Browsing by Author "Liedtke, Wolfgang B"
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Item Open Access Functional Analysis of Ion Selectivity and Permeation Mechanisms of the C. elegans TRPV Channel OSM-9(2011) Lindy, Amanda SueFor all organisms, the ability to sense and react to noxious environments is fundamental to their survival. For multi-celled organisms this process generally involves a nervous system and an extensive network of signal transduction pathways. TRPV ion channels have been shown to participate in signal transduction in response to noxious stimuli. At the cellular level these channels function in sensing of mechanical, thermal, and osmotic stimuli, and at the organismal level they function in homeostasis and nociception. TRPV ion channels participate in nociceptive signal transduction via cation influx, but exactly how these channels function at a mechanistic level and lead to activation of the cell or induction of a specific behavior is elusive. Previous research has shown that the pore-forming unit of an ion channel is critical for channel regulation, gating, ion selectivity, and ion permeation. Various regulatory domains have been identified to date in the pore-forming unit of TRP channels and a clearer picture of channel gating is beginning to emerge, but less is known about ion permeation.
To better understand the specific domains that are critical to ion capture, selectivity, and permeation in TRPV channels, we investigated the function of these regions using the C. elegans TRPV channel OSM-9 in vivo, and the mammalian TRPV channel TRPV4 in heterologous cell culture. OSM-9 is the functional ortholog of mammalian TRPV4 and it is likely that critical domains identified in OSM-9 are functionally conserved in TRPV4 and play a similar role in other TRPV channels. OSM-9 is expressed in the ASH neurons and is responsible for all of the behaviors initiated by that cell. The stereotypical avoidance behavior mediated by ASH, in response to noxious stimuli, serves as a model for nociception in vertebrates. As OSM-9 is necessary for all of these behavioral responses, activation of ASH acts as a read-out for OSM-9 function.
Through targeted mutagenesis of the OSM-9 loop domains and transgenic expression directed to the ASH head sensory neurons in an osm-9 null background, we discovered a critical role for the amino acids both N- and C- terminal to the pore helix in osmotic avoidance behavior. We confirmed the existence of a selectivity filter C-terminal to the pore helix and revealed that the turret is critical for channel function, possibly as a component of the inactivation gate.
We first identified the boundaries of the selectivity filter to be M601-F609. We also determined what properties of those residues were critical to Ca2+ and Na+ selectivity. In vivo Ca2+ imaging strongly suggested that residues Y604, D605, and F609 are critical for Ca2+ entry into the cell. Patch-clamp electrophysiology of a chimeric ion channel consisting largely of rat TRPV4, but encompassing transmembranes 5 through 6 of OSM-9, revealed that OSM-9 conducts both Ca2+ and Na+. Mutation Y604G disrupted both Ca2+ and Na+ conductance, whereas mutations Y604F and Y606A increased or maintained Na+ conductance and severely reduced Ca2+ conductance, while maintaining avoidance behavior. Homology modeling of OSM-9, based on an alignment of OSM-9 to Kv1.2, suggests that Y604 and F609 serve structural roles in maintaining filter constraints. Thus, aromatic and negative residues in the OSM-9 selectivity filter are critical to ion permeation and selectivity.
Our studies involving the selectivity filter support previous research that the selectivity filter is critical for TRP channel function. We also provide evidence that the selectivity filter is critical for nocifensive animal behavior. Fewer studies, however, have investigated the TM5-pore helix linker, known as the turret. The turret is believed to function in the binding of ligands and toxins in K+ channels, and more recently was suggested to be critical for temperature sensing in TRPV1. We investigated the function of the turret residues in several sensory submodalities of the OSM-9 channel and found that all deletions tested result in channel defects, including gain- and loss-of-function phenotypes. Several charge reversal mutations in the OSM-9 turret also resulted in partial defects. The discovery of a gain-of-function mutation indicates that the turret functions in gating. When the turret is mutated in this way, the channel is unable to enter into the inactivated state, allowing continued ion influx after repeated stimulation. The loss-of-function phenotypes indicate that the secondary structure of the turret is critical to the function of the channel, and perhaps gating. These findings, combined with the observed charge-reversal defects, support the conclusion that the turret is necessary for transducing conformational changes in response to stimuli.
Our in vivo findings on the external pore forming structures increase the understanding of ion permeation in TRP channels and clarify mechanisms of activation in nociceptor neurons in vivo. Furthermore, these studies enhance our insights into evolution of mammalian nociception in view of the established functional orthology of OSM-9 and TRPV4.
Item Open Access Inflammatory signaling sensitizes Piezo1 mechanotransduction in articular chondrocytes as a pathogenic feed-forward mechanism in osteoarthritis.(Proceedings of the National Academy of Sciences of the United States of America, 2021-03) Lee, Whasil; Nims, Robert J; Savadipour, Alireza; Zhang, Qiaojuan; Leddy, Holly A; Liu, Fang; McNulty, Amy L; Chen, Yong; Guilak, Farshid; Liedtke, Wolfgang BOsteoarthritis (OA) is a painful and debilitating condition of synovial joints without any disease-modifying therapies [A. M. Valdes, T. D. Spector, Nat. Rev. Rheumatol. 7, 23-32 (2011)]. We previously identified mechanosensitive PIEZO channels, PIEZO1 and PIEZO2, both expressed in articular cartilage, to function in chondrocyte mechanotransduction in response to injury [W. Lee et al., Proc. Natl. Acad. Sci. U.S.A. 111, E5114-E5122 (2014); W. Lee, F. Guilak, W. Liedtke, Curr. Top. Membr. 79, 263-273 (2017)]. We therefore asked whether interleukin-1-mediated inflammatory signaling, as occurs in OA, influences Piezo gene expression and channel function, thus indicative of maladaptive reprogramming that can be rationally targeted. Primary porcine chondrocyte culture and human osteoarthritic cartilage tissue were studied. We found that interleukin-1α (IL-1α) up-regulated Piezo1 in porcine chondrocytes. Piezo1 expression was significantly increased in human osteoarthritic cartilage. Increased Piezo1 expression in chondrocytes resulted in a feed-forward pathomechanism whereby increased function of Piezo1 induced excess intracellular Ca2+ at baseline and in response to mechanical deformation. Elevated resting state Ca2+ in turn rarefied the F-actin cytoskeleton and amplified mechanically induced deformation microtrauma. As intracellular substrates of this OA-related inflammatory pathomechanism, in porcine articular chondrocytes exposed to IL-1α, we discovered that enhanced Piezo1 expression depended on p38 MAP-kinase and transcription factors HNF4 and ATF2/CREBP1. CREBP1 directly bound to the proximal PIEZO1 gene promoter. Taken together, these signaling and genetic reprogramming events represent a detrimental Ca2+-driven feed-forward mechanism that can be rationally targeted to stem the progression of OA.Item Open Access Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage.(Proc Natl Acad Sci U S A, 2014-11-25) Lee, Whasil; Leddy, Holly A; Chen, Yong; Lee, Suk Hee; Zelenski, Nicole A; McNulty, Amy L; Wu, Jason; Beicker, Kellie N; Coles, Jeffrey; Zauscher, Stefan; Grandl, Jörg; Sachs, Frederick; Guilak, Farshid; Liedtke, Wolfgang BDiarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca(2+) signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca(2+) transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.Item Open Access Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch.(J Biol Chem, 2016-05-06) Chen, Yong; Fang, Quan; Wang, Zilong; Zhang, Jennifer Y; MacLeod, Amanda S; Hall, Russell P; Liedtke, Wolfgang BTRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of "forefront" signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.Item Open Access TRPV channel-mediated calcium transients in nociceptor neurons are dispensable for avoidance behaviour.(Nat Commun, 2014-09-02) Lindy, Amanda S; Parekh, Puja K; Zhu, Richard; Kanju, Patrick; Chintapalli, Sree V; Tsvilovskyy, Volodymyr; Patterson, Randen L; Anishkin, Andriy; van Rossum, Damian B; Liedtke, Wolfgang BAnimals need to sense and react to potentially dangerous environments. TRP ion channels participate in nociception, presumably via Ca(2+) influx, in most animal species. However, the relationship between ion permeation and animals' nocifensive behaviour is unknown. Here we use an invertebrate animal model with relevance for mammalian pain. We analyse the putative selectivity filter of OSM-9, a TRPV channel, in osmotic avoidance behaviour of Caenorhabditis elegans. Using mutagenized OSM-9 expressed in the head nociceptor neuron, ASH, we study nocifensive behaviour and Ca(2+) influx. Within the selectivity filter, M(601)-F(609), Y604G strongly reduces avoidance behaviour and eliminates Ca(2+) transients. Y604F also abolishes Ca(2+) transients in ASH, while sustaining avoidance behaviour, yet it disrupts behavioral plasticity. Homology modelling of the OSM-9 pore suggests that Y(604) may assume a scaffolding role. Thus, aromatic residues in the OSM-9 selectivity filter are critical for pain behaviour and ion permeation. These findings have relevance for understanding evolutionary roots of mammalian nociception.Item Open Access UVB radiation generates sunburn pain and affects skin by activating epidermal TRPV4 ion channels and triggering endothelin-1 signaling.(Proc Natl Acad Sci U S A, 2013-08-20) Moore, Carlene; Cevikbas, Ferda; Pasolli, H Amalia; Chen, Yong; Kong, Wei; Kempkes, Cordula; Parekh, Puja; Lee, Suk Hee; Kontchou, Nelly-Ange; Yeh, Iwei; Jokerst, Nan Marie; Fuchs, Elaine; Steinhoff, Martin; Liedtke, Wolfgang BAt our body surface, the epidermis absorbs UV radiation. UV overexposure leads to sunburn with tissue injury and pain. To understand how, we focus on TRPV4, a nonselective cation channel highly expressed in epithelial skin cells and known to function in sensory transduction, a property shared with other transient receptor potential channels. We show that following UVB exposure mice with induced Trpv4 deletions, specifically in keratinocytes, are less sensitive to noxious thermal and mechanical stimuli than control animals. Exploring the mechanism, we find that epidermal TRPV4 orchestrates UVB-evoked skin tissue damage and increased expression of the proalgesic/algogenic mediator endothelin-1. In culture, UVB causes a direct, TRPV4-dependent Ca(2+) response in keratinocytes. In mice, topical treatment with a TRPV4-selective inhibitor decreases UVB-evoked pain behavior, epidermal tissue damage, and endothelin-1 expression. In humans, sunburn enhances epidermal expression of TRPV4 and endothelin-1, underscoring the potential of keratinocyte-derived TRPV4 as a therapeutic target for UVB-induced sunburn, in particular pain.