Chanhyung Bae

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. 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. DOI: http://dx.doi.org/10.7554/eLife.11273.001

Tarantula toxins use common surfaces for interacting with Kv and ASIC ion channels

Tarantula toxins that bind to voltage-sensing domains of voltage-activated ion channels are thought to partition into the membrane and bind to the channel within the bilayer. While no structures of a voltage-sensor toxin bound to a channel have been solved, a structural homolog, psalmotoxin (PcTx1), was recently crystalized in complex with the extracellular domain of an acid sensing ion channel (ASIC). In the present study we use spectroscopic, biophysical and computational approaches to compare membrane interaction properties and channel binding surfaces of PcTx1 with the voltage-sensor toxin guangxitoxin (GxTx-1E). Our results show that both types of tarantula toxins interact with membranes, but that voltage-sensor toxins partition deeper into the bilayer. In addition, our results suggest that tarantula toxins have evolved a similar concave surface for clamping onto α-helices that is effective in aqueous or lipidic physical environments. DOI: http://dx.doi.org/10.7554/eLife.06774.001

Solution structure of kurtoxin: a gating modifier selective for Cav3 voltage-gated Ca(2+) channels.

Kurtoxin is a 63-amino acid polypeptide isolated from the venom of the South African scorpion Parabuthus transvaalicus. It is the first and only peptide ligand known to interact with Cav3 (T-type) voltage-gated Ca2+ channels with high affinity and to modify the voltage-dependent gating of these channels. Here we describe the nuclear magnetic resonance (NMR) solution structure of kurtoxin determined using two- and three-dimensional NMR spectroscopy with dynamical simulated annealing calculations. The molecular structure of the toxin was highly similar to those of scorpion α-toxins and contained an α-helix, three β-strands, and several turns stabilized by four disulfide bonds. This so-called “cysteine-stabilized α-helix and β-sheet (CSαβ)” motif is found in a number of functionally varied small proteins. A detailed comparison of the backbone structure of kurtoxin with those of the scorpion α-toxins revealed that three regions [first long loop (Asp8–Ile15), β-hairpin loop (Gly39–Leu42), and C-terminal segment (Arg57–Ala63)] in kurtoxin significantly differ from the corresponding regions in scorpion α-toxins, suggesting that these regions may be important for interacting with Cav3 (T-type) Ca2+ channels. In addition, the surface profile of kurtoxin shows a larger and more focused electropositive patch along with a larger hydrophobic surface compared to those seen on scorpion α-toxins. These distinct surface properties of kurtoxin could explain its binding to Cav3 (T-type) voltage-gated Ca2+ channels.

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. Here, 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 the 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. DOI: http://dx.doi.org/10.7554/eLife.13356.001

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. DOI: http://dx.doi.org/10.7554/eLife.16409.001

Heat activation is intrinsic to the pore domain of TRPV1

Significance The TRPV1 channel is an important detector of noxious heat, yet the location of the heat sensor and the mechanism of heat activation remain poorly understood. Here we used structure-based engineering between the heat-activated TRPV1 channel and the Shaker Kv channel to demonstrate that transplantation of the pore domain of TRPV1 into Shaker gives rise to functional channels that can be activated by a TRPV1-selective tarantula toxin and by noxious heat, demonstrating that the pore of TRPV1 contains the structural elements sufficient for activation by temperature. The TRPV1 channel is a sensitive detector of pain-producing stimuli, including noxious heat, acid, inflammatory mediators, and vanilloid compounds. Although binding sites for some activators have been identified, the location of the temperature sensor remains elusive. Using available structures of TRPV1 and voltage-activated potassium channels, we engineered chimeras wherein transmembrane regions of TRPV1 were transplanted into the Shaker Kv channel. Here we show that transplanting the pore domain of TRPV1 into Shaker gives rise to functional channels that can be activated by a TRPV1-selective tarantula toxin that binds to the outer pore of the channel. This pore-domain chimera is permeable to Na+, K+, and Ca2+ ions, and remarkably, is also robustly activated by noxious heat. Our results demonstrate that the pore of TRPV1 is a transportable domain that contains the structural elements sufficient for activation by noxious heat.

TRPM channels come into focus

The structures of TRPM channels help to explain how they can sense intracellular calcium Transient receptor potential (TRP) channels were first identified in photoreceptors of the fruit fly (1, 2). In mammals, six major families of TRP channels play key roles in sensing stimuli such as light, temperature, membrane lipids, and intracellular Ca2+. In 2013, two landmark publications revealed the cryo–electron microscopy (cryo-EM) structure of the heat- and capsaicin-activated TRPV1 channel (3, 4). Two articles in this issue report cryo-EM structures of cation-selective TRPM channels. On page 228, Autzen et al. (5) describe TRPM4, which is activated by intracellular Ca2+ and involved in controlling arterial tone, cardiac rhythm, and the immune response (6). On page 237, Yin et al. (7) report on TRPM8, which senses cold and menthol and may serve as a cancer biomarker (8).

Surface Characterization and Membrane Interaction of Double-Knot Toxin, an Activator of TRPV1 Channels

Double-knot toxin (DkTx) is a novel tarantula toxin that activates TRPV1 channels by binding to the extracellular pore domain of the channel, and is composed of two lobes named knot1(K1) and knot2(K2). Previous studies have shown that both lobes can be synthesized separately and activate the channel with different affinities. Recently, near atomic resolution structures of TRPV1 in distinct states (apo, capsaicin bound and DkTx&RTx bound) were reported using electron cryo-microscopy. These structures show that TRPV1 adopts a structure that is similar to Kv channels, and that the pore domain undergoes distributed conformational change upon activation in response to binding of DkTx and RTx. Although these structures show where DkTx binds, they do not have sufficient resolution to reveal the structural basis of the toxin-channel interaction. Here we solved the solution NMR structure of DkTx and dock that structure into the DkTx/RTx bound electron density maps using the Xplor-NIH program. Our results show that the toxin binds to a perimeter of the pore domain at the interface between the pore helix and S6 of neighboring subunit of the channel, and demonstrate that the toxin-channel interface is dominated by hydrophobic interactions. Interestingly, when bound to the channel, several residues on the toxin extend over the edge of the pore domain where they would be expected in interact with the surrounding membrane. To explore this possibility we tested whether DkTx can interact with membranes using a tryptophan fluorescence assay (each lobe contains a single conserved tryptophan). Indeed, each lobe of DkTx interacts with membranes, and the interaction is energetically more favorable in the bivalent toxin.

Structural Characterization of Double-Knot Toxin, an Activator of TRPV1 Channels

Venom from poisonous organisms is a rich source of peptide toxins interacting with different ion channels proteins. These peptide toxins modulate ion channels by different mechanisms, and have been widely used as tools for investigating ion channel mechanisms. Double-knot toxin (DkTx) is a novel peptide toxin that activates TRPV1 channels, and contains two inhibitory cysteine knot (ICK) motifs, as its name suggests. Previous studies show that DkTx activates TRPV1 channels, and suggest that the avidity of the toxin (slow unbinding) arises from its bivalent nature. Here we use solid-phase peptide synthesis to individually produce the two knots of DkTx (K1 and K2), fold each in vitro, and find that they exhibit different binding affinities for the channel even though they share high sequence homology. As a first step toward understanding the structural and functional relationship of DkTx binding to TRPV1 channels, we determined solution structures of each knot in using NMR. The structures show that DkTx is composed of two notably amphipathic ICK motifs (each with two beta-strands) that are connected by a flexible linker, and that K2 has a larger hydrophobic surface compared to K1. In addition, the single conserved Trp residue in each knot show different orientations, with that in K1 exhibiting greater solvent exposure. Interestingly, using intrinsic Trp fluorescence, we observe strong partitioning of DkTx and K1, but see no evidence of membrane partitioning for K2. We also made a series of K1/K2 chimeras, and identified variant residues in two loops and the C-terminus that are responsible for the higher activity of K2. From these results we propose that membrane interactions are involved in the mechanisms of DkTx activation of TRPV1, and identify surfaces of the two knots that likely involved in binding to the channel.