The transient receptor potential vanilloid type 1 (TRPV1) channel has emerged as a complex integrator of signals that can lead to chronic pain and hyperalgesia. Yet even as the list of agonists and modulators of this heat- and capsaicin-sensing channel has grown, uncertainty persists about the channel’s thermosensing properties and how phospholipids regulate TRPV1 function. Research led by David Julius, University of California, San Francisco, US, now shows that purified TRPV1 channels are fully functional and display intrinsic heat sensitivity when reconstituted into artificial liposomes. The study further indicates that channel activity does not require phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane lipid known to regulate TRP channel family members, including TRPV1. The current work sheds light on how various modulators of TRPV1 might lead to channel sensitization and hyperalgesia.
The study was published February 20 in Neuron.
An intrinsic heat sensor
A number of modulators interact with TRPV1, including protons (low pH), ingredients in the “inflammatory soup,” and phospholipids. G protein-coupled receptors also play a role in TRPV1 signaling, by mediating, for example, the sensitizing effect of the inflammatory mediator bradykinin on the channel. And phosphorylation of TPRV1 by kinases also alters channel activity.
To study TRPV1 independent of other cellular proteins and molecules that might modulate its activity, the researchers reconstituted purified recombinant TRPV1 expressed in insect cells into artificial liposomes—tiny, empty vesicles made of nothing but lipid components. “We wanted to examine this issue from a different perspective by using a very reductionist approach, investigating the purified protein in a fully defined system,” said Julius.
In the current work, first author Erhu Cao and colleagues at UCSF, along with co-investigators at the State University of New York at Buffalo, US, placed strict controls on the makeup of the artificial membrane, stripping it down to a short list of known lipid components, none of which affect TRPV1. They first wanted to determine whether the heat sensitivity was intrinsic to the channel or instead depended on other cellular elements. In excised patches from the artificial liposomes, said Julius, the answer was clear. “We saw exactly the same biophysical properties as we’ve seen in native cells: a beautiful, steep temperature activation curve.” In essence, the experiment showed that “you don’t need an auxiliary protein or molecule for TRPV1 heat sensitivity; you just need the channel.” As in native cells, the TRPV1 antagonist capsazepine blocked heat-dependent currents, and extracellular protons potentiated them.
D.P. Mohapatra, who studies TRPV1 at the University of Iowa, Iowa City, US, and was not involved in the current work, was encouraged by the team’s success at reconstituting the purified receptor protein in liposomes and by TRPV1’s native functional behavior in the artificial system. “That can actually lead to helping us understand the channel’s structure,” he said, an important step on the path to new therapeutics.
The role of PIP2
The team next looked to the artificial liposomes in order to better understand the role of PIP2 in TRPV1 regulation. PIP2 is a phosphoinositide lipid component of cell membranes with the ability to interact with and modulate ion channels, including TRPV1. When the enzyme phospholipase C (PLC) becomes activated by proinflammatory mediators, PLC cleaves PIP2, thereby depleting PIP2 from the membrane and potentially decreasing its association with TRPV1. Previous investigations had shown that depletion or addition of PIP2 did affect TRPV1 activity, but it remained unclear whether the phosphoinositide positively or negatively modulated TRPV1 sensitivity (Sowa et al., 2010; Ufret-Vincenty et al., 2011). Much of that research had been performed in animals or native cells, where other signaling molecules may have contributed to the apparent effects of PIP2 on the channel.
The finding that TRPV1 responded normally to heat, capsaicin, and other agonists, even in the minimalist membranes, ruled out the possibility that PIP2 acts as an obligate co-agonist. “This result says that you don’t need PIP2 in the membrane for channel activity,” said Julius, a sentiment with which Mohapatra agreed. In contrast, when the investigators added PIP2 as a component of the membrane, TRPV1 currents were slightly reduced, suggesting that PIP2 (and several other phospholipids tested) negatively modulates channel activity. Julius pointed out that this is but one of many ways the channel can be subtly affected by cellular events. The researchers showed, for instance, that a number of other bioactive lipid molecules either directly activated or sensitized TRPV1, including arachidonic acid and anandamide.
Interestingly, in previous work by another group using reconstituted purified TRPM8, a channel that senses cold and menthol, showed that that channel’s activity does rely upon PIP2, demonstrating diversity among TRP channels in their response to membrane lipids (Zakharian et al., 2010).
Where does PIP2 bind to TRPV1?
Julius was originally drawn back to the issue of phospholipid interactions with TRPV1 channels following his lab’s recent observations on the TRPV1 orthologue from vampire bats (see PRF related news story). The bats sense infrared heat by using a variant of the channel that is activated by lower temperatures than usual. The bat variant has a truncation in the C-terminal region, where Julius and colleagues previously identified mutations in the mammalian channel that shift its heat threshold. This led them to propose that the C-terminal region might be the site of phospholipid interactions, and it might determine the channel’s heat sensitivity (Prescott and Julius, 2003).
To further explore that issue, in the new study, the investigators chemically tethered the protein’s C-terminus to the artificial membrane—much as it would be during a PIP2 interaction in a native cell. That manipulation caused a slight shift in the heat threshold to higher temperatures, adding further support to the idea that the C-terminal region interacts with PIP2 and sets the channel’s sensitivity. Mohapatra noted that he would like to see future work nail down the precise PIP2-TRPV1 interaction sites within that region, which also contains sites for other modifications, such as phosphorylation. The different modifications, he said, could interact with one another; for example, might the channel’s phosphorylation state influence the PIP2-TRPV1 interaction? “We need to understand more detail about structure-function and how channel gating is affected” by these multiple modifications, Mohapatra said.
Ultimately, said Julius, the reason to study these details of the channel is to determine optimal ways to manipulate TRPV1 to prevent or treat pain. “You don’t want a drug to shut down the channel completely, because the heat sensitivity works as a defense mechanism against injury,” he said. “But you want to prevent inflammatory mediators from hypersensitizing the channel.”
Mohapatra suggested that an effective strategy might be to selectively target channels that have reached that hypersensitized state. “Can we block the channel only when it is modulated, and leave unmodulated channels alone?” he wondered.
For his part, Julius is struck by the intricacy of TRPV1 structure and function. “The molecule has evolved over time to recognize a number of inflammatory molecules. The channel has become even more beautiful and complex,” he said. Perhaps by understanding that complexity, researchers can keep the channel in check.
Stephani Sutherland, PhD, is a freelance neuroscience writer based in Southern California, US.