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Acute Heat-Evoked Temperature Sensation Is Impaired but Not Abolished in Mice Lacking TRPV1 and TRPV3 Channels  [PDF]
Irène Marics, Pascale Malapert, Ana Reynders, Stéphane Gaillard, Aziz Moqrich
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0099828
Abstract: The discovery of heat-sensitive Transient Receptor Potential Vanilloid ion channels (ThermoTRPVs) greatly advanced our molecular understanding of acute and injury-evoked heat temperature sensation. ThermoTRPV channels are activated by partially overlapping temperatures ranging from warm to supra-threshold noxious heat. TRPV1 is activated by noxious heat temperature whereas TRPV3 can be activated by warm as well as noxious heat temperatures. Loss-of-function studies in single TRPV1 and TRPV3 knock-out mice have shown that heat temperature sensation is not completely abolished suggesting functional redundancies among these two channels and highlighting the need of a detailed analysis of TRPV1::TRPV3 double knock-out mice (V1V3dKO) which is hampered by the close proximity of the loci expressing the two channels. Here we describe the generation of a novel mouse model in which trpv1 and trpv3 genes have been inactivated using bacterial artificial chromosome (BAC)-based homologous recombination in embryonic stem cells. In these mice, using classical thermosensory tests such hot plate, tail flick and the thermotaxis gradient paradigms, we confirm that TRPV1 is the master channel for sensing noxious heat temperatures and identify a cooperative role of TRPV1 and TRPV3 for sensing a well-defined window of acute moderate heat temperature. Using the dynamic hot plate assay, we unravel an intriguing and unexpected pronounced escape behavior in TRPV1 knock-out mice that was attenuated in the V1V3dKO. Together, and in agreement with the temperature activation overlap between TRPV1 and TRPV3 channels, our data provide in vivo evidence of a cooperative role between skin-derived TRPV3 and primary sensory neurons-enriched TRPV1 in modulation of moderate and noxious heat temperature sensation and suggest that other mechanisms are required for heat temperature sensation.
High Yield Production and Refolding of the Double-Knot Toxin, an Activator of TRPV1 Channels  [PDF]
Chanhyung Bae, Jeet Kalia, Inhye Song, JeongHeon Yu, Ha Hyung Kim, Kenton J. Swartz, Jae Il Kim
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0051516
Abstract: A unique peptide toxin, named double-knot toxin (DkTx), was recently purified from the venom of the tarantula Ornithoctonus huwena and was found to stably activate TRPV1 channels by targeting the outer pore domain. DkTx has been shown to consist of two inhibitory cysteine-knot (ICK) motifs, referred to as K1 and K2, each containing six cysteine residues. Beyond this initial characterization, however, the structural and functional details about DkTx remains elusive in large part due to the lack of a high yielding methodology for the synthesis and folding of this cysteine-rich peptide. Here, we overcome this obstacle by generating pure DkTx in quantities sufficient for structural and functional analyses. Our methodology entails expression of DkTx in E. coli followed by oxidative folding of the isolated linear peptide. Upon screening of various oxidative conditions for optimizing the folding yield of the toxin, we observed that detergents were required for efficient folding of the linear peptide. Our synthetic DkTx co-eluted with the native toxin on HPLC, and irreversibly activated TRPV1 in a manner identical to native DkTx. Interestingly, we find that DkTx has two interconvertible conformations present in a 1:6 ratio at equilibrium. Kinetic analysis of DkTx folding suggests that the K1 and K2 domains influence each other during the folding process. Moreover, the CD spectra of the toxins shows that the secondary structures of K1 and K2 remains intact even after separating the two knots. These findings provide a starting point for detailed studies on the structural and functional characterization of DkTx and utilization of this toxin as a tool to explore the elusive mechanisms underlying the polymodal gating of TRPV1.
Differential expression of the capsaicin receptor TRPV1 and related novel receptors TRPV3, TRPV4 and TRPM8 in normal human tissues and changes in traumatic and diabetic neuropathy
Paul Facer, Maria A Casula, Graham D Smith, Christopher D Benham, Iain P Chessell, Chas Bountra, Marco Sinisi, Rolfe Birch, Praveen Anand
BMC Neurology , 2007, DOI: 10.1186/1471-2377-7-11
Abstract: We have studied TRPV1, TRPV3, TRPV4, and TRPM8 in nerves (n = 14) and skin from patients with nerve injury, avulsed dorsal root ganglia (DRG) (n = 11), injured spinal nerve roots (n = 9), diabetic neuropathy skin (n = 8), non-diabetic neuropathic nerve biopsies (n = 6), their respective control tissues, and human post mortem spinal cord, using immunohistological methods.TRPV1 and TRPV3 were significantly increased in injured brachial plexus nerves, and TRPV1 in hypersensitive skin after nerve repair, whilst TRPV4 was unchanged. TRPM8 was detected in a few medium diameter DRG neurons, and was unchanged in DRG after avulsion injury, but was reduced in axons and myelin in injured nerves. In diabetic neuropathy skin, TRPV1 expressing sub- and intra-epidermal fibres were decreased, as was expression in surviving fibres. TRPV1 was also decreased in non-diabetic neuropathic nerves. Immunoreactivity for TRPV3 was detected in basal keratinocytes, with a significant decrease of TRPV3 in diabetic skin. TRPV1-immunoreactive nerves were present in injured dorsal spinal roots and dorsal horn of control spinal cord, but not in ventral roots, while TRPV3 and TRPV4 were detected in spinal cord motor neurons.The accumulation of TRPV1 and TRPV3 in peripheral nerves after injury, in spared axons, matches our previously reported changes in avulsed DRG. Reduction of TRPV1 levels in nerve fibres in diabetic neuropathy skin may result from the known decrease of nerve growth factor (NGF) levels. The role of TRPs in keratinocytes is unknown, but a relationship to changes in NGF levels, which is produced by keratinocytes, deserves investigation. TRPV1 represents a more selective therapeutic target than other TRPs for pain and hypersensitivity, particularly in post-traumatic neuropathy.The cloning of the vanilloid receptor-1 (TRPV1) [1,2] has led to greater understanding of the mechanisms of thermosensation and the effects of capsaicin, the noxious component from chilli peppers. TRPV1 is a non
Increased capsaicin receptor TRPV1 in skin nerve fibres and related vanilloid receptors TRPV3 and TRPV4 in keratinocytes in human breast pain
Preethi Gopinath, Elaine Wan, Anita Holdcroft, Paul Facer, John B Davis, Graham D Smith, Chas Bountra, Praveen Anand
BMC Women's Health , 2005, DOI: 10.1186/1472-6874-5-2
Abstract: Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF).TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness (TRPV1 fibres / mm epidermis, median [range] – no pain group, n = 8, 0.69 [0–1.27]; pain group, n = 10, 2.15 [0.77–4.38]; p = 0.0009). Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] – no pain group, n = 6, 0.75 [0–2]; pain group, n = 11, 2 [1-3], p = 0.008; TRPV4, median [range] – no pain group, n = 6, [0–1]; pain group, n = 11, 1 [0.5–2], p = 0.014).Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study.Breast pain is a common problem, which can affect up to 70% of women [1]. Breast pain or mastalgia can be cyclical or non-cyclical. The cyclical type of breast pain has been attributed to sex hormonal changes through the menstrual cycle that may increase the size of the breast tissue, which stretches the internal structures and causes pain or soreness. Numerous studies have demonstrated variation in pain perception during the menstrual cycle [2-5]. Heat sensitivity is increased in th
Effect of cholesterol depletion on the pore dilation of TRPV1  [cached]
Jansson Erik T,Trkulja Carolina L,Ahemaiti Aikeremu,Millingen Maria
Molecular Pain , 2013, DOI: 10.1186/1744-8069-9-1
Abstract: The TRPV1 ion channel is expressed in nociceptors, where pharmacological modulation of its function may offer a means of alleviating pain and neurogenic inflammation processes in the human body. The aim of this study was to investigate the effects of cholesterol depletion of the cell on ion-permeability of the TRPV1 ion channel. The ion-permeability properties of TRPV1 were assessed using whole-cell patch-clamp and YO-PRO uptake rate studies on a Chinese hamster ovary (CHO) cell line expressing this ion channel. Prolonged capsaicin-induced activation of TRPV1 with N-methyl-D-glucamine (NMDG) as the sole extracellular cation, generated a biphasic current which included an initial outward current followed by an inward current. Similarly, prolonged proton-activation (pH 5.5) of TRPV1 under hypocalcemic conditions also generated a biphasic current including a fast initial current peak followed by a larger second one. Patch-clamp recordings of reversal potentials of TRPV1 revealed an increase of the ion-permeability for NMDG during prolonged activation of this ion channel under hypocalcemic conditions. Our findings show that cholesterol depletion inhibited both the second current, and the increase in ion-permeability of the TRPV1 channel, resulting from sustained agonist-activation with capsaicin and protons (pH 5.5). These results were confirmed with YO-PRO uptake rate studies using laser scanning confocal microscopy, where cholesterol depletion was found to decrease TRPV1 mediated uptake rates of YO-PRO. Hence, these results propose a novel mechanism by which cellular cholesterol depletion modulates the function of TRPV1, which may constitute a novel approach for treatment of neurogenic pain.
Acidification of rat TRPV1 alters the kinetics of capsaicin responses
Torben R Neelands, Michael F Jarvis, Ping Han, Connie R Faltynek, Carol S Surowy
Molecular Pain , 2005, DOI: 10.1186/1744-8069-1-28
Abstract: The vanilloid receptor 1 (TRPV1) is a member of the transient receptor potential family (TRP) of non-selective cation channels [1]. These receptors are activated by a variety of lipids, acidic conditions and temperatures above 42°C. TRPV1 channels are tetramers composed of subunits with six transmembrane spanning domains, a pore loop between TM5 and TM6, and large N- and C-terminal intracellular domains [2]. An intracellular domain just C-terminal to TM6 has been characterized as being important in the tetramerization of the channel and is coincident, in part, with the TRP box that is common among this family of ion channels [3].The structural features of TRPV1 suggest that the primary ligand interaction site(s) and important regulatory mechanisms for the channel are intracellular. Indeed, multiple mutagenesis studies have shown that distinct intracellular regions are necessary for the binding of the exogenous TRPV1 agonist, capsaicin [4-6] although an extracellular site may also contribute to capsaicin binding [7]. In contrast, extracellular site acidic residues have been implicated in proton activation (at pH < 6) and sensitization of TRPV1 [8]. Further evidence that TRPV1 activation mechanisms are different for capsaicin and protons is provided by site-directed mutagenesis studies that disrupt capsaicin activation of the channel but leave proton actions intact [9]. Despite these differences, there is evidence of some commonality in the gating of the channel in response to capsaicin or acidic pH activation [10].Under pathological conditions multiple agents may simultaneously influence the activity of TRPV1 receptors. For instance, inflammation, ischemia, and infections result in elevated proton concentrations that can reduce the pH below 6 in the surrounding tissues [11]. Acidic pH has been shown to stimulate a subpopulation of sensory nerves that are also activated by capsaicin [12]. In addition, disruption of the TRPV1 gene attenuates proton-induced excitation o
TRPV3 and TRPV4 ion channels are not major contributors to mouse heat sensation
Susan M Huang, Xiaoxin Li, YinYin Yu, Juan Wang, Michael J Caterina
Molecular Pain , 2011, DOI: 10.1186/1744-8069-7-37
Abstract: TRPV3 knockout mice on the C57BL6 background exhibited no obvious alterations in thermal preference behavior. On the 129S6 background, absence of TRPV3 resulted in a more restrictive range of occupancy centered around cooler floor temperatures. TRPV3 knockout mice showed no deficits in acute heat nociception on either background. Mice deficient in both TRPV3 and TRPV4 on a C57BL6 background showed thermal preference behavior similar to wild-type controls on the thermal gradient, and little or no change in acute heat nociception or inflammatory heat hyperalgesia. Masking of TRPV1 by the TRPV1 antagonist JNJ-17203212 did not reveal differences between C57BL6 animals deficient in TRPV3 and TRPV4, compared to their wild-type counterparts.Our results support the notion that TRPV3 and TRPV4 likely make limited and strain-dependent contributions to innocuous warm temperature perception or noxious heat sensation, even when TRPV1 is masked. These findings imply the existence of other significant mechanisms for heat perception.TRPV1 is a non-selective cation channel that can be activated by heat (at > ~42°C) or a wide range of chemical agonists such as capsaicin and acid [1]. TRPV1 is highly expressed in small diameter primary sensory neurons. Mice deficient for TRPV1 show blunted noxious heat perception in tests of acute heat nociception and inflammatory heat hyperalgesia [2]. Although these responses are impaired, mice devoid of TRPV1 are still able to respond to heat. For example, although TRPV1 knockout mice exhibit a 4-fold longer tail withdrawal latency at 50°C, they still withdraw their tails in response to hot water. In one TRPV1 knockout line, behavioral deficits were reported in thermal hyperalgesia but not in acute heat nociception [3]. Moreover, thermal selection behavior on a thermal gradient is normal in the absence of TRPV1 [4]. Significant residual responses to heat have also been observed in skin-nerve explants derived from TRPV1 knockout mice [2,5,6]. Thus,
Lattice model of protein conformations  [PDF]
S. Albeverio,S. V. Kozyrev
Quantitative Biology , 2012,
Abstract: We introduce a lattice model of protein conformations which is able to reproduce second structures of proteins (alpha--helices and beta--sheets). This model is based on the following two main ideas. First, we model backbone parts of amino acid residues in a peptide chain by edges in the cubic lattice which are not parallel to the coordinate axes. Second, we describe possible contacts of amino acid residues using a discrete model of the Ramachandran plot. This model allows to describe hydrogen bonds between the residues in the backbone of the peptide chain. In particular the lattice secondary structures have the correct structure of hydrogen bonds. We also take into account the side chains of amino acid residues and their interaction. The expression for the energy of conformation of a lattice protein which contains contributions from hydrogen bonds in the backbone of the peptide chain and from interaction of the side chains is proposed. The lattice secondary structures are local minima of the introduced energy.
Cooperative Transition between Open and Closed Conformations in Potassium Channels  [PDF]
Turkan Haliloglu ,Nir Ben-Tal
PLOS Computational Biology , 2008, DOI: 10.1371/journal.pcbi.1000164
Abstract: Potassium (K+) ion channels switch between open and closed conformations. The nature of this important transition was revealed by comparing the X-ray crystal structures of the MthK channel from Methanobacterium thermoautotrophicum, obtained in its open conformation, and the KcsA channel from Streptomyces lividans, obtained in its closed conformation. We analyzed the dynamic characteristics and energetics of these homotetrameric structures in order to study the role of the intersubunit cooperativity in this transition. For this, elastic models and in silico alanine-scanning mutagenesis were used, respectively. Reassuringly, the calculations manifested motion from the open (closed) towards the closed (open) conformation. The calculations also revealed a network of dynamically and energetically coupled residues. Interestingly, the network suggests coupling between the selectivity filter and the gate, which are located at the two ends of the channel pore. Coupling between these two regions was not observed in calculations that were conducted with the monomer, which emphasizes the importance of the intersubunit interactions within the tetrameric structure for the cooperative gating behavior of the channel.
Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes  [PDF]
Yulia Kushnareva,Alexander Y. Andreyev,Tomomi Kuwana,Donald D. Newmeyer
PLOS Biology , 2012, DOI: 10.1371/journal.pbio.1001394
Abstract: Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ~28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event.
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