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Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin.

Bae C, Anselmi C, Kalia J, Jara-Oseguera A, Schwieters CD, Krepkiy D, Won Lee C, Kim EH, Kim JI, Faraldo-Gómez JD, Swartz KJ - Elife (2016)

Bottom Line: We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes.Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation.Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States.

ABSTRACT
Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve the structure of double-knot toxin (DkTx), a tarantula toxin that activates the heat-activated TRPV1 channel. We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes. We find that DkTx binds to the outer edge of the external pore of TRPV1 in a counterclockwise configuration, using a limited protein-protein interface and inserting hydrophobic residues into the bilayer. We also show that DkTx partitions naturally into membranes, with the two lobes exhibiting opposing energetics for membrane partitioning and channel activation. Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation. Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.

No MeSH data available.


Related in: MedlinePlus

Validation of the proposed structural model of the TRPV1-DkTx complex.Close-up of K1 (A) and K2 (B) in the top-ranking ROSETTA model of DkTx bound to TRPV1. Side chains of TRPV1 residues where mutation disrupts DkTx-induced channel activation (I599, F649, A657 and F659) (Bohlen et al., 2010) are highlighted in purple. (C) Representative time courses of TRPV1 activation at +120 mV by either DkTx (left panel), K1K1 (middle panel) or K2K2 (right panel) measured from a train of voltage-ramps in the whole-cell configuration with the TRPV1 expressed in HEK cells. In the case of K1K1, toxin was reapplied to verify that the observed decrease in current upon removal of the toxin was due to toxin dissociation from the channel rather than desensitization. Voltage ramps were from −120 to +140 mV over 1 s and were applied every 3 s from a holding potential of −90 mV. The colored horizontal lines denote the application of toxins. The dotted gray line denotes the zero-current level. Fractional dissociation 1 min after toxin removal was quantified across cells as the current amplitude 1 min after toxin removal divided by the steady-state current amplitude in the presence of the toxin. Fractional dissociation values (mean ± sem) were 0.8 ± 0.05 (n = 8) for DkTx, 0.3 ± 0.04 (n = 5) for K1K1 and 0.96 ± 0.03 for K2K2 (n = 6). (D) Mean normalized I-V relations obtained from voltage ramps at steady-state for control solution (gray), toxins (yellow, green or blue) and saturating capsaicin (10 µM; black) in the whole-cell configuration using transiently transfected HEK293 cells. The thin colored curves represent the mean and the thicker envelope the standard error (n = 5–6 for each panel). (E) Primary sequence of K1 without linker (K1-ΔL), K2 and gain-of-function chimeras between K1 and K2. Residues of K1-ΔL and K2 are shown in green and cyan, respectively, and cysteines are shown in yellow. Loops between cysteine residues are labeled as L1, L2, L3 or L4. CT denotes C-terminal region. (F) Concentration-dependence for activation of TRPV1 by K1, K2 and chimeras in two-electrode voltage clamp recordings of oocytes expressing TRPV1. Activation of the full-length TRPV1 channel by the toxin at different concentrations was measured in oocytes at a holding voltage of −60 mV. Toxin-induced currents (ITx) were normalized against the current activated by a saturating concentration of capsaicin (Icap at 10 μM), in the same cell. Note that the data for K1(D1N) and K1(K2L3) are obscured by the data for K1-ΔL. Error bars represent SEM (n = 3). (G) Close-up of the structure of K2 bound to TRPV1 with regions of the toxin colored with the same scheme as in (F). Side chains of K603 and Y627 indicate the region where the pore turret (residues 604–626) would likely reside if not deleted in the construct used for cryo-EM and in our model.DOI:http://dx.doi.org/10.7554/eLife.11273.015
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fig3: Validation of the proposed structural model of the TRPV1-DkTx complex.Close-up of K1 (A) and K2 (B) in the top-ranking ROSETTA model of DkTx bound to TRPV1. Side chains of TRPV1 residues where mutation disrupts DkTx-induced channel activation (I599, F649, A657 and F659) (Bohlen et al., 2010) are highlighted in purple. (C) Representative time courses of TRPV1 activation at +120 mV by either DkTx (left panel), K1K1 (middle panel) or K2K2 (right panel) measured from a train of voltage-ramps in the whole-cell configuration with the TRPV1 expressed in HEK cells. In the case of K1K1, toxin was reapplied to verify that the observed decrease in current upon removal of the toxin was due to toxin dissociation from the channel rather than desensitization. Voltage ramps were from −120 to +140 mV over 1 s and were applied every 3 s from a holding potential of −90 mV. The colored horizontal lines denote the application of toxins. The dotted gray line denotes the zero-current level. Fractional dissociation 1 min after toxin removal was quantified across cells as the current amplitude 1 min after toxin removal divided by the steady-state current amplitude in the presence of the toxin. Fractional dissociation values (mean ± sem) were 0.8 ± 0.05 (n = 8) for DkTx, 0.3 ± 0.04 (n = 5) for K1K1 and 0.96 ± 0.03 for K2K2 (n = 6). (D) Mean normalized I-V relations obtained from voltage ramps at steady-state for control solution (gray), toxins (yellow, green or blue) and saturating capsaicin (10 µM; black) in the whole-cell configuration using transiently transfected HEK293 cells. The thin colored curves represent the mean and the thicker envelope the standard error (n = 5–6 for each panel). (E) Primary sequence of K1 without linker (K1-ΔL), K2 and gain-of-function chimeras between K1 and K2. Residues of K1-ΔL and K2 are shown in green and cyan, respectively, and cysteines are shown in yellow. Loops between cysteine residues are labeled as L1, L2, L3 or L4. CT denotes C-terminal region. (F) Concentration-dependence for activation of TRPV1 by K1, K2 and chimeras in two-electrode voltage clamp recordings of oocytes expressing TRPV1. Activation of the full-length TRPV1 channel by the toxin at different concentrations was measured in oocytes at a holding voltage of −60 mV. Toxin-induced currents (ITx) were normalized against the current activated by a saturating concentration of capsaicin (Icap at 10 μM), in the same cell. Note that the data for K1(D1N) and K1(K2L3) are obscured by the data for K1-ΔL. Error bars represent SEM (n = 3). (G) Close-up of the structure of K2 bound to TRPV1 with regions of the toxin colored with the same scheme as in (F). Side chains of K603 and Y627 indicate the region where the pore turret (residues 604–626) would likely reside if not deleted in the construct used for cryo-EM and in our model.DOI:http://dx.doi.org/10.7554/eLife.11273.015

Mentions: The model of the TRPV1-DkTx complex reveals that K1 and K2 engage the outer pore of TRPV1 using loop 2 and loop 4 (Figure 3A,B). These two loops straddle the interface between TRPV1 subunits, with loop 2 capping the S6 helix from one subunit, while loop 4 localizes near the N-terminus of the re-entrant pore helix from the adjacent subunit. Scanning mutagenesis previously identified four residues within the outer pore domain of TRPV1, namely I599A at the top of S5, F649A in the pore loop, and both A657P and F659A at the top of S6, where mutations disrupt activation of the channel by DkTx (Bohlen et al., 2010); two additional mutants in this region (V595A in S5, and T650A in the pore loop) also display diminished activation by the toxin, whereas Y631A, near the N-terminus of the pore helix, enhances the effect of DkTx. Our model indicates that among these residues, only F649, A657, T650 and Y631 could interact directly with the toxin, suggesting that the other mutations influence toxin activation of the channel through an indirect mechanism (see below). Interestingly, the model also shows that both K1 and K2 drape over the top of S6 at the presumed interface with the surrounding membrane, and position several conserved hydrophobic residues (W11, F27 and I28 in K1, and W53, F67 and I68 in K2) where they would interact directly with lipids in the bilayer.10.7554/eLife.11273.015Figure 3.Validation of the proposed structural model of the TRPV1-DkTx complex.


Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin.

Bae C, Anselmi C, Kalia J, Jara-Oseguera A, Schwieters CD, Krepkiy D, Won Lee C, Kim EH, Kim JI, Faraldo-Gómez JD, Swartz KJ - Elife (2016)

Validation of the proposed structural model of the TRPV1-DkTx complex.Close-up of K1 (A) and K2 (B) in the top-ranking ROSETTA model of DkTx bound to TRPV1. Side chains of TRPV1 residues where mutation disrupts DkTx-induced channel activation (I599, F649, A657 and F659) (Bohlen et al., 2010) are highlighted in purple. (C) Representative time courses of TRPV1 activation at +120 mV by either DkTx (left panel), K1K1 (middle panel) or K2K2 (right panel) measured from a train of voltage-ramps in the whole-cell configuration with the TRPV1 expressed in HEK cells. In the case of K1K1, toxin was reapplied to verify that the observed decrease in current upon removal of the toxin was due to toxin dissociation from the channel rather than desensitization. Voltage ramps were from −120 to +140 mV over 1 s and were applied every 3 s from a holding potential of −90 mV. The colored horizontal lines denote the application of toxins. The dotted gray line denotes the zero-current level. Fractional dissociation 1 min after toxin removal was quantified across cells as the current amplitude 1 min after toxin removal divided by the steady-state current amplitude in the presence of the toxin. Fractional dissociation values (mean ± sem) were 0.8 ± 0.05 (n = 8) for DkTx, 0.3 ± 0.04 (n = 5) for K1K1 and 0.96 ± 0.03 for K2K2 (n = 6). (D) Mean normalized I-V relations obtained from voltage ramps at steady-state for control solution (gray), toxins (yellow, green or blue) and saturating capsaicin (10 µM; black) in the whole-cell configuration using transiently transfected HEK293 cells. The thin colored curves represent the mean and the thicker envelope the standard error (n = 5–6 for each panel). (E) Primary sequence of K1 without linker (K1-ΔL), K2 and gain-of-function chimeras between K1 and K2. Residues of K1-ΔL and K2 are shown in green and cyan, respectively, and cysteines are shown in yellow. Loops between cysteine residues are labeled as L1, L2, L3 or L4. CT denotes C-terminal region. (F) Concentration-dependence for activation of TRPV1 by K1, K2 and chimeras in two-electrode voltage clamp recordings of oocytes expressing TRPV1. Activation of the full-length TRPV1 channel by the toxin at different concentrations was measured in oocytes at a holding voltage of −60 mV. Toxin-induced currents (ITx) were normalized against the current activated by a saturating concentration of capsaicin (Icap at 10 μM), in the same cell. Note that the data for K1(D1N) and K1(K2L3) are obscured by the data for K1-ΔL. Error bars represent SEM (n = 3). (G) Close-up of the structure of K2 bound to TRPV1 with regions of the toxin colored with the same scheme as in (F). Side chains of K603 and Y627 indicate the region where the pore turret (residues 604–626) would likely reside if not deleted in the construct used for cryo-EM and in our model.DOI:http://dx.doi.org/10.7554/eLife.11273.015
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fig3: Validation of the proposed structural model of the TRPV1-DkTx complex.Close-up of K1 (A) and K2 (B) in the top-ranking ROSETTA model of DkTx bound to TRPV1. Side chains of TRPV1 residues where mutation disrupts DkTx-induced channel activation (I599, F649, A657 and F659) (Bohlen et al., 2010) are highlighted in purple. (C) Representative time courses of TRPV1 activation at +120 mV by either DkTx (left panel), K1K1 (middle panel) or K2K2 (right panel) measured from a train of voltage-ramps in the whole-cell configuration with the TRPV1 expressed in HEK cells. In the case of K1K1, toxin was reapplied to verify that the observed decrease in current upon removal of the toxin was due to toxin dissociation from the channel rather than desensitization. Voltage ramps were from −120 to +140 mV over 1 s and were applied every 3 s from a holding potential of −90 mV. The colored horizontal lines denote the application of toxins. The dotted gray line denotes the zero-current level. Fractional dissociation 1 min after toxin removal was quantified across cells as the current amplitude 1 min after toxin removal divided by the steady-state current amplitude in the presence of the toxin. Fractional dissociation values (mean ± sem) were 0.8 ± 0.05 (n = 8) for DkTx, 0.3 ± 0.04 (n = 5) for K1K1 and 0.96 ± 0.03 for K2K2 (n = 6). (D) Mean normalized I-V relations obtained from voltage ramps at steady-state for control solution (gray), toxins (yellow, green or blue) and saturating capsaicin (10 µM; black) in the whole-cell configuration using transiently transfected HEK293 cells. The thin colored curves represent the mean and the thicker envelope the standard error (n = 5–6 for each panel). (E) Primary sequence of K1 without linker (K1-ΔL), K2 and gain-of-function chimeras between K1 and K2. Residues of K1-ΔL and K2 are shown in green and cyan, respectively, and cysteines are shown in yellow. Loops between cysteine residues are labeled as L1, L2, L3 or L4. CT denotes C-terminal region. (F) Concentration-dependence for activation of TRPV1 by K1, K2 and chimeras in two-electrode voltage clamp recordings of oocytes expressing TRPV1. Activation of the full-length TRPV1 channel by the toxin at different concentrations was measured in oocytes at a holding voltage of −60 mV. Toxin-induced currents (ITx) were normalized against the current activated by a saturating concentration of capsaicin (Icap at 10 μM), in the same cell. Note that the data for K1(D1N) and K1(K2L3) are obscured by the data for K1-ΔL. Error bars represent SEM (n = 3). (G) Close-up of the structure of K2 bound to TRPV1 with regions of the toxin colored with the same scheme as in (F). Side chains of K603 and Y627 indicate the region where the pore turret (residues 604–626) would likely reside if not deleted in the construct used for cryo-EM and in our model.DOI:http://dx.doi.org/10.7554/eLife.11273.015
Mentions: The model of the TRPV1-DkTx complex reveals that K1 and K2 engage the outer pore of TRPV1 using loop 2 and loop 4 (Figure 3A,B). These two loops straddle the interface between TRPV1 subunits, with loop 2 capping the S6 helix from one subunit, while loop 4 localizes near the N-terminus of the re-entrant pore helix from the adjacent subunit. Scanning mutagenesis previously identified four residues within the outer pore domain of TRPV1, namely I599A at the top of S5, F649A in the pore loop, and both A657P and F659A at the top of S6, where mutations disrupt activation of the channel by DkTx (Bohlen et al., 2010); two additional mutants in this region (V595A in S5, and T650A in the pore loop) also display diminished activation by the toxin, whereas Y631A, near the N-terminus of the pore helix, enhances the effect of DkTx. Our model indicates that among these residues, only F649, A657, T650 and Y631 could interact directly with the toxin, suggesting that the other mutations influence toxin activation of the channel through an indirect mechanism (see below). Interestingly, the model also shows that both K1 and K2 drape over the top of S6 at the presumed interface with the surrounding membrane, and position several conserved hydrophobic residues (W11, F27 and I28 in K1, and W53, F67 and I68 in K2) where they would interact directly with lipids in the bilayer.10.7554/eLife.11273.015Figure 3.Validation of the proposed structural model of the TRPV1-DkTx complex.

Bottom Line: We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes.Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation.Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology and Biophysics Section, Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, United States.

ABSTRACT
Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. Here, we solve the structure of double-knot toxin (DkTx), a tarantula toxin that activates the heat-activated TRPV1 channel. We also provide improved structures of TRPV1 with and without the toxin bound, and investigate the interactions of DkTx with the channel and membranes. We find that DkTx binds to the outer edge of the external pore of TRPV1 in a counterclockwise configuration, using a limited protein-protein interface and inserting hydrophobic residues into the bilayer. We also show that DkTx partitions naturally into membranes, with the two lobes exhibiting opposing energetics for membrane partitioning and channel activation. Finally, we find that the toxin disrupts a cluster of hydrophobic residues behind the selectivity filter that are critical for channel activation. Collectively, our findings reveal a novel mode of toxin-channel recognition that has important implications for the mechanism of thermosensation.

No MeSH data available.


Related in: MedlinePlus