title: Design and construction of conformational biosensors to monitor ion channel activation: A prototype FlAsH/BRET-approach to Kir3 channels.
abstract: Ion channels play a vital role in numerous physiological functions and drugs that target them are actively pursued for development of novel therapeutic agents. Here we report a means for monitoring in real time the conformational changes undergone by channel proteins upon exposure to pharmacological stimuli. The approach relies on tracking structural rearrangements by monitoring changes in bioluminescence energy transfer (BRET). To provide proof of principle we have worked with Kir3 neuronal channels producing 10 different constructs which were combined into 17 donor-acceptor BRET pairs. Among these combinations, pairs bearing the donor Nano-Luc (NLuc) at the C-terminal end of Kir3.2 subunits and the FlAsH acceptor at the N-terminal end (NT) or the interfacial helix (N70) of Kir3.1 subunits were identified as potential tools. These pairs displayed significant changes in energy transfer upon activation with direct channel ligands or via stimulation of G protein-coupled receptors. Conformational changes associated with channel activation followed similar kinetics as channel currents. Dose response curves generated by different agonists in FlAsH-BRET assays displayed similar rank order of potency as those obtained with conventional BRET readouts of G protein activation and ion flux assays. Conformational biosensors as the ones reported herein should prove a valuable complement to other methodologies currently used in channel drug discovery.
authors.affiliation: CHU Ste-Justine Centre de Recherche, Montreal, Quebec, Canada.
authors.forename: Derek N
authors.initials: DN
authors.lastname: Robertson
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
authors.forename: Rory
authors.initials: R
authors.lastname: Sleno
authors.affiliation: CHU Ste-Justine Centre de Recherche, Montreal, Quebec, Canada; Department of Pharmacology, Université de Montréal, Montreal, Quebec, Canada.
authors.forename: Karim
authors.initials: K
authors.lastname: Nagi
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
authors.forename: Darlaine
authors.initials: D
authors.lastname: Pétrin
authors.affiliation: Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
authors.forename: Terence E
authors.initials: TE
authors.lastname: Hébert
authors.affiliation: CHU Ste-Justine Centre de Recherche, Montreal, Quebec, Canada; Department of Psychiatry, Université de Montréal, Montreal, Quebec, Canada; Department of Pharmacology, Université de Montréal, Montreal, Quebec, Canada; Department of Neuroscience, Université de Montréal, Montreal, Quebec, Canada. Electronic address: graciela.pineyro.filpo@umontreal.ca.
authors.forename: Graciela
authors.initials: G
authors.lastname: Pineyro
mesh_terms: D000701:Analgesics, Opioid / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D060807:Bioluminescence Resonance Energy Transfer Techniques / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D015195:Drug Design; D019793:Fluorescein / Q000138:chemical synthesis* / Q000378:metabolism*; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology; D051379:Mice; D011487:Protein Conformation
chemical_list: D000701:Analgesics, Opioid; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D019793:Fluorescein
keywords: BRET; Biosensor; Fluxor®; High throughput screening; Kir3 channels; Nano-Luc (NLuc®)