Sara L. Morales-Lázaro

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.

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.

Expression of LPP3 in Bergmann glia is required for proper cerebellar sphingosine‐1‐phosphate metabolism/signaling and development

Bioactive lipids serve as intracellular and extracellular mediators in cell signaling in normal and pathological conditions. Here we describe that an important regulator of some of these lipids, the lipid phosphate phosphatase‐3 (LPP3), is abundantly expressed in specific plasma membrane domains of Bergmann glia (BG), a specialized type of astrocyte with key roles in cerebellum development and physiology. Mice selectively lacking expression of LPP3/Ppap2b in the nervous system are viable and fertile but exhibit defects in postnatal cerebellum development and modifications in the cytoarchitecture and arrangement of BG with a mild non‐progressive motor coordination defect. Lipid and gene profiling studies in combination with pharmacological treatments suggest that most of these effects are associated with alterations in sphingosine‐1‐phosphate (S1P) metabolism and signaling. Altogether our data indicate that LPP3 participates in several aspects of neuron‐glia communication required for proper cerebellum development.

A role for β-dystroglycan in the organization and structure of the nucleus in myoblasts

We recently characterized a nuclear import pathway for β-dystroglycan; however, its nuclear role remains unknown. In this study, we demonstrate for the first time, the interaction of β-dystroglycan with distinct proteins from different nuclear compartments, including the nuclear envelope (NE) (emerin and lamins A/C and B1), splicing speckles (SC35), Cajal bodies (p80-coilin), and nucleoli (Nopp140). Electron microscopy analysis revealed that β-dystroglycan localized in the inner nuclear membrane, nucleoplasm, and nucleoli. Interestingly, downregulation of β-dystroglycan resulted in both mislocalization and decreased expression of emerin and lamin B1, but not lamin A/C, as well in disorganization of nucleoli, Cajal bodies, and splicing speckles with the concomitant decrease in the levels of Nopp140, and p80-coilin, but not SC35. Quantitative reverse transcription PCR and cycloheximide-mediated protein arrest assays revealed that β-dystroglycan deficiency did not change mRNA expression of NE proteins emerin and lamin B1 bud did alter their stability, accelerating protein turnover. Furthermore, knockdown of β-dystroglycan disrupted NE-mediated processes including nuclear morphology and centrosome-nucleus linkage, which provides evidence that β-dystroglycan association with NE proteins is biologically relevant. Unexpectedly, β-dystroglycan-depleted cells exhibited multiple centrosomes, a characteristic of cancerous cells. Overall, these findings imply that β-dystroglycan is a nuclear scaffolding protein involved in nuclear organization and NE structure and function, and that might be a contributor to the biogenesis of nuclear envelopathies.

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

Background: The TRPV1 ion channel can be regulated by negatively charged lipids. Results: TRPV1 shows specificity for LPA analogs containing monounsaturated hydrocarbon chains with a negatively charged phosphate, cyclic phosphate, and thiophosphate headgroup. Conclusion: TRPV1 activation is highly restricted to natural lipids with oleyl or oleoyl side chains. Significance: Production of endogenous C18:1 lysophospholipids can selectively trigger activation of TRPV1 and nociceptive neuronal responses. 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.

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.

Lack of lipid phosphate phosphatase-3 in embryonic stem cells compromises neuronal differentiation and neurite outgrowth

Background: Bioactive lipids such as lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P) have been recently described as important regulators of pluripotency and differentiation of embryonic stem (ES) cells and neural progenitors. Due to the early lethality of LPP3, an enzyme that regulates the levels and biological activities of the aforementioned lipids, it has been difficult to assess its participation in early neural differentiation and neuritogenesis. Results: We find that Ppap2b−/− (Lpp3−/−) ES cells differentiated in vitro into spinal neurons show a considerable reduction in the amount of neural precursors and young neurons formed. In addition, differentiated Lpp3−/− neurons exhibit impaired neurite outgrowth. Surprisingly, when Lpp3−/− ES cells were differentiated, an unexpected appearance of smooth muscle actin‐positive cells was observed, an event that was partially dependent upon phosphorylated sphingosines. Conclusions: Our data show that LPP3 plays a fundamental role during spinal neuron differentiation from ES and that it also participates in regulating neurite and axon outgrowth. Developmental Dynamics 241:953–964, 2012.

Role for the TRPV1 Channel in Insulin Secretion from Pancreatic Beta Cells

Transient receptor potential channels have been put forward as regulators of insulin secretion. A role for the TRPV1 ion channel in insulin secretion has been suggested in pancreatic beta cell lines. We explored whether TRPV1 is functionally expressed in RINm5F and primary beta cells from neonate and adult rats. We examined if capsaicin could activate cationic non-selective currents. Our results show that TRPV1 channels are not functional in insulin-secreting cells, since capsaicin did not produce current activation, not even under culture conditions known to induce the expression of other ion channels in these cells. Although TRPV1 channels seem to be irrelevant for the physiology of isolated beta cells, they may play a role in glucose homeostasis acting through the nerve fibers that regulate islet function. At the physiological level, we observed that Trpv1−/− mice presented lower fasting insulin levels than their wild-type littermates, however, we did not find differences between these experimental groups nor in the glucose tolerance test or in the insulin secretion. However, we did find that the Trpv1−/− mice exhibited a higher insulin sensitivity compared to their wild-type counterparts. Our results demonstrate that TRPV1 does not contribute to glucose-induced insulin secretion in beta cells as was previously thought, but it is possible that it may control insulin sensitivity.

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

Significance The TRPV1 ion channel has been widely associated with the generation of painful responses. The responses of cells expressing this ion channel and, presumably, the overall pain response of an organism may be regulated by controlling the amount of TRPV1 channels in the plasma membrane. TRPV1 levels can be regulated by its interaction with intracellular proteins, but there are no studies describing TRPV1 or any other mammalian TRP channel’s association with chaperones or how these interactions may affect the perception of pain. Here, we show that TRPV1-dependent pain is decreased through Sig-1R antagonism by progesterone and determine the presence of a physical interaction between these two proteins that may reduce pain under physiological conditions such as pregnancy. 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.

Organic Toxins as Tools to Understand Ion Channel Mechanisms and Structure

Ion channels constitute a varied class of membrane proteins with pivotal roles in cellular physiology and that are fundamental for neuronal signaling, hormone secretion and muscle contractility. Hence, it is not unanticipated that toxins from diverse organisms have evolved to modulate the activity of ion channels. For instance, animals such as cone snails, scorpions, spiders and snakes use toxins to immobilize and capture their prey by affecting ion channel function. This is a beautiful example of an evolutionary process that has led to the development of an injection apparatus from predators and to the existence of toxins with high affinity and specificity for a given target. Toxins have been used in the field of ion channel biophysics for several decades to gain insight into the gating mechanisms and the structure of ion channels. Through the use of these peptides, much has been learned about the ion conduction pathways, voltage-sensing mechanisms, pore sizes, kinetics, inactivation processes, etc. This review examines an assortment of toxins that have been used to study different ion channels and describes some key findings about the structure-function relationships in these proteins through the details of the toxin-ion channel interactions.