Modes of action of lysophospholipids as endogenous activators of the TRPV4 ion channel.

The Transient Receptor Potential Vanilloid 4 (TRPV4) channel has been shown to function in many physiological and pathophysiological processes. Despite abundant information on its importance in physiology, very few endogenous agonists for this channel have been described, and very few underlying mechanisms for its activation have been clarified. TRPV4 is expressed by several types of cells, such as vascular endothelial, and skin and lung epithelial cells, where it plays pivotal roles in their function. In the present study, we show that TRPV4 is activated by lysophosphatidic acid (LPA) in both endogenous and heterologous expression systems, pinpointing this molecule as one of the few known endogenous agonists for TRPV4. Importantly, LPA is a bioactive glycerophospholipid, relevant in several physiological conditions, including inflammation and vascular function, where TRPV4 has also been found to be essential. Here we also provide mechanistic details of the activation of TRPV4 by LPA and another glycerophospholipid, lysophosphatidylcholine (LPC), and show that LPA directly interacts with both the N- and C-terminal regions of TRPV4 to activate this channel. Moreover, we show that LPC activates TRPV4 by producing an open state with a different single-channel conductance to that observed with LPA. Our data suggest that the activation of TRPV4 can be finely tuned in response to different endogenous lipids, highlighting this phenomenon as a regulator of cell and organismal physiology. KEY POINTS: The Transient Receptor Potential Vaniloid (TRPV) 4 ion channel is a widely distributed protein with important roles in normal and disease physiology for which few endogenous ligands are known. TRPV4 is activated by a bioactive lipid, lysophosphatidic acid (LPA) 18:1, in a dose-dependent manner, in both a primary and a heterologous expression system. Activation of TRPV4 by LPA18:1 requires residues in the N- and C-termini of the ion channel. Single-channel recordings show that TRPV4 is activated with a decreased current amplitude (conductance) in the presence of lysophosphatidylcholine (LPC) 18:1, while LPA18:1 and GSK101 activate the channel with a larger single-channel amplitude. Distinct single-channel amplitudes produced by LPA18:1 and LPC18:1 could differentially modulate the responses of the cells expressing TRPV4 under different physiological conditions.

TRPV1 channels and the progesterone receptor Sig-1R interact to regulate pain.

The Transient Receptor Potential Vanilloid 1 (TRPV1) ion channel is expressed in nociceptors where, when activated by chemical or thermal stimuli, it functions as an important transducer of painful and itch-related stimuli. Although the interaction of TRPV1 with proteins that regulate its function has been previously explored, their modulation by chaperones has not been elucidated, as is the case for other mammalian TRP channels. Here we show that TRPV1 physically interacts with the Sigma 1 Receptor (Sig-1R), a chaperone that binds progesterone, an antagonist of Sig-1R and an important neurosteroid associated to the modulation of pain. Antagonism of Sig-1R by progesterone results in the down-regulation of TRPV1 expression in the plasma membrane of sensory neurons and, consequently, a decrease in capsaicin-induced nociceptive responses. This is observed both in males treated with a synthetic antagonist of Sig-1R and in pregnant females where progesterone levels are elevated. This constitutes a previously undescribed mechanism by which TRPV1-dependent nociception and pain can be regulated.

Structural determinants of the transient receptor potential 1 (TRPV1) channel activation by phospholipid analogs.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a polymodal protein that responds to various stimuli, including capsaicin (the pungent compound found in chili peppers), extracellular acid, and basic intracellular pH, temperatures close to 42 °C, and several lipids. Lysophosphatidic acid (LPA), an endogenous lipid widely associated with neuropathic pain, is an agonist of the TRPV1 channel found in primary afferent nociceptors and is activated by other noxious stimuli. Agonists or antagonists of lipid and other chemical natures are known to possess specific structural requirements for producing functional effects on their targets. To better understand how LPA and other lipid analogs might interact and affect the function of TRPV1, we set out to determine the structural features of these lipids that result in the activation of TRPV1. By changing the acyl chain length, saturation, and headgroup of these LPA analogs, we established strict requirements for activation of TRPV1. Among the natural LPA analogs, we found that only LPA 18:1, alkylglycerophosphate 18:1, and cyclic phosphatidic acid 18:1, all with a monounsaturated C18 hydrocarbon chain activate TRPV1, whereas polyunsaturated and saturated analogs do not. Thus, TRPV1 shows a more restricted ligand specificity compared with LPA G-protein-coupled receptors. We synthesized fatty alcohol phosphates and thiophosphates and found that many of them with a single double bond in position Δ9, 10, or 11 and Δ9 cyclopropyl group can activate TRPV1 with efficacy similar to capsaicin. Finally, we developed a pharmacophore and proposed a mechanistic model for how these lipids could induce a conformational change that activates TRPV1.

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.

Unconventional interactions of the TRPV4 ion channel with beta-adrenergic receptor ligands.

The transient receptor potential vanilloid 4 (TRPV4) ion channel is present in different tissues including those of the airways. This channel is activated in response to stimuli such as changes in temperature, hypoosmotic conditions, mechanical stress, and chemicals from plants, lipids, and others. TRPV4's overactivity and/or dysfunction has been associated with several diseases, such as skeletal dysplasias, neuromuscular disorders, and lung pathologies such as asthma and cardiogenic lung edema and COVID-19-related respiratory malfunction. TRPV4 antagonists and blockers have been described; nonetheless, the mechanisms involved in achieving inhibition of the channel remain scarce, and the search for safe use of these molecules in humans continues. Here, we show that the widely used bronchodilator salbutamol and other ligands of ß-adrenergic receptors inhibit TRPV4's activation. We also demonstrate that inhibition of TRPV4 by salbutamol is achieved through interaction with two residues located in the outer region of the pore and that salbutamol leads to channel closing, consistent with an allosteric mechanism. Our study provides molecular insights into the mechanisms that regulate the activity of this physiopathologically important ion channel.

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-Ile(585) 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.

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.

Different agonists induce distinct single-channel conductance states in TRPV1 channels.

The TRPV1 ion channel is a membrane protein that is expressed in primary afferent nociceptors, where it is activated by a diverse array of stimuli. Our prior work has shown that this channel is activated by lysophosphatidic acid (LPA), an unsaturated lysophospholipid that is produced endogenously and released under certain pathophysiological conditions, resulting in the sensation of pain. Macroscopic currents activated by saturating concentrations of LPA applied to excised membrane patches are larger in magnitude than those activated by saturating concentrations of capsaicin, which causes near-maximal TRPV1 open probability. Here we show that activation of TRPV1 by LPA is associated with a higher single-channel conductance than activation by capsaicin. We also observe that the effects of LPA on TRPV1 are not caused by an increase in the surface charge nor are they mimicked by a structurally similar lipid, ruling out the contribution of change in membrane properties. Finally, we demonstrate that the effects of LPA on the unitary conductance of TRPV1 depend upon the presence of a positively charged residue in the C terminus of the channel, suggesting that LPA induces a distinct conformational change.

Inhibition of TRPV1 channels by a naturally occurring omega-9 fatty acid reduces pain and itch.

The transient receptor potential vanilloid 1 (TRPV1) ion channel is mainly found in primary nociceptive afferents whose activity has been linked to pathophysiological conditions including pain, itch and inflammation. Consequently, it is important to identify naturally occurring antagonists of this channel. Here we show that a naturally occurring monounsaturated fatty acid, oleic acid, inhibits TRPV1 activity, and also pain and itch responses in mice by interacting with the vanilloid (capsaicin)-binding pocket and promoting the stabilization of a closed state conformation. Moreover, we report an itch-inducing molecule, cyclic phosphatidic acid, that activates TRPV1 and whose pruritic activity, as well as that of histamine, occurs through the activation of this ion channel. These findings provide insights into the molecular basis of oleic acid inhibition of TRPV1 and also into a way of reducing the pathophysiological effects resulting from its activation.

The helical character of the S6 segment of TRPV1 channels.

The era of molecular structure of ion channels has revealed that their transmembrane segments are alpha helices, as was suspected from hydropathy analysis and experimental data. TRP channels are recent additions to the known families of ion channels, and little structural data is available. In a recent work, we explored the conformational changes occurring at the putative S6 segment of TRPV1 channels; and we observed a periodicity of chemical modification of residues suggestive of an alpha helical structure. Further analysis of the periodicity of the disposition of hydrophobic residues in the S6 segment, suggests that the general architecture of the TRPV1 S6 segment, is very similar to that of voltage-dependent channels of known structure--an aqueous cavity lined by an amphipathic alpha helix, with most of the hydrophobic residues pointing into it.

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.

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 channel's 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 A for the inner pore of TRPV1 channels.