Andrés Jara-Oseguera

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 Allosteric Coupling on Thermal Activation of Thermo-TRP Channels

Thermo-transient receptor potential channels display outstanding temperature sensitivity and can be directly gated by low or high temperature, giving rise to cold- and heat-activated currents. These constitute the molecular basis for the detection of changes in ambient temperature by sensory neurons in animals. The mechanism that underlies the temperature sensitivity in thermo-transient receptor potential channels remains unknown, but has been associated with large changes in standard-state enthalpy (ΔH(o)) and entropy (ΔS(o)) upon channel gating. The magnitude, sign, and temperature dependence of ΔH(o) and ΔS(o), the last given by an associated change in heat capacity (ΔCp), can determine a channels temperature sensitivity and whether it is activated by cooling, heating, or both, if ΔCp makes an important contribution. We show that in the presence of allosteric gating, other parameters, besides ΔH(o) and ΔS(o), including the gating equilibrium constant, the strength- and temperature dependence of the coupling between gating and the temperature-sensitive transitions, as well as the ΔH(o)/ΔS(o) ratio associated with them, can also determine a channels temperature-dependent activity, and even give rise to channels that respond to both cooling and heating in a ΔCp-independent manner.

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.

TRP Channel Gating Physiology

Transient Receptor Potential (TRP) cation channels participate in several processes of vital importance in cell and organism physiology, and have been demonstrated to participate in the detection of sensory stimuli. The thermo TRPs reviewed: TRPV1 (vanilloid 1), TRPM8 (melastatin 8) and TRPA1 (ankyrin-like 1) are known to integrate different chemical and physical stimuli such as changes in temperature and sensing different irritant or pungent compounds. However, despite the physiological importance of these channels the mechanisms by which they detect incoming stimuli, how the sensing domains are coupled to channel gating and how these processes are connected to specific structural regions in the channel are not fully understood, but valuable information is available. Many sites involved in agonist detection have been characterized and gating models that describe many features of the channels behavior have been put forward. In this review we will survey some of the key findings concerning the structural and molecular mechanisms of TRPV1, TRPA1 and TRPM8 activation.

Uncoupling Charge Movement from Channel Opening in Voltage-gated Potassium Channels by Ruthenium Complexes

The Kv2.1 channel generates a delayed-rectifier current in neurons and is responsible for modulation of neuronal spike frequency and membrane repolarization in pancreatic β-cells and cardiomyocytes. As with other tetrameric voltage-activated K+-channels, it has been proposed that each of the four Kv2.1 voltage-sensing domains activates independently upon depolarization, leading to a final concerted transition that causes channel opening. The mechanism by which voltage-sensor activation is coupled to the gating of the pore is still not understood. Here we show that the carbon-monoxide releasing molecule 2 (CORM-2) is an allosteric inhibitor of the Kv2.1 channel and that its inhibitory properties derive from the CORM-2 ability to largely reduce the voltage dependence of the opening transition, uncoupling voltage-sensor activation from the concerted opening transition. We additionally demonstrate that CORM-2 modulates Shaker K+-channels in a similar manner. Our data suggest that the mechanism of inhibition by CORM-2 may be common to voltage-activated channels and that this compound should be a useful tool for understanding the mechanisms of electromechanical coupling.

Molecular Mechanisms of TRPV1 Channel Activation

Transient Receptor Potential (TRP) cation channels participate in various fundamental processes in cell- and organism-physiology in unicellular eukaryotes, invertebrates and vertebrates. Interestingly, many TRP channels function as detectors of sensory stimuli. The TRPV1 (vanilloid 1) channel serves as an integrator of noxious chemical and physical stimuli known to cause irritation and pain, such as elevated temperatures, acids, and irritant chemical compounds, and its activation has been linked to acute nociceptive pain and neurogenic inflammation. The mechanisms by which the channel detects incoming stimuli, how the sensing domains are coupled to channel gating and how these processes are connected to specific structural regions in the channel are not fully understood, but valuable information is available. Many sites in- volved in agonist detection have been characterized and gating models that describe many features of the channels behav- ior have been put forward. Structural and functional information indicates TRP channels are similar to voltage-activated potassium channels, with a tetrameric organization and six-transmembrane-region subunits, a pore domain with multi-ion binding properties and an intracellular S6 gate that seems to be the point of convergence of the many activation modalities leading to the opening of the ion conduction pathway. Furthermore, TRPV1 expression is altered in various disease states and TRPV1 gene polymorphism was speculated to play a role in pain sensation. The complex activation and regulation of TRPV1 may have important implications for drug development.