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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2020)

TRPA1 - also known as the ‘wasabi receptor’ - is activated by pungent natural products from mustard and allium plants and is responsible for the sinus clearing or eye stinging pain that one experiences when eating horse radish or chopping an onion. More importantly, TRPA1 is a receptor for an astoundingly broad class of volatile environmental irritants and endogenous pro-algesic agents that elicit or exacerbate tissue damage, pain, and neurogenic inflammation. Many of these irritants are chemically reactive electrophiles that activate TRPA1 through a fascinating mechanism involving covalent modification of the channel’s cytoplasmic N-terminus, a phenomenon that is not well understood at the biochemical or structural level. TRPA1 also functions as a ‘receptor-operated’ channel that is activated downstream of phospholipase C signaling pathways, such as those involved in the transduction of pruritic signals. Together, these actions make TRPA1 a major contributor to a broad range of chemo-nociceptive actions include pain and itch.

In this work we have exploited natural product pharmacology, functional recording, and structural methods to understand how TRPA1 is activated by a range of stimuli (reactive and non-reactive, direct and indirect). Collectively, these data draw a detailed mechanistic picture of how this important chemo-nociceptive ion channel detects physiological stimuli, transduces this information into neuronal excitation, and is regulated by cellular events. In light of TRPA1’s well recognized role in acute and persistent pain and itch, our findings are of both investigational and therapeutic import. Beyond TRPA1, they are of general relevance to illuminating the gating mechanisms other sensory TRP channels, several of which are also regulated downstream of metabotropic receptors and/or by cellular calcium.

    Cover page: Stinging Sensations: Activation Mechanisms of the Wasabi Receptor, TRPA1
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2022)
    Transient receptor potential (TRP) ion channels are sophisticated signaling machines that detect a wide variety of environmental and physiological signals. Every cell in the body expresses one or more members of the extended TRP channel family, which consists of over 30 subtypes, each likely possessing distinct pharmacological, biophysical, and/or structural attributes. While the function of some TRP subtypes remains enigmatic, those involved in sensory signaling are perhaps best characterized and have served as models for understanding how these excitatory ion channels serve as polymodal signal integrators. With the recent resolution revolution in cryo-electron microscopy, these and other TRP channel subtypes are now yielding their secrets to detailed atomic analysis, which is beginning to reveal structural underpinnings of stimulus detection and gating, ion permeation, and allosteric mechanisms governing signal integration. These insights are providing a framework for designing and evaluating modality-specific pharmacological agents for treating sensory and other TRP channel-associated disorders.
      Cover page: Sensory TRP Channels in Three Dimensions
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2020)
      The transient receptor potential ion channel TRPA1 is expressed by primary afferent nerve fibres, in which it functions as a low-threshold sensor for structurally diverse electrophilic irritants, including small volatile environmental toxicants and endogenous algogenic lipids1. TRPA1 is also a 'receptor-operated' channel whose activation downstream of metabotropic receptors elicits inflammatory pain or itch, making it an attractive target for novel analgesic therapies2. However, the mechanisms by which TRPA1 recognizes and responds to electrophiles or cytoplasmic second messengers remain unknown. Here we use strutural studies and electrophysiology to show that electrophiles act through a two-step process in which modification of a highly reactive cysteine residue (C621) promotes reorientation of a cytoplasmic loop to enhance nucleophilicity and modification of a nearby cysteine (C665), thereby stabilizing the loop in an activating configuration. These actions modulate two restrictions controlling ion permeation, including widening of the selectivity filter to enhance calcium permeability and opening of a canonical gate at the cytoplasmic end of the pore. We propose a model to explain functional coupling between electrophile action and these control points. We also characterize a calcium-binding pocket that is highly conserved across TRP channel subtypes and accounts for all aspects of calcium-dependent TRPA1 regulation, including potentiation, desensitization and activation by metabotropic receptors. These findings provide a structural framework for understanding how a broad-spectrum irritant receptor is controlled by endogenous and exogenous agents that elicit or exacerbate pain and itch.
        Cover page: Irritant-evoked activation and calcium modulation of the TRPA1 receptor
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2019)
        TRPA1 is a chemosensory ion channel that functions as a sentinel for structurally diverse electrophilic irritants. Channel activation occurs through an unusual mechanism involving covalent modification of cysteine residues clustered within an amino-terminal cytoplasmic domain. Here, we describe a peptidergic scorpion toxin (WaTx) that activates TRPA1 by penetrating the plasma membrane to access the same intracellular site modified by reactive electrophiles. WaTx stabilizes TRPA1 in a biophysically distinct active state characterized by prolonged channel openings and low Ca2+ permeability. Consequently, WaTx elicits acute pain and pain hypersensitivity but fails to trigger efferent release of neuropeptides and neurogenic inflammation typically produced by noxious electrophiles. These findings provide a striking example of convergent evolution whereby chemically disparate animal- and plant-derived irritants target the same key allosteric regulatory site to differentially modulate channel activity. WaTx is a unique pharmacological probe for dissecting TRPA1 function and its contribution to acute and persistent pain.
          Cover page: A Cell-Penetrating Scorpion Toxin Enables Mode-Specific Modulation of TRPA1 and Pain
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2022)
          Presequence protease (PreP), a 117 kDa mitochondrial M16C metalloprotease vital for mitochondrial proteostasis, degrades presequence peptides cleaved off from nuclear-encoded proteins and other aggregation-prone peptides, such as amyloid β (Aβ). PreP structures have only been determined in a closed conformation; thus, the mechanisms of substrate binding and selectivity remain elusive. Here, we leverage advanced vitrification techniques to overcome the preferential denaturation of one of two ~55 kDa homologous domains of PreP caused by air-water interface adsorption. Thereby, we elucidate cryoEM structures of three apo-PreP open states along with Aβ- and citrate synthase presequence-bound PreP at 3.3-4.6 Å resolution. Together with integrative biophysical and pharmacological approaches, these structures reveal the key stages of the PreP catalytic cycle and how the binding of substrates or PreP inhibitor drives a rigid body motion of the protein for substrate binding and catalysis. Together, our studies provide key mechanistic insights into M16C metalloproteases for future therapeutic innovations.
            Cover page: Structural basis for the mechanisms of human presequence protease conformational switch and substrate recognition.
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