title: Fluorescence-Based Automated Screening Assay for the Study of the pH-Sensitive Channel ASIC1a.
abstract: Acid-sensing ion channel 1a (ASIC1a) is involved in several pathologies, including neurodegenerative and neuroinflammatory disorders, stroke, epilepsy, and inflammatory pain. ASIC1a has been the subject of intense drug discovery programs devoted to the development of new pharmacological tools for its modulation. However, these efforts to generate new compounds have faced the lack of an efficient screening procedure. In the past decades, improvements in screening technologies and fluorescent sensors for the study of ion channels have provided new opportunities in this field. Unfortunately, ASIC1a is mainly a Na(+) permeable channel and undergoes desensitization after its activation, two features that make the use of the available screening procedures problematic. We propose here a novel screening approach for the study of ASIC1a activity in full automation. Our method is based on the stimulation of ASIC1a-expressing cells by protons and the use of electrochromic fluorescent voltage sensors as a readout of ion channel activation. This method will prove to be useful for drug screening programs aimed at ASIC1a modulation.
authors.affiliation: Advanced Light and Electron Microscopy Bio-Imaging Centre, Experimental Imaging Centre, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Nausicaa
authors.initials: N
authors.lastname: Mazzocchi
authors.affiliation: Neuroimmunolgy Unit, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: De Ceglia
authors.affiliation: Advanced Fluorescence Microscopy and Nanoscopy Research Unit, Experimental Imaging Center, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Mazza
authors.affiliation: Center for Neuroscience and Dept. of Theoretical and Applied Sciences (DiSTA), Biomedical Division, University of Insubria, Busto Arsizio (VA), Italy.
authors.forename: Lia
authors.initials: L
authors.lastname: Forti
authors.affiliation: Neuroimmunolgy Unit, Institute of Experimental Neurology (INSPE), Division of Neuroscience, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Muzio
authors.affiliation: Advanced Light and Electron Microscopy Bio-Imaging Centre, Experimental Imaging Centre, San Raffaele Scientific Institute, Milan, Italy menegon.andrea@hsr.it.
authors.forename: Andrea
authors.initials: A
authors.lastname: Menegon
mesh_terms: D062565:Acid Sensing Ion Channels / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D057205:Automation, Laboratory; D016466:CHO Cells; D002414:Cations, Monovalent; D002460:Cell Line; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D003412:Cricetulus; D004133:Diminazene / Q000031:analogs & derivatives / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D057166:High-Throughput Screening Assays; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D061251:Primary Cell Culture; D011714:Pyrans / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D013111:Spider Venoms / Q000494:pharmacology; D013343:Styrenes / Q000378:metabolism*; D019076:Transgenes; D056969:Voltage-Sensitive Dye Imaging / Q000379:methods*
chemical_list: C568267:ASIC1 protein, mouse; D062565:Acid Sensing Ion Channels; D002414:Cations, Monovalent; D005456:Fluorescent Dyes; C413209:PcTX1 protein, Psalmopoeus cambridgei; D010455:Peptides; D011714:Pyrans; D011726:Pyridinium Compounds; D013111:Spider Venoms; D013343:Styrenes; C050019:1-(3-sulfonatopropyl)-4-(beta)(2-(di-n-butylamino)-6-naphthylvinyl)pyridinium betaine; D012964:Sodium; C082825:4-(dicyanomethylene)-2-methyl-6-(4-(dimethylamino)styryl)-4H-pyran; C003915:diminazene aceturate; D004133:Diminazene
keywords: ASIC1a; screening; voltage sensitive dyes