Tamara Rosenbaum

Ca2+/Calmodulin Modulates TRPV1 Activation by Capsaicin

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159–212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487–517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816–824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015–1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525–3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of ∼30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin.

A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic

Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.

Lysophosphatidic acid directly activates TRPV1 through a C-terminal binding site

Since 1992, there has been growing evidence that the bioactive phospholipid lysophosphatidic acid (LPA), whose amounts are increased upon tissue injury, activates primary nociceptors resulting in neuropathic pain. The TRPV1 ion channel is expressed in primary afferent nociceptors and is activated by physical and chemical stimuli. Here we show that in control mice LPA produces acute pain-like behaviors, which are substantially reduced in Trpv1-null animals. Our data also demonstrate that LPA activates TRPV1 through a unique mechanism that is independent of G protein-coupled receptors, contrary to what has been widely shown for other ion channels, by directly interacting with the C terminus of the channel. We conclude that TRPV1 is a direct molecular target of the pain-producing molecule LPA and that this constitutes, to our knowledge, the first example of LPA binding directly to an ion channel to acutely regulate its function.

TRPV1: On the Road to Pain Relief

Historically, drug research targeted to pain treatment has focused on trying to prevent the propagation of action potentials in the periphery from reaching the brain rather than pinpointing the molecular basis underlying the initial detection of the nociceptive stimulus: the receptor itself. This has now changed, given that many receptors of nociceptive stimuli have been identified and/or cloned. Transient Receptor Potential (TRP) channels have been implicated in several physiological processes such as mechanical, chemical and thermal stimuli detection. Ten years after the cloning of TRPV1, compelling data has been gathered on the role of this channel in inflammatory and neuropathic states. TRPV1 activation in nociceptive neurons, where it is normally expressed, triggers the release of neuropeptides and transmitters resulting in the generation of action potentials that will be sent to higher CNS areas where they will often be perceived as pain. Its activation also will evoke the peripheral release of pro-inflammatory compounds that may sensitize other neurons to physical, thermal or chemical stimuli. For these reasons as well as because its continuous activation causes analgesia, TRPV1 has become a viable drug target for clinical use in the management of pain. This review will provide a general picture of the physiological and pathophysiological roles of the TRPV1 channel and of its structural, pharmacological and biophysical properties. Finally, it will provide the reader with an overall view of the status of the discovery of potential therapeutic agents for the management of chronic and neuropathic pain.

Identification of a Binding Motif in the S5 Helix That Confers Cholesterol Sensitivity to the TRPV1 Ion Channel

The TRPV1 ion channel serves as an integrator of noxious stimuli with its activation linked to pain and neurogenic inflammation. Cholesterol, a major component of cell membranes, modifies the function of several types of ion channels. Here, using measurements of capsaicin-activated currents in excised patches from TRPV1-expressing HEK cells, we show that enrichment with cholesterol, but not its diastereoisomer epicholesterol, markedly decreased wild-type rat TRPV1 currents. Substitutions in the S5 helix, rTRPV1-R579D, and rTRPV1-F582Q, decreased this cholesterol response and rTRPV1-L585I was insensitive to cholesterol addition. Two human TRPV1 variants, with different amino acids at position 585, had different responses to cholesterol with hTRPV1-Ile585 being insensitive to this molecule. However, hTRPV1-I585L was inhibited by cholesterol addition similar to rTRPV1 with the same S5 sequence. In the absence of capsaicin, cholesterol enrichment also inhibited TRPV1 currents induced by elevated temperature and voltage. These data suggest that there is a cholesterol-binding site in TRPV1 and that the functions of TRPV1 depend on the genetic variant and membrane cholesterol content.

Structural determinants of gating in the TRPV1 channel

Transient receptor potential vanilloid 1 (TRPV1) channels mediate several types of physiological responses. Despite the importance of these channels in pain detection and inflammation, little is known about how their structural components convert different types of stimuli into channel activity. To localize the activation gate of these channels, we inserted cysteines along the S6 segment of mutant TRPV1 channels and assessed their accessibility to thiol-modifying agents. We show that access to the pore of TRPV1 is gated by S6 in response to both capsaicin binding and increases in temperature, that the pore-forming S6 segments are helical structures and that two constrictions are present in the pore: one that impedes the access of large molecules and the other that hampers the access of smaller ions and constitutes an activation gate of these channels.

The role of endogenous molecules in modulating pain through transient receptor potential vanilloid 1 (TRPV1).

•  TRPV1 (transient receptor potential vanilloid 1) channels are found throughout the body in epithelial cells and in peripheral and central terminals in neurons. They exert a variety of functions ranging from inflammation, to nociception and pain. •  TRPV1 is a molecular integrator in that it can be activated by different endogenous stimuli. These interact to alter the channels’ properties, thereby changing the threshold to a given stimulus and resulting in sensitization. •  TRPV1 has numerous agonists and antagonists, including lipids and their metabolites, as well as gases and ions. Here, we detail what is known about the mechanisms used by endogenous molecules to modulate the activity of this important transducer for environmental and painful stimuli. •  Understanding how these compounds modify TRPV1 activity will allow us to comprehend how some pathologies are associated with its deregulation.

Dissecting Intersubunit Contacts in Cyclic Nucleotide-Gated Ion Channels

In cyclic nucleotide-gated (CNG) ion channels, binding of cGMP or cAMP drives a conformational change that leads to opening of an ion-conducting pore. One region implicated in the coupling of ligand binding to opening of the pore is the C linker region. Here, we used crosslinking of endogenous cysteines to study interregion proximity. We demonstrate that an individual amino acid--C481--in the C linker region of each of two neighboring subunits can form a disulfide bond. Further, using tandem dimers, we show that a disulfide bond between C35 in the N-terminal region and C481 in the C linker region can form either within a subunit or between subunits. From our data on proximity between individual amino acids and previous studies, a picture emerges of the C linker as a potential dimerization interface.

Subunit modification and association in VR1 ion channels

BackgroundThe capsaicin (vanilloid) receptor, VR1, is an agonist-activated ion channel expressed by sensory neurons that serves as a detector of chemical and thermal noxious stimuli.ResultsIn the present study we investigated the properties of VR1 ion channels expressed in Xenopus oocytes. A VR1 subunit with a FLAG epitope tag at the C-terminus was constructed. When examined for size on an SDS gel, VR1-expressing oocytes produced a doublet corresponding to the size of the monomer and a band at about twice the molecular weight of the monomer. A consensus site for N-linked glycosylation was identified in the primary sequence at position 604. In channels in which the putative glycosylation site was mutated from asparagine to serine (N604S), the larger of the two monomer bands could no longer be detected on the gel. Electrophysiological experiments showed these unglycosylated channels to be functional. The high molecular weight band observed on the gel could represent either a dimer or a monomer conjugated to an unknown factor. To distinguish between these possibilities, we coexpressed a truncated VR1 subunit with full-length VR1. A band of intermediate molecular weight (composed of one full-length and one truncated subunit) was observed. This dimer persisted under strongly reducing conditions, was not affected by capsaicin or calcium, and was refractory to treatment with transglutaminase inhibitors.ConclusionsThe persistence of this dimer even under harsh denaturing and reducing conditions indicates a strong interaction among pairs of subunits. This biochemical dimerization is particularly intriguing given that functional channels are almost certainly tetramers.

Properties of the Inner Pore Region of TRPV1 Channels Revealed by Block with Quaternary Ammoniums

The transient receptor potential vanilloid 1 (TRPV1) nonselective cationic channel is a polymodal receptor that activates in response to a wide variety of stimuli. To date, little structural information about this channel is available. Here, we used quaternary ammonium ions (QAs) of different sizes in an effort to gain some insight into the nature and dimensions of the pore of TRPV1. We found that all four QAs used, tetraethylammonium (TEA), tetrapropylammonium (TPrA), tetrabutylammonium, and tetrapentylammonium, block the TRPV1 channel from the intracellular face of the channel in a voltage-dependent manner, and that block by these molecules occurs with different kinetics, with the bigger molecules becoming slower blockers. We also found that TPrA and the larger QAs can only block the channel in the open state, and that they interfere with the channels activation gate upon closing, which is observed as a slowing of tail current kinetics. TEA does not interfere with the activation gate, indicating that this molecule can reside in its blocking site even when the channel is closed. The dependence of the rate constants on the size of the blocker suggests a size of around 10 Å for the inner pore of TRPV1 channels.