title: Privileged crosstalk between TRPV1 channels and mitochondrial calcium shuttling machinery controls nociception.
abstract: The nociceptive noxious heat-activated receptor - TRPV1, conducts calcium and sodium, thus producing a depolarizing receptor potential, leading to activation of nociceptive neurons. TRPV1-mediated calcium and sodium influx is negatively modulated by calcium, via calcium-dependent desensitization of TRPV1 channels. A mitochondrial Ca2+uniporter - MCU, controls mitochondrial Ca2+entry while a sodium/calcium transporter - NCLX shapes calcium and sodium transients by mediating sodium entry into and removing calcium from the mitochondria. The functional interplay between TRPV1, MCU and NCLX, in controlling the cytosolic and mitochondrial calcium and sodium transients and subsequently the nociceptive excitability, is poorly understood. Here, we used cytosolic and mitochondrial fluorescent calcium and sodium imaging together with electrophysiological recordings of TRPV1-induced currents in HEK293T cells and nociceptor-like dissociated rat dorsal root ganglion neurons, while modulating NCLX or MCU expression using specific small interfering RNA (siNCLX). We show that the propagation of the TRPV1-induced cytosolic calcium and sodium fluxes into mitochondria is dependent on coordinated activity of NCLX and MCU. Thus, knocking-down of NCLX triggers down regulation of MCU dependent mitochondrial Ca2+uptake. This in turn decreases rate and amplitude of TRPV1-mediated cytosolic calcium, which inhibits capsaicin-induced inward current and neuronal firing. TRPV1-mediated currents were fully rescued by intracellular inclusion of the fast calcium chelator BAPTA. Finally, NCLX controls capsaicin-induced cell death, by supporting massive mitochondrial Ca2+shuttling. Altogether, our results suggest that NCLX, by regulating cytosolic and mitochondrial ionic transients, modulates calcium-dependent desensitization of TRPV1 channels, thereby, controlling nociceptive signaling.
authors.affiliation: Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Israel.
authors.forename: Iulia I
authors.initials: II
authors.lastname: Nita
authors.affiliation: Department of Medical Neurobiology, Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel; The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University, Jerusalem 91120, Israel.
authors.forename: Yaki
authors.initials: Y
authors.lastname: Caspi
authors.affiliation: Department of Medical Neurobiology, Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel; The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University, Jerusalem 91120, Israel.
authors.forename: Sagi
authors.initials: S
authors.lastname: Gudes
authors.affiliation: Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Israel.
authors.forename: Dimitri
authors.initials: D
authors.lastname: Fishman
authors.affiliation: Department of Medical Neurobiology, Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel; The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University, Jerusalem 91120, Israel.
authors.forename: Shaya
authors.initials: S
authors.lastname: Lev
authors.affiliation: Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Israel.
authors.forename: Michal
authors.initials: M
authors.lastname: Hersfinkel
authors.affiliation: Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University of the Negev, 84105, Israel. Electronic address: israel.sekler@gmail.com.
authors.forename: Israel
authors.initials: I
authors.lastname: Sekler
authors.affiliation: Department of Medical Neurobiology, Institute for Medical Research Israel-Canada, Faculty of Medicine, The Hebrew University, Jerusalem 91120, Israel; The Edmond and Lily Safra Center for Brain Sciences, The Hebrew University, Jerusalem 91120, Israel. Electronic address: alexander.binshtok@ekmd.huji.ac.il.
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Binshtok
mesh_terms: D000200:Action Potentials / Q000187:drug effects / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002211:Capsaicin / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology; D024101:Mitochondrial Proteins; D057054:Molecular Imaging; D009619:Nociceptors / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D061251:Primary Cell Culture; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D059010:Single-Cell Analysis; D012964:Sodium / Q000378:metabolism*; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; C483046:SLC8B1 protein, human; D019831:Sodium-Calcium Exchanger; D050916:TRPV Cation Channels; C494482:TRPV1 protein, human; C034703:mitochondrial calcium uniporter; D004533:Egtazic Acid; D012964:Sodium; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002211:Capsaicin; D002118:Calcium
keywords: Capsaicin; Mitochondrial calcium signaling; NCLX; Nociception; Pain; TRPV1