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.
Comments
Sid Simon, Duke University
The Regulation of TRPV1 by
The Regulation of TRPV1 by Phosphoinositides in Liposomes
This comment is coauthored by Tamara Rosenbaum, Universidad Nacional Autónoma de México, Distrito Federal, México, and Sidney A. Simon, Duke University, Durham, North Carolina, USA
The TRPV1 channel is a versatile protein expressed in the central and peripheral nervous system as well as in several other non-neuronal tissues (Jara-Oseguera and Rosenbaum, 2012). Several endogenous and natural molecules have been identified as activators and modulators of TRPV1 function. One well-studied activator is the vanilloid capsaicin, the principal ingredient in chili pepper that gives rise to a burning sensation. It is well established that repeated or long-term application of capsaicin will cause a decrease in TRPV1 activity with subsequent application. This phenomenon is known as desensitization. Over the past years the mechanisms leading to TRPV1 sensitization and desensitization have been extensively investigated (Jara-Oseguera et al., 2008; Szallasi et al., 2006). Among the molecules known to play important roles in sensitization and desensitization of TRPV1 are amphiphiles, such as lipids and their metabolites. Although several lipids have been shown to modulate TRPV1’s activity, for the phosphoinositide phosphatidylinositol-4,5-bisphosphate (PIP2), a lipid that is localized to the cytosolic leaflet of plasma membranes, controversy has surrounded its mode of action. The recent paper by Cao et al. (2013) has addressed this controversy by studying the responses of TRPV1 ion channels in liposomes.
In 2003, evidence from whole-cell patch-clamp experiments in TRPV1-containing DRG neurons revealed that PIP2 inhibited the response of TRPV1 to capsaicin, since increased channel activity was observed upon the activation of phospholipase C (PLC) that resulted in the depletion of PIP2 (Prescott and Julius, 2003). In contrast, in 2006 Stein et al. (Stein et al., 2006) applied PIP2 directly to the cytoplasmic face of an isolated membrane patch of DRG neurons, and rather than inhibiting TRPV1, PIP2 potentiated the activation of TRPV1 by capsaicin. In addition, by depleting the patches of endogenous PIP2, these researchers found that TRPV1 lost its ability to be activated by capsaicin, a quality that was recovered after exposure to a soluble analogue of PIP2. In yet another patch clamp study with DRG neurons, it was found that prolonged applications of capsaicin (in the presence of extracellular Ca2+) resulted in nearly complete desensitization of TRPV1, and that recovery from desensitization could be prevented by depleting PIP2. In this regard, replenishment of PIP2 resulted in the recovery of TRPV1 from desensitization, allowing the channel to respond again to the next capsaicin application (Liu et al., 2005). The question then arises, could the same molecule prevent TRPV1 from opening under one condition and yet be necessary for it to be activated under a different condition?
The recently published study by Cao et al. in Neuron addressed the role of PIP2 in TRPV1 regulation by reconstituting TRPV1 into liposomes containing several lipid components, including PIP2. It is important to mention that in this bilayer system, the channels may be oriented in either direction, which would rationalize why some of the presented I-V plots showed less rectification than others, and also why PIP2 was present in both monolayers of the bilayer. What is important to the understanding of how TRPV1 is modulated by phosphoinositides is that Cao et al. found that the responses to capsaicin (and resiniferatoxin), 2-APB, extracellular acid, lysophosphatic acid (LPA), the spider toxin DkTx, and heat (temperature) are reasonably similar to those found in biological membranes such as DRG neurons. Their data also demonstrate that despite the six ankyrins in the N-terminal of TRPV1, the interaction with cytoskeletal elements is not necessary for the normal functioning of the channel. Interestingly, the investigators also found that the expression of TRPV1 in different lipid bilayer systems results in modified sensitivities to its agonists (EC50s), which suggests that the presence of certain cellular components might alter the sensitivity of the channel to different stimuli.
With regard to the inclusion of PIP2 (and like molecules; see below) in the liposome, Cao et al. found that they inhibited the activation of TRPV1 by many of the above-mentioned activators. Specifically, they found that when TRPV1 was reconstituted into liposomes where PIP2 was not present, the channel’s response to capsaicin or heat was not affected, suggesting that this phosphoinositide is not required for TRPV1 activation. In contrast, when the channel was incorporated into liposomes where phospholipids such as phosphatidylinositol (PI), PIP2, or phosphatidylinositol 4-phosphate (PI4P) were incorporated, TRPV1 exhibited a reduced sensitivity to capsaicin, clearly demonstrating that in lipid bilayers, PIP2, PI, and PI4P are negative regulators of TRPV1. Specifically, Cao et al. found that the inclusion of various phosphoinositides produced marked rightward shifts in the thermal and capsaicin response profiles.
It is noteworthy that PIP3, although very similar in structure to PIP2 but having one additional negative charge, did not affect TRPV1 activation by capsaicin, a result that deserves further investigation, since the addition of the extra charge may have affected the hydrophilic-hydrophobic balance, causing a reduction in its membrane concentration (McLaughlin and Aderem, 1995). An important future experiment would be to add the exogenous lipids from the aqueous phase, although we are aware that this may only affect those channels with the exposed cytoplasmic surface, and may induce a bending moment in the bilayer (Evans, 1974; McIntosh and Simon, 2006).
One aspect of this otherwise elegant work that could be refined was the study of how membrane elastic properties such as their compressibility modulus (as modulated by cholesterol) and elastic bending energy (as influenced by lysolipids) might alter TRPV1 function (Chernomordik and Zimmerberg, 1995; McIntosh and Simon, 2006). The use of liposomes containing many components including sphingomyelin (which sequesters cholesterol; Harder and Simons, 1997) and the relatively low mole fractions used do not, in our opinion, adequately explore the effects of these parameters.
Cao et al. also addressed the region of the channel where PIP2 is bound. It was previously suggested that the region in TRPV1 conferring PIP2 sensitivity was located at positively charged residues R701 and K710 at the proximal part of the C-terminal (Brauchi et al., 2006; Brauchi et al., 2007). These results were recently confirmed (Ufret-Vincenty et al., 2011). Using truncated constructs of the C-terminus of TRPV1, Cao et al. showed that such channels were unresponsive to PIP2. Moreover, when liposomes containing the truncated C-terminus were used, and the interaction between this region and the liposomal membrane containing phosphoinositides was reestablished through chemical strategies, the channel regained sensitivity to PIP2. Although it has been recently proposed that the N-terminus of TRPV1 might also participate in the interactions with PIP2 (Grycova et al., 2012), the data obtained by Cao and collaborators show that the C-terminus is sufficient for TRPV1’s response to PIP2.
In summary, the study by Cao et al. represents a significant advance in the understanding of the role of PIP2 in the modulation of TRPV1. We now look forward to obtaining an understanding of its role in the physiology and pathology of the cells where TRPV1 is expressed.
Acknowledgment: Work in the Rosenbaum lab is supported by grants from the Marcos Moshinsky Foundation, CONACyT 129474 and PAPIIT IN204111/22.
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