Using melanopsin to study G protein signaling in cortical neurons.
Our understanding of G protein-coupled receptors (GPCRs) in the central nervous system (CNS) has been hampered by the limited availability of tools allowing for the study of their signaling with precise temporal control. To overcome this, we tested the utility of the bistable mammalian opsin melanopsin to examine G protein signaling in CNS neurons. Specifically, we used biolistic (gene gun) approaches to transfect melanopsin into cortical pyramidal cells maintained in organotypic slice culture. Whole cell recordings from transfected neurons indicated that application of blue light effectively activated the transfected melanopsin to elicit the canonical biphasic modulation of membrane excitability previously associated with the activation of GPCRs coupling to Gαq-11 Remarkably, full mimicry of exogenous agonist concentration could be obtained with pulses as short as a few milliseconds, suggesting that their triggering required a single melanopsin activation-deactivation cycle. The resulting temporal control over melanopsin activation allowed us to compare the activation kinetics of different components of the electrophysiological response. We also replaced the intracellular loops of melanopsin with those of the 5-HT2A receptor to create a light-activated GPCR capable of interacting with the 5-HT2A receptor interacting proteins. The resulting chimera expressed weak activity but validated the potential usefulness of melanopsin as a tool for the study of G protein signaling in CNS neurons.
Department of Pharmacology, Wayne State University, Detroit, Michigan; and.
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McGregor
Institut de Génomique Fonctionnelle, CNRS UMR5203, INSERM U1191, Université de Montpellier, Montpellier, France.
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Bécamel
Institut de Génomique Fonctionnelle, CNRS UMR5203, INSERM U1191, Université de Montpellier, Montpellier, France.
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Marin
Department of Pharmacology, Wayne State University, Detroit, Michigan; and randrade@med.wayne.edu.
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Andrade
D000109:Acetylcholine / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D002540:Cerebral Cortex / Q000166:cytology*; D066298:In Vitro Techniques; D008027:Light; D055537:Light Signal Transduction / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D009474:Neurons / Q000187:drug effects / Q000502:physiology*; D018377:Neurotransmitter Agents / Q000494:pharmacology; D009924:Organ Culture Techniques; D018408:Patch-Clamp Techniques; D010775:Photic Stimulation; D011810:Quinoxalines / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D017299:Rod Opsins / Q000235:genetics / Q000378:metabolism*; D013779:Tetrodotoxin / Q000494:pharmacology; D014162:Transfection; D000099210:Melanopsin
D000109:Acetylcholine; D018377:Neurotransmitter Agents; D011810:Quinoxalines; D043562:Receptors, G-Protein-Coupled; D017299:Rod Opsins; D013779:Tetrodotoxin; D000099210:Melanopsin; C056723:FG 9041
5-HT2A receptor; Gαq-11 signaling; melanopsin; pyramidal neuron