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.

Differential role of STIM1 and STIM2 during transient inward (T in) current generation and the maturation process in the Xenopus oocyte.

BACKGROUND: The Xenopus oocyte is a useful cell model to study Ca2+ homeostasis and cell cycle regulation, two highly interrelated processes. Here, we used antisense oligonucleotides to investigate the role in the oocyte of stromal interaction molecule (STIM) proteins that are fundamental elements of the store-operated calcium-entry (SOCE) phenomenon, as they are both sensors for Ca2+ concentration in the intracellular reservoirs as well as activators of the membrane channels that allow Ca2+ influx. RESULTS: Endogenous STIM1 and STIM2 expression was demonstrated, and their synthesis was knocked down 48-72 h after injecting oocytes with specific antisense sequences. Selective elimination of their mRNA and protein expression was confirmed by PCR and Western blot analysis, and we then evaluated the effect of their absence on two endogenous responses: the opening of SOC channels elicited by G protein-coupled receptor (GPCR)-activated Ca2+ release, and the process of maturation stimulated by progesterone. Activation of SOC channels was monitored electrically by measuring the T in response, a Ca2+-influx-dependent Cl- current, while maturation was assessed by germinal vesicle breakdown (GVBD) scoring and electrophysiology. CONCLUSIONS: It was found that STIM2, but not STIM1, was essential in both responses, and T in currents and GVBD were strongly reduced or eliminated in cells devoid of STIM2; STIM1 knockdown had no effect on the maturation process, but it reduced the T in response by 15 to 70%. Thus, the endogenous SOCE response in Xenopus oocytes depended mainly on STIM2, and its expression was necessary for entry into meiosis induced by progesterone.

Agonist-activated Ca2+ influx and Ca2+ -dependent Cl- channels in Xenopus ovarian follicular cells: functional heterogeneity within the cell monolayer.

Xenopus follicles are endowed with specific receptors for ATP, ACh, and AII, transmitters proposed as follicular modulators of gamete growth and maturation in several species. Here, we studied ion-current responses elicited by stimulation of these receptors and their activation mechanisms using the voltage-clamp technique. All agonists elicited Cl(-) currents that depended on coupling between oocyte and follicular cells and on an increase in intracellular Ca(2+) concentration ([Ca(2+) ](i)), but they differed in their activation mechanisms and in the localization of the molecules involved. Both ATP and ACh generated fast Cl(-) (F(Cl)) currents, while AII activated an oscillatory response; a robust Ca(2+) influx linked specifically to F(Cl) activation elicited an inward current (I(iw,Ca)) which was carried mainly by Cl(-) ions, through channels with a sequence of permeability of SCN(-) > I(-) > Br(-) > Cl(-). Like F(Cl), I(iw,Ca) was not dependent on oocyte [Ca(2+) ](i) ; instead both were eliminated by preventing [Ca(2+) ](i) increase in the follicular cells, and also by U73122 and 2-APB, drugs that inhibit the phospolipase C (PLC) pathway. The results indicated that F(Cl) and I(iw,Ca) were produced by the expected, PLC-stimulated Ca(2+) -release and Ca(2+) -influx, respectively, and by the opening of I(Cl(Ca)) channels located in the follicular cells. Given their pharmacological characteristics and behavior in conditions of divalent cation deprivation, Ca(2+) -influx appeared to be driven through store-operated, calcium-like channels. The AII response, which is also known to require PLC activation, did not activate I(iw,Ca) and was strictly dependent on oocyte [Ca(2+) ](i) increase; thus, ATP and ACh receptors seem to be expressed in a population of follicular cells different from that expressing AII receptors, which were coupled to the oocyte through distinct gap-junction channels.

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.

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.