RESUMO
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.
Assuntos
Canais de Potencial de Receptor Transitório , Microscopia Crioeletrônica , Transdução de Sinais , Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/genética , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
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.
Assuntos
Venenos de Escorpião/farmacologia , Canal de Cátion TRPA1/metabolismo , Animais , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Ratos Sprague-Dawley , Escorpiões/metabolismoRESUMO
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.
Assuntos
Cálcio/metabolismo , Cálcio/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Canal de Cátion TRPA1/química , Canal de Cátion TRPA1/metabolismo , Sequência de Aminoácidos , Cisteína/metabolismo , Condutividade Elétrica , Humanos , Iodoacetamida/farmacologia , Modelos Moleculares , Mutação , Oximas/farmacologia , Canal de Cátion TRPA1/genéticaRESUMO
Adult hippocampal neurogenesis is critical for learning and memory, and aberrant adult neurogenesis has been implicated in cognitive decline associated with aging and neurological diseases [J. T. Gonçalves, S. T. Schafer, F. H. Gage, Cell 167, 897914 (2016)]. In previous studies, we observed that the delayed-rectifier voltage-gated potassium channel Kv1.1 controls the membrane potential of neural stem and progenitor cells and acts as a brake on neurogenesis during neonatal hippocampal development [S. M. Chou et al., eLife 10, e58779 (2021)]. To assess the role of Kv1.1 in adult hippocampal neurogenesis, we developed an inducible conditional knockout mouse to specifically remove Kv1.1 from adult neural stem cells via tamoxifen administration. We determined that Kv1.1 deletion in adult neural stem cells causes overproliferation and depletion of radial glia-like neural stem cells, prevents proper adult-born granule cell maturation and integration into the dentate gyrus, and moderately impairs hippocampus-dependent contextual fear learning and memory. Taken together, these findings support a critical role for this voltage-gated ion channel in adult neurogenesis.
Assuntos
Condicionamento Clássico , Hipocampo , Canal de Potássio Kv1.1 , Células-Tronco Neurais , Neurogênese , Neurônios , Animais , Medo , Hipocampo/citologia , Hipocampo/crescimento & desenvolvimento , Canal de Potássio Kv1.1/genética , Canal de Potássio Kv1.1/fisiologia , Camundongos , Camundongos Knockout , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/fisiologiaRESUMO
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 ß (Aß). 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 Aß- 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.
Assuntos
Peptídeos beta-Amiloides , Mitocôndrias , Peptídeos beta-Amiloides/metabolismo , Microscopia Crioeletrônica , Humanos , Metaloproteases/metabolismo , Mitocôndrias/metabolismo , Conformação Molecular , Conformação Proteica , Especificidade por SubstratoRESUMO
Human presequence protease (hPreP) is an M16 metalloprotease localized in mitochondria. There, hPreP facilitates proteostasis by utilizing an â¼13,300-Å(3) catalytic chamber to degrade a diverse array of potentially toxic peptides, including mitochondrial presequences and ß-amyloid (Aß), the latter of which contributes to Alzheimer disease pathogenesis. Here, we report crystal structures for hPreP alone and in complex with Aß, which show that hPreP uses size exclusion and charge complementation for substrate recognition. These structures also reveal hPreP-specific features that permit a diverse array of peptides, with distinct distributions of charged and hydrophobic residues, to be specifically captured, cleaved, and have their amyloidogenic features destroyed. SAXS analysis demonstrates that hPreP in solution exists in dynamic equilibrium between closed and open states, with the former being preferred. Furthermore, Aß binding induces the closed state and hPreP dimerization. Together, these data reveal the molecular basis for flexible yet specific substrate recognition and degradation by hPreP.