300 173 3668 1455 3

N658A 1.94 0.28 128 44 2705 308 4

V659A 1.14 0.20 859 306 ND 426 4 1E435A/E437A/E438A/E444A/C445A/C449A; 2E435R/E437R/E438R/P440R *Itail measured at −120 mV. ND, not determined (deactivation too slow to measure accurately)

Supplemental Table 3. Hydrogen bond contacts to Rg3 (% over simulation time)

activated activated1 run1 run2 run1 run2

Y420 - - - - T421 - 2,5 - - T425 0,5 7,2 - - S428 49, 6 8,1 30,6 13,5 E435 4,6 0,4 72,5 76,4 D460 - - - - D509 - - - - K525 0,8 9,78 50,4 - R528 1,3 43,65 61,0 62,5 Lipid 1 42,1 33,5 71,7 71,9 Lipid 2 19,3 28,4 44,7 32,9