Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels
- National Institutes of Health, United States
- Gwangju Institute of Science and Technology, Republic of Korea
- University of Missouri, United States
- University of California, Davis, United States
Figures
Figure 1
Structural comparison between tarantula toxins targeting ASIC and Kv channels.
(A) Crystal structure of PcTx1 bound to the ASIC1a channel (left, PDB 4FZ0) and of the Kv1.2-Kv2.1 paddle chimera channel (right, PDB 2R9R) viewed from within the membrane. Voltage-sensor paddles …
Figure 2
Interaction of GxTx-1E(Nle) and PcTx1 with lipid vesicles detected with intrinsic Trp fluorescence.
(A, B) Fluorescence emission spectra of GxTx-1E(Nle) and PcTx1 in the absence (black) or presence of lipid vesicles composed of a 1:1 mix of POPC:POPG (blue). Lipid concentration was 1.0 mM. (C, D) …
Figure 3
Interaction of GxTx-1E(Nle) and PcTx1 with lipid vesicles using acrylamide dequenching and quenching with brominated lipids.
(A, B) Fluorescence emission spectra of GxTx-1E(Nle) and PcTx1 in the absence (black) or presence of lipid vesicles composed of 1:1 mix of POPC:POPG (blue) in a solution containing 0.2 M acrylamide. …
Figure 4 with 1 supplement
Determination of apparent affinity for mutants of GxTx-1E.
(A) Families of macroscopic ionic currents elicited by test depolarizations before (black) and after (red) addition of 454 nM GxTx-1E(Nle) for an oocyte expressing the Kv2.1 channel. (B) G-V …
Figure 4—figure supplement 1
Influence of PcTx1 on chimeras between Kv2.1 and ASIC1a.
(A) Sequence alignment of S3 helices in Kv2.1 (blue) replaced with helix-5 from ASIC1a (red) in the chimeric constructs. (B–G) Normalized G-V relations in the absence (black circles) or presence …
Figure 5 with 3 supplements
Comparison of residues on PcTx1 and GxTx-1E involved in receptor binding.
(A) GxTx-1E residues colored by perturbation in apparent affinity (ΔΔGo in kcal mol−1). Several side-chains with the largest perturbation energies are labeled. P16 was not colored since its mutation …
Figure 5—figure supplement 1
Interaction of tryptophan mutants of GxTx-1E(Nle) with lipid vesicles detected with intrinsic Trp fluorescence.
(A–D) Fluorescence emission spectra of GxTx-1E(Nle), W8A, W9A and W28A in the absence (black) or presence of lipid vesicles composed of a 1:1 mix of POPC:POPG (blue). The lipid concentration was 1.0 …
Figure 5—figure supplement 2
Depth-dependent quenching of tryptophan fluorescence by brominated (diBr) phosphatidylcholines.
(A–D) Fluorescence emission spectra of GxTx-1E(Nle) W8A, W9A and W28A in the absence (black) or presence of unlabeled (gray) or brominated (blue) lipids present at a concentration of 1.2 mM.
Figure 5—figure supplement 3
Comparison of the effects of GxTx-1E and SGTx1 mutation on apparent affinity and membrane partitioning.
(A) Sequence alignment between GxTx-1E and SGTx1. Conserved residues are highlighted in yellow. (B) GxTx-1E residues colored by perturbation in apparent affinity (ΔΔGo). (C and D) SGTx1 residues …
Figure 6 with 2 supplements
Model of GxTx-1E bound to the S3b helix of the Kv1.2/2.1 paddle chimera.
The preferred model of GxTx-1E bound to the X-ray structure of the voltage-sensing domain of the Kv1.2/2.1 paddle chimera generated as described in ‘Materials and methods’. The model maintains a …
Figure 6—figure supplement 1
Model structures of GxTx-1E bound to the voltage-sensing domain of a Kv channel.
(A) Side chains of GxTx-1E (left; PDB 2WH9) and the voltage-sensing domain of the Kv1.2/Kv2.1 paddle chimera (right; PDB 2R9R) were colored by free energy perturbation values obtained by alanine …
Figure 6—figure supplement 2
Effects of GxTx-1E and Kv2.1 mutations on the energetics of toxin-channel interactions.
(A) Perturbations in the free energy of GxTx-1E interactions with the Kv2.1 channel assessed from alanine scans of the toxin (Table 1) and channel (Milescu et al., 2009). Gray circles in the results …
Tables
Table 1
Affinities and perturbation energies for mutants of GxTx-1E
| Toxin | Kd (nM) | Kdmut/Kdwt | ∆∆G (kcal mol−1) |
|---|---|---|---|
| Gxtx-1E(Nle) | 224 ± 25 | 1.0 | – |
| E1A | 414 ± 38 | 1.8 | 0.36 |
| G2A | 297 ± 50 | 1.3 | 0.17 |
| E3A | 125 ± 62 | 0.6 | −0.35 |
| G5A | 13,652 ± 320 | 60.9 | 2.44 |
| G6A | 379 ± 51 | 1.7 | 0.31 |
| F7A | 89,033 ± 9413 | 397.4 | 3.55 |
| W8A | 9641 ± 504 | 43.0 | 2.23 |
| W9A | 5837 ± 1850 | 26.1 | 1.93 |
| K10A | 984 ± 90 | 4.4 | 0.88 |
| G12A | 21,755 ± 1086 | 97.1 | 2.71 |
| S13A | 545 ± 56 | 2.4 | 0.53 |
| G14A | 461 ± 89 | 2.1 | 0.43 |
| K15A | 823 ± 127 | 3.7 | 0.77 |
| P16A | 22,967 ± 2812 | 102.5 | 2.75 |
| P20A | 652 ± 73 | 2.9 | 0.63 |
| K21A | 440 ± 82 | 2.0 | 0.40 |
| Y22A | 35,337 ± 7464 | 157.7 | 3.00 |
| V23A | 516 ± 60 | 2.3 | 0.50 |
| S25A | 1169 ± 64 | 5.2 | 0.98 |
| P26A | 813 ± 99 | 3.6 | 0.76 |
| K27A | 763 ± 49 | 3.4 | 0.73 |
| W28A | 44,298 ± 2471 | 197.7 | 3.14 |
| L30A | 2474 ± 278 | 11.0 | 1.42 |
| N32A | 1228 ± 166 | 5.5 | 1.01 |
| F33A | 491 ± 18 | 2.2 | 0.47 |
| P34A | 348 ± 60 | 1.6 | 0.26 |
| Nle35A | 306 ± 19.8 | 1.4 | 0.19 |
| P36A | 250 ± 22 | 1.1 | 0.07 |
