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Principal Investigator: Toshimitsu Kawate
Organization: CORNELL UNIVERSITY
Fiscal Year: 2024
Award: $327,314
Funding agency: National Institute of General Medical Sciences
ABSTRACT
Pannexins comprise a unique family of heptameric large-pore channels that are emerging as novel targets
for treating common, yet hard to cure diseases such as hypertension and chronic pain. Previous studies
indicate that Panx1 is activated through stimulation of structurally unrelated receptors such as G protein-
coupled receptors, ligand-gated ion channels, and tumor necrosis factor receptors. However, it remains un-
clear what cellular mechanism(s) actually open and close the Panx1 channel downstream of such seemingly
unrelated stimuli. Furthermore, Panx2 and 3 are severely understudied and essentially nothing is known about
the activation mechanisms of these subtypes. The long-term goal is to elucidate the mechanisms underlying
pannexin gating, regulation, and physiological signaling pathways. The specific objectives for this proposal
are to identify the physiological pannexin activators and elucidate the subtype-specific activation mechanisms.
The central hypothesis is that both Panx1 and 2 are directly activated by naturally occurring signaling mole-
cules in living cells and that Panx1 specifically requires posttranslational modifications to be "primed" for its
activation. The rationale for the proposed research is that once the direct activation-stimuli and the subtype-
specific mechanisms are identified, it will enable us to fill the critical gap in the pannexin-dependent signaling
pathway by connecting the upstream cell-stimulation and the downstream ATP-permeable membrane pore
formation. To attain the overall objectives, the following three specific aims will be performed:1) Identify the
direct pannexin activators for living cells; 2) Elucidate the role of the N-terminal domain (NTD) in pannexin
activation; and 3) Uncover the subtype-specific structural features of pannexins. These research aims will be
executed by using a combination of a cell-based pannexin activity assay, electrophysiology, functional recon-
stitution, and cryo-EM. The research proposed in this application is innovative because it introduces a novel
concept that pannexins—including the understudied Panx2—are directly activated by signaling molecules
produced downstream of various stimuli in living cells. It is also innovative because it will provide important
insights into the structure of the open channel and why Panx2 and 3 behave differently from Panx1. The
proposed research is significant because it will provide concrete molecular mechanisms for the missing link
in the pannexin signaling function. The proposed research is also expected to provide a strong structural
foundation for subtype specific mechanisms of pannexin channels. These results are expected to have pro-
found positive impact not only because they provide detailed basic mechanisms, but also because they will
open a new door for screening/designing pannexin-specific inhibitors—much-needed molecular tools that
have great potentials to serve as novel therapeutics for a variety of currently uncurable diseases.
Terms: <Adrenergic Receptor><Adrenoceptors><Assay><Bioassay><Biological Assay><Blood Vessels><Body Tissues><C-terminal><Cachectin Receptors><Caspase><Caspase Gene><Cell Body><Cell Communication and Signaling><Cell Line><Cell Membrane Permeability><Cell Signaling><Cell-Death Protease><CellLine><Cells><Cerebid><Cerespan><Cryo-electron Microscopy><Cryoelectron Microscopy><Cysteine Endopeptidases><Cysteine Protease><Cysteine Proteinases><Dephosphorylation><Disease><Disorder><EC 3.1.1.4><Electron Cryomicroscopy><Electrophysiology><Electrophysiology (science)><Epinephrine Receptors><External Domain><Extracellular Domain><Family><Foundations><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G-Protein-Coupled Receptors><GPCR><Genetic><Goals><Human Figure><Human body><Hypertension><ICE-like protease><Immune response><Immunological response><Inflammation><Inflammatory><Intracellular Communication and Signaling><Ion Channel><Ion Channel Gating><Ion Channel Gatings><Ionic Channels><Kidney Tubules><Kinases><Knock-out><Knockout><Knowledge><Lecithinase A2><Ligands><Link><Mediating><Membrane Channels><Molecular><Molecular Configuration><Molecular Conformation><Molecular Stereochemistry><N-Methyl-D-Aspartate Receptors><N-Methylaspartate Receptors><N-terminal><NH2-terminal><NMDA Receptor-Ionophore Complex><NMDA Receptors><Nerve Impulse Transmission><Nerve Transmission><Nervous System Diseases><Nervous System Disorder><Neural Development><Neurologic Disorders><Neurological Disorders><Neuronal Transmission><Neurophysiology / Electrophysiology><PLA2><Papaverine><Pavabid><Pavacap><Pavatym><Phosphatases><Phosphohydrolases><Phospholipase A2><Phosphomonoesterases><Phosphoric Monoester Hydrolases><Phosphorylation><Phosphotransferase Gene><Phosphotransferases><Physiologic><Physiological><Play><Post-Translational Modification Protein/Amino Acid Biochemistry><Post-Translational Modifications><Post-Translational Protein Modification><Post-Translational Protein Processing><Posttranslational Modifications><Posttranslational Protein Processing><Process><Protein Dephosphorylation><Protein Modification><Protein Phosphorylation><Receptor Protein><Regulation><Renal tubule structure><Research><Resolution><Role><Signal Pathway><Signal Transduction><Signal Transduction Systems><Signaling><Signaling Molecule><Stimulus><Strains Cell Lines><Structure><Synapses><Synaptic><TNF Receptor Family Protein><TNF Receptor Superfamily><TNF Receptors><TNFR><Tissues><Transphosphorylases><Tumor Necrosis Factor Receptor><Tumor Necrosis Factor Receptor Family><Tumor Necrosis Factor Receptor Superfamily><Tyrosine Phosphorylation><Unicelles><Vasal><Vascular Hypertensive Disease><Vascular Hypertensive Disorder><adenoreceptor><axon signaling><axon-glial signaling><axonal signaling><biological signal transduction><brain tissue><cell type><chronic pain><conformation><conformational><conformational state><conformationally><conformations><cryo-EM><cryoEM><cryogenic electron microscopy><cultured cell line><cystein protease><cystein proteinase><cysteine endopeptidase><design><designing><electrophysiological><glia signaling><glial signaling><high blood pressure><host response><hyperpiesia><hyperpiesis><hypertensive disease><hypertensive disorder><immune system response><immunoresponse><inhibitor><innovate><innovation><innovative><insight><knock-down><knockdown><lecithinase A><lymphatic vasculature><membrane permeability><nerve signaling><neural signaling><neurodevelopment><neurological disease><neuronal signaling><neurotransmission><new drug treatments><new drugs><new pharmacological therapeutic><new therapeutics><new therapy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel therapeutics><novel therapy><pharmacologic><phosphatidase><phosphatidolipase><phosphatidylcholine 2 acylhydrolase><receptor><reconstitute><reconstitution><reconstruction><renal tubule><resolutions><screening><screenings><shear stress><social role><synapse><tool><vascular>