1) Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin Venom toxins are invaluable tools for exploring the structure and mechanisms of ion channels. We solved 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. 2) An external sodium ion binding site controls allosteric gating in TRPV1 channels TRPV1 channels in sensory neurons are integrators of painful stimuli and heat, yet how they integrate diverse stimuli and sense temperature remains elusive. We show that external sodium ions stabilize the TRPV1 channel in a closed state, such that removing the external ion leads to channel activation. In studying the underlying mechanism, we find that the temperature sensors in TRPV1 activate in two steps to favor opening, and that binding of sodium to an extracellular site exerts allosteric control over temperature-sensor activation and opening of the pore. The binding of a tarantula toxin to the external pore also exerts control over temperature-sensor activation, whereas binding of vanilloids influences temperature-sensitivity by largely affecting the open/closed equilibrium. Our results reveal a fundamental role of the external pore in the allosteric control of TRPV1 channel gating and provide essential constraints for understanding how these channels can be tuned by diverse stimuli. 3) Engineering vanilloid-sensitivity into the rat TRPV2 channel The TRPV1 channel is a detector of noxious stimuli, including heat, acidosis, vanilloid compounds and lipids. The gating mechanisms of the related TRPV2 channel are poorly understood because selective high affinity ligands are not available, and the threshold for heat activation is extremely high (> 50 C). Cryo-EM structures of TRPV1 and TRPV2 reveal that they adopt similar structures, and identify a putative vanilloid binding pocket near the internal side of TRPV1. Here we use biochemical and electrophysiological approaches to investigate the resiniferatoxin (RTx) binding site in TRPV1 and to explore the functional relationships between TRPV1 and TRPV2. Collectively, our results support the interaction of vanilloids with the proposed RTx binding pocket, and demonstrate an allosteric influence of a tarantula toxin on vanilloid binding. Moreover, we show that sensitivity to RTx can be engineered into TRPV2, demonstrating that the gating and permeation properties of this channel are similar to TRPV1.
| Ballesteros, Angela; Swartz, Kenton J (2018) Lipids surf the groove in scramblases. Proc Natl Acad Sci U S A 115:7648-7650 |
| Ballesteros, Angela; Fenollar-Ferrer, Cristina; Swartz, Kenton Jon (2018) Structural relationship between the putative hair cell mechanotransduction channel TMC1 and TMEM16 proteins. Elife 7: |
| Bae, Chanhyung; Jara-Oseguera, Andres; Swartz, Kenton J (2018) TRPM channels come into focus. Science 359:160-161 |
| Matthies, Doreen; Bae, Chanhyung; Toombes, Gilman Es et al. (2018) Single-particle cryo-EM structure of a voltage-activated potassium channel in lipid nanodiscs. Elife 7: |
| Zhang, Feng; Jara-Oseguera, Andres; Chang, Tsg-Hui et al. (2018) Heat activation is intrinsic to the pore domain of TRPV1. Proc Natl Acad Sci U S A 115:E317-E324 |
| Chavan, Tanmay; Maduke, Merritt; Swartz, Kenton (2017) Protein ligands for studying ion channel proteins. J Gen Physiol 149:407-411 |
| Toombes, Gilman E S; Swartz, Kenton J (2016) STRUCTURAL BIOLOGY. Twists and turns in gating ion channels with voltage. Science 353:646-7 |
| Jara-Oseguera, Andres; Bae, Chanhyung; Swartz, Kenton J (2016) An external sodium ion binding site controls allosteric gating in TRPV1 channels. Elife 5: |
| Bae, Chanhyung; Anselmi, Claudio; Kalia, Jeet et al. (2016) Structural insights into the mechanism of activation of the TRPV1 channel by a membrane-bound tarantula toxin. Elife 5: |
| Zhang, Feng; Hanson, Sonya M; Jara-Oseguera, Andres et al. (2016) Engineering vanilloid-sensitivity into the rat TRPV2 channel. Elife 5: |
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