Inhibition of TRPV1 by an antagonist in clinical trials is dependent on cholesterol binding.

TRP Vanilloid 1 (TRPV1) channel, one of the major members of the TRP family was discovered to play a critical role in pain sensation, particularly inflammatory pain, and is associated with hyperalgesia, an enhanced sensitivity to pain. A new study by Fanet al."Structural basis of TRPV1 inhibition by SAF312 and cholesterol" sheds new light on the mechanistic structural basis of TRPV1 inhibition by SAF312 (Libvatrep), a TRPV1 antagonist, currently in phase II clinical trials.

Membrane lipid modulations by methyl-β-cyclodextrin uncouple the Drosophila light-activated phospholipase C from TRP and TRPL channel gating.

Sterols are hydrophobic molecules, known to cluster signaling membrane-proteins in lipid rafts, while methyl-β-cyclodextrin (MβCD) has been a major tool for modulating membrane-sterol content for studying its effect on membrane proteins, including the transient receptor potential (TRP) channels. The Drosophila light-sensitive TRP channels are activated downstream of a G-protein-coupled phospholipase Cβ (PLC) cascade. In phototransduction, PLC is an enzyme that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol, inositol-tris-phosphate, and protons, leading to TRP and TRP-like (TRPL) channel openings.

Proteome-wide quantitative analysis of redox cysteine availability in the Drosophila melanogaster eye reveals oxidation of phototransduction machinery during blue light exposure and age.

The retina is one of the highest oxygen-consuming tissues because visual transduction and light signaling processes require large amounts of ATP. Thus, because of the high energy demand, oxygen-rich environment, and tissue transparency, the eye is susceptible to excess production of reactive oxygen species (ROS) resulting in oxidative stress. Oxidative stress in the eye is associated with the development and progression of ocular diseases including cataracts, glaucoma, age-related macular degeneration, and diabetic retinopathy.

Diacylglycerol Activates the Light Sensitive Channel TRPL Expressed in HEK Cells.

Physiological activation by light of the TRP and TRP-like (TRPL) channels requires the activation of phospholipase Cβ (PLC). The hydrolysis of phosphatidylinositol 4,5, bisphosphate (PIP) by PLC is a crucial step in the still-unclear light activation, while the generation of Diacylglycerol (DAG) by PLC seems to be involved. In this study, we re-examined the ability of a DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) to activate the TRPL channels expressed in HEK cells.

Nociception and pain in humans lacking a functional TRPV1 channel.

BACKGROUNDChronic pain is a debilitating illness with currently limited therapy, in part due to difficulties in translating treatments derived from animal models to patients. The transient receptor potential vanilloid 1 (TRPV1) channel is associated with noxious heat detection and inflammatory pain, and reports of adverse effects in human trials have hindered extensive efforts in the clinical development of TRPV1 antagonists as novel pain relievers.METHODSWe examined 2 affected individuals (A1 and A2) carrying a homozygous missense mutation in TRPV1, rendering the channel nonfunctional.

The light-activated TRP channel: the founding member of the TRP channel superfamily.

The light-activated Transient Receptor Potential (TRP) channel is the founding member of a large and diverse family of channel proteins. The TRP (dTRP) channel, which generates the electrical response to light has been investigated in a great detail two decades before the first mammalian TRP channel was discovered. Thus, dTRP is unique among members of the TRP channel superfamily because its physiological role and the enzymatic cascade underlying its activation are established.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors.

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies.

The Role of Membrane Lipids in Light-Activation of TRP Channels.

Transient Receptor Potential (TRP) channels constitute a large superfamily of polymodal channel proteins with diverse roles in many physiological and sensory systems that function both as ionotropic and metabotropic receptors. From the early days of TRP channel discovery, membrane lipids were suggested to play a fundamental role in channel activation and regulation. A prominent example is the TRP and TRP-like (TRPL) channels, which are predominantly expressed in the visual system of .

Knockdown of Dehydrodolichyl Diphosphate Synthase in the Retina Leads to a Unique Pattern of Retinal Degeneration.

Dehydrodolichyl diphosphate synthase (DHDDS) is a ubiquitously expressed enzyme that catalyzes -prenyl chain elongation to produce the poly-prenyl backbone of dolichol. It appears in all tissues including the nervous system and it is a highly conserved enzyme that can be found in all animal species. Individuals who have biallelic missense mutations in the gene are presented with non-syndromic retinitis pigmentosa with unknown underlying mechanism.

Modulation of Transient Receptor Potential C Channel Activity by Cholesterol.

Changes of cholesterol level in the plasma membrane of cells have been shown to modulate ion channel function. The proposed mechanisms underlying these modulations include association of cholesterol to a single binding site at a single channel conformation, association to a highly flexible cholesterol binding site adopting multiple poses, and perturbation of lipid rafts. These perturbations have been shown to induce reversible targeting of mammalian transient receptor potential C (TRPC) channels to the cholesterol-rich membrane environment of lipid rafts.

Comparative docking analysis of cholesterol analogs to ion channels to discriminate between stereospecific binding vs. stereospecific response.

Cholesterol is a major component of the membrane and a key regulator of many ion channels. Multiple studies showed that cholesterol regulates ion channels in a stereospecific manner, with cholesterol but not its chiral isomers having a functional effect. This stereospecificity has been universally attributed to the specificity of cholesterol binding, with the assumption that only native cholesterol binds to the channels whereas its isomers do not.

The Drosophila light-activated TRP and TRPL channels - Targets of the phosphoinositide signaling cascade.

The Drosophila light-activated Transient Receptor Potential (TRP) channel is the founding member of a large and diverse family of channel proteins. It is now established that TRP channels are evolutionarily conserved and are found in many organisms and tissues. This review outlines the progress made in our understanding of Drosophila phototransduction with a focus on the light sensitive TRP channels.

Depletion of Membrane Cholesterol Suppresses Drosophila Transient Receptor Potential-Like (TRPL) Channel Activity.

Cholesterol is an essential compound of higher eukaryotic cell membranes and a known modulator of ion channel activity. Changes in phospholipids and cholesterol composition of cell membranes are known to alter the activity of ion channels. However, there is little knowledge on the effects of cholesterol on transient receptor potential (TRP) channels.

The latency of the light response is modulated by the phosphorylation state of Drosophila TRP at a specific site.

Drosophila photoreceptors respond to oscillating light of high frequency (∼100 Hz), while increasing the oscillating light intensity raises the maximally detected frequency. Recently, we reported that dephosphorylation of the light-activated TRP ion channel at S936 is a fast, graded, light-, and Ca-dependent process. We further found that this process affects the detection limit of high frequency oscillating light.

Electrophysiological Method for Whole-cell Voltage Clamp Recordings from Drosophila Photoreceptors.

Whole-cell voltage clamp recordings from Drosophila melanogaster photoreceptors have revolutionized the field of invertebrate visual transduction, enabling the use of D. melanogaster molecular genetics to study inositol-lipid signaling and Transient Receptor Potential (TRP) channels at the single-molecule level. A handful of labs have mastered this powerful technique, which enables the analysis of the physiological responses to light under highly controlled conditions.

The Phosphorylation State of the TRP Channel Modulates the Frequency Response to Oscillating Light .

photoreceptors respond to oscillating light of high frequency (∼100 Hz), while the detected maximal frequency is modulated by the light rearing conditions, thus enabling high sensitivity to light and high temporal resolution. However, the molecular basis for this adaptive process is unclear. Here, we report that dephosphorylation of the light-activated transient receptor potential (TRP) ion channel at S936 is a fast, graded, light-dependent, and Ca-dependent process that is partially modulated by the rhodopsin phosphatase retinal degeneration C (RDGC).

Ectopic Expression of Mouse Melanopsin in Photoreceptors Reveals Fast Response Kinetics and Persistent Dark Excitation.

The intrinsically photosensitive M1 retinal ganglion cells (ipRGC) initiate non-image-forming light-dependent activities and express the melanopsin (OPN4) photopigment. Several features of ipRGC photosensitivity are characteristic of fly photoreceptors. However, the light response kinetics of ipRGC is much slower due to unknown reasons.

Next Generation RNA Sequencing Analysis Reveals Expression of a Transient EMT Profile During Early Organization of MCF10A Cells in 3D.

RNA sequencing is a technique widely used to identify and characterize gene expression patterns. We demonstrate that this method can be applied to screen expression profiles in mammary epithelial cells cultured in 3D, supported by a natural laminin-rich extracellular matrix, but requires several specific steps in the preparation of the RNA samples. Here we describe the use of RNA sequencing to analyze mRNA patterns in MCF10A human mammary epithelial cells cultured under 3D conditions in a laminin-rich extracellular matrix.

Electroretinogram (ERG) Recordings from .

Phototransduction is a process in which light is converted into electrical signals used by the central nervous system. Invertebrate phototransduction is a process mediated by the phosphoinositide signaling cascade, characterized by Phospholipase C (PLC) as the effector enzyme and the Transient Receptor Potential (TRP) channels as its target. The great advantage of using invertebrate photoreceptors is the simplicity of the preparation, the ease of light stimulation, the robust expression of key molecular components, and most importantly, the ability to apply the power of molecular genetics.

Functional cooperation between the IP3 receptor and phospholipase C secures the high sensitivity to light of Drosophila photoreceptors in vivo.

Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. We have recently shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cβ (PLCβ) activity level required for bump generation.

A new genetic model for calcium induced autophagy and ER-stress in Drosophila photoreceptor cells.

Cytoplasmic Ca2+ overload is known to trigger autophagy and ER-stress. Furthermore, ER-stress and autophagy are commonly associated with degenerative pathologies, but their role in disease progression is still a matter of debate, in part, owing to limitations of existing animal model systems. The Drosophila eye is a widely used model system for studying neurodegenerative pathologies.

Systems biology-embedded target validation: improving efficacy in drug discovery.

The pharmaceutical industry is faced with a range of challenges with the ever-escalating costs of drug development and a drying out of drug pipelines. By harnessing advances in -omics technologies and moving away from the standard, reductionist model of drug discovery, there is significant potential to reduce costs and improve efficacy. Embedding systems biology approaches in drug discovery, which seek to investigate underlying molecular mechanisms of potential drug targets in a network context, will reduce attrition rates by earlier target validation and the introduction of novel targets into the currently stagnant market.