title: Evidence for the role of lipid rafts and sphingomyelin in Ca2+-gating of Transient Receptor Potential channels in trigeminal sensory neurons and peripheral nerve terminals.
abstract: Transient Receptor Potential (TRP) cation channels, such as TRP Vanilloid 1 and TRP Ankyrin repeat domain 1 (TRPV1 and TRPA1) are nocisensors playing important role to signal pain. Two "melastatin" TRP receptors, like TRPM8 and TRPM3 are also expressed in a subgroup of primary sensory neurons. These channels serve as thermosensors with unique thermal sensitivity ranges and are activated also by several exogenous and endogenous chemical ligands inducing conformational changes from various allosteric ("multisteric") sites. We analysed the role of plasma membrane microdomains of lipid rafts on isolated trigeminal (TRG) neurons and TRPV1-expressing CHO cell line by measuring agonist-induced Ca2+ transients with ratiometric technique. Stimulation-evoked calcitonin gene related peptide (CGRP) release from sensory nerve endings of the isolated rat trachea by radioimmunoassay was also measured. Lipid rafts were disrupted by cleaving sphingomyelin (SM) with sphingomyelinase (SMase), cholesterol depletion with methyl β-cyclodextrin (MCD) and ganglioside breakdown with myriocin. It has been revealed that intracellular Ca2+ increase responses evoked by the TRPV1 agonist capsaicin, the TRPA1 agonsits allyl isothiocyanate (AITC) and formaldehyde as well as the TRPM8 activator icilin were inhibited after SMase, MCD and myriocin incubation but the response to the TRPM3 agonist pregnenolon sulphate was not altered. Extracellular SMase treatment did not influence the thapsigargin-evoked Ca2+-release from intracellular stores. Besides the cell bodies, SMase also inhibited capsaicin- or AITC-evoked CGRP release from peripheral sensory nerve terminals, this provides the first evidence for the importance of lipid raft integrity in TRPV1 and TRPA1 gating on capsaicin-sensitive nerve terminals. SM metabolites, ceramide and sphingosine, did not influence TRPA1 and TRPV1 activation on TRG neurons, TRPV1-expressing CHO cell line, and nerve terminals. We suggest, that the hydrophobic interactions between TRP receptors and membrane lipid raft interfaces modulate the opening properties of these channels and therefore, targeting this interaction might be a promising tool for drug developmental purposes.
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, Hungary; Szentágothai Research Center, University of Pécs, Hungary.
authors.forename: Éva
authors.initials: É
authors.lastname: Sághy
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, Hungary; Szentágothai Research Center, University of Pécs, Hungary; MTA-PTE Chronic Pain Research Group, Pécs, Hungary. Electronic address: eva.szoke@aok.pte.hu.
authors.forename: Éva
authors.initials: É
authors.lastname: Szőke
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, Hungary; Szentágothai Research Center, University of Pécs, Hungary.
authors.forename: Maja
authors.initials: M
authors.lastname: Payrits
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, Hungary; Szentágothai Research Center, University of Pécs, Hungary; MTA-PTE Chronic Pain Research Group, Pécs, Hungary.
authors.forename: Zsuzsanna
authors.initials: Z
authors.lastname: Helyes
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, Hungary; Szentágothai Research Center, University of Pécs, Hungary.
authors.forename: Rita
authors.initials: R
authors.lastname: Börzsei
authors.affiliation: Szentágothai Research Center, University of Pécs, Hungary; Department of Experimental Physics, University of Pécs, Pécs, Hungary.
authors.forename: János
authors.initials: J
authors.lastname: Erostyák
authors.affiliation: Szentágothai Research Center, University of Pécs, Hungary; Department of Experimental Physics, University of Pécs, Pécs, Hungary.
authors.forename: Tibor Zoltán
authors.initials: TZ
authors.lastname: Jánosi
authors.affiliation: Szentágothai Research Center, University of Pécs, Hungary; Department of Medical Biology, Faculty of Medicine, University of Pécs, Pécs, Hungary.
authors.forename: György
authors.initials: G
authors.lastname: Sétáló
authors.affiliation: Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Pécs, Pécs, Hungary; Szentágothai Research Center, University of Pécs, Hungary.
authors.forename: János
authors.initials: J
authors.lastname: Szolcsányi
mesh_terms: D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D003412:Cricetulus; D005732:Gangliosides / Q000378:metabolism; D015640:Ion Channel Gating / Q000502:physiology; D021962:Membrane Microdomains / Q000378:metabolism*; D009411:Nerve Endings / Q000378:metabolism*; D010525:Peripheral Nerves / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D011984:Sensory Receptor Cells / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism*; D014276:Trigeminal Nerve / Q000378:metabolism*; D047392:beta-Cyclodextrins / Q000378:metabolism
chemical_list: D005732:Gangliosides; D013109:Sphingomyelins; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D002118:Calcium
keywords: Lipid rafts; Methyl β-cyclodextrin; Nerve terminal; Sensory neuron; Sphingomyelin; Transient Receptor Potential channel