The cloning of transient receptor potential channel V1 (TRPV1) in 1997 by David Julius and colleagues (Caterina et al., 1997) ushered in a new age of molecular pain research. A heat-activated channel and the receptor for capsaicin (the pungent heat in chili peppers), TRPV1 (then called VR1), was already appreciated as a key molecule in pain and a viable target for analgesic therapies, and its genetic identification gave researchers the ability to study the enigmatic channel’s relation to pain sensation. Now Julius and collaborator Yifan Cheng, both at the University of California, San Francisco, US, have hit another milestone in the TRPV1 story. In two papers published online December 5 in Nature, the researchers report the first-ever atomic-level structure of the channel.
In the new work, first authors Erhu Cao and Maofu Liao pushed the envelope with single-particle cryo-electron microscopy (cryo-EM) techniques to achieve a high-resolution view of a rat TRPV1 channel. They determined the channel structure to nearly 3 angstroms of detail in the closed state and in two open conformations, one with capsaicin and the other with two potent activators: the plant-based resiniferatoxin (RTX) and double-knot toxin (DkTx), a component of tarantula spider venom.
TRPV1 is not only the key sensor of acute heat pain, but also—and perhaps more importantly in terms of pain treatment—serves as a signal integrator that contributes to chronic pain sensitivity. The newly solved structure will give researchers tools to probe the complexities of TRPV1 regulation and design better TRPV1-targeted therapies.
Among the most exciting discoveries from the three structures was confirmation of the idea that TRPV1 has two separate gates that control current flow. Moreover, said Julius, the structure “shows that the gates can communicate with one another, which is important for understanding structural mechanisms underlying TRPV1 modulation and pain hypersensitivity.”
Zooming in
The work marks the first time that cryo-EM has achieved such a high-resolution look at a small, transmembrane protein, thanks to improvements that Cheng, UCSF microscopist David Agard, and colleagues recently made to the microscopy and data analysis methods (Li et al., 2013). The structure is “a landmark both in the evolution of the single-particle cryo-EM method and in its use for tackling the structure of a macromolecular complex that has been difficult to study by other means,” wrote Richard Henderson of Cambridge University, UK, in a News & Views article accompanying the two papers. (Henderson was not involved in the study.)
“Many of us were looking at trees, and now we see the forest,” said Rachelle Gaudet of Harvard University, Cambridge, US, who was also not involved in the new work. Gaudet was referring to TRP channel researchers who have been studying details of the channels piecemeal. With the new structure, she said, “we can reinterpret previous data and change the way we design experiments, because we have a view of the whole channel, even if we’re interested in its parts.”
“The findings reach beyond pain,” Julius said. TRPV1 is one of 27 members of the diverse transient receptor potential (TRP) family of channels, and the first to have its structure solved. The channels are found throughout the body, and TRP channel abnormalities confer a number of human maladies, so this structure will be helpful for thinking about other TRP channel-based physiological disease targets, Julius said.
Structural revelations
Although TRP channels bear little functional or sequence resemblance to their voltage-gated kin, scientists had predicted that the overall structure of TRPV1 would resemble potassium and sodium channels, which have been solved by X-ray crystallography (MacKinnon, 2003; Catterall, 2012). The new structure confirmed that, much like those channels, TRPV1 is composed of four symmetrical subunits, with six transmembrane helices (S1-S6) each. Ions flow through a central pore formed by the four subunits’ S5 and S6 helices together with a small loop that dips in and out of the membrane between them.
The images of the channel’s closed and open states captured unique elements of TRPV1 topography, too. The charged amino acids in S1-S4 that serve as voltage sensors in voltage-gated channels are replaced in TRPV1 by aromatic residues, which seem to stabilize the channel core. Rather than moving like voltage sensors do in response to depolarization, the TRPV1 S1-S4 helices provide an anchor for movements within the pore itself. “When ligands bind, the S4-S5 linker acts as a fulcrum to move the pore domain relative to stable parts,” Julius said.
This video shows a three-dimensional model of the TRPV1 protein at 3.4 angstroms, bound by both spider toxin (pink) and resiniferatoxin (red spheres). Resiniferatoxin occupies the same binding pocket as capsaicin. The protein’s four subunits are colored light yellow, blue, green, and red. As the model rotates to show top and bottom views, the central channel through which ions pass into the cell is clearly visible. Credit: Yifan Cheng, University of California, San Francisco, US.
The multiple structures revealed distinctive dual gates, or restriction points, in the channel pore. Previous studies had suggested such duplicity, including one paper that proposed dual-gating processes in the related TRPV4 channel (Loukin et al., 2010). The new findings showed a large, funnel-shaped cytoplasmic entry leading to a constriction point that appeared to function as a selectivity filter for ions. Further into the pore, the structures revealed a second hydrophobic constriction point. Both gates underwent structural rearrangements in the active state consistent with channel opening.
Perhaps the biggest surprise from the cryo-EM structures came in the form of a detectable signal showing where RTX or capsaicin bound—in a hydrophobic pocket deep in the protein just outside the S1-S4 helices. “Although we knew which residues are key for agonist binding, you don’t know what the pocket looks like until you can see it in a structure,” said Julius, who described that detail as “a beautiful little nugget, a little gift.”
The structure and location of the C-terminal TRP domain—unique to TRP channels and found in about half of them—also illuminated the channel’s inner workings. The authors described the TRP domain as “strategically located” to influence the channel’s open state in response to stimuli. Gaudet ranked these structural details among the findings’ most enticing. “This small region has been implicated in allosteric regulation,” she said, alluding to the way that channel subunits and other protein domains interact with one another. “Now we can hypothesize about how it works. That is really exciting, because it has broad repercussions for the field,” such as how to target overactive TRPV1 channels.
Protons interact with the channel at TRPV1’s outer pore to potentiate current flow. “Our structure now shows hydrogen bonding there, which could be disrupted to facilitate gating,” Julius said. “That gives us a clearer picture for how protons might work, consistent with previous biophysical findings,” he said. “That’s very important for regulation of the channel.”
The structure also showed four of the six ankyrin repeats—intracellular scaffold domains that host protein-protein interactions both known and unknown. The ankyrin repeat domain and its linker to the protein’s N-terminus served to lock the N- and C-termini of individual subunits to one another and also interacted with neighboring subunits. “This shows that their role is to help stabilize the subunits together and probably to mediate other modulations,” Julius said, which might occur at the domain’s “cytoplasmic skirt” region. “We knew the repeats were important for the overall structural integrity of the channel; now we can begin to think more precisely about what they do in regard to functionality,” he said.
Gaudet agreed, and added that many in the field “had anticipated an overall organization of those repeats, and it is exciting to see those details.”
Many questions remain about TRPV1, such as how it gates in response to temperature. Julius’ team plans to tackle that question next by heating TRPV1 channels to the heat sensor’s activation range and then freezing them in hopes of catching a glimpse of that conformational structure. Such an endeavor “will be a technical tour de force, but if it can be done, cryo-EM is the way to do it,” Julius said.
Seeing clearly
As pain therapies, TRPV1 blockers carry the danger that they interfere with normal, protective heat sensation (Rowbotham et al., 2011). But Julius still has hope for TRPV1 modulators as analgesics. “The best drug would not perturb the channel’s core heat-sensing ability, but instead would focus on its modulation,” Julius said. Past studies have suggested that the outer pore domain might be the best locus to target pain sensitization without affecting the heat sensor. “Those are the drugs you want,” Julius said, “and perhaps the new structure will allow researchers to dock compounds in the channel and see where to best target hypersensitivity.”
In his News & Views piece, Henderson expressed the same hope, writing that the findings may “herald the dawn of cryo-EM as a technique to aid rational drug design.”
Stephani Sutherland, PhD, is a neuroscientist, yogi, and freelance writer in Southern California, US.
Image: From Liao et al. Reprinted by permission from Macmillan Publishers Ltd: Nature 504: 107-112, copyright 2013.
