title: Fluorophore-assisted light inactivation produces both targeted and collateral effects on N-type calcium channel modulation in rat sympathetic neurons.
abstract: Fluorophore-assisted light inactivation (FALI) is a method to inactivate specific proteins on a time scale of seconds to minutes using either diffuse or coherent light. Here we examine a novel FALI modality that utilizes a fluorescein-conjugated polypeptide, alpha-bungarotoxin (BTX) and a 13 amino acid BTX-binding site engineered into the N-terminus of metabotropic glutamate receptor 8a (mGluR8a), a class C G-protein-coupled receptor (GPCR). The tagged mGluR8a was expressed in rat sympathetic neurons and labelled with fluorescein-conjugated BTX (FL-BTX). The efficacy of FALI was evaluated by monitoring mGluR8a-mediated inhibition of calcium currents (I(Ca)) using whole-cell voltage-clamp techniques. Following either wide-field or laser illumination of FL-BTX-labelled neurons, mGluR8a-mediated I(Ca) inhibition was greatly attenuated whereas holding current and basal I(Ca), measures of non-specific effects, were minimally affected. Sodium azide, a collision quencher of singlet oxygen, reduced the magnitude of FALI-mediated effects supporting a role for reactive oxygen species in the process. Although these results were consistent with an acute inactivation of mGluR8a, the intended target, two findings confounded this interpretation. First, effects on a natively expressed signalling pathway, alpha(2)-adrenergic receptor-mediated I(Ca) modulation, were observed following illumination of neurons expressing FL-BTX-labelled sodium channel beta2 subunits or ionotropic 5-HT(3) receptors, proteins with no overt relationship to GPCR signalling pathways. Second, GPCR-independent I(Ca) modulation induced with intracellular guanylyl imidophosphate was also attenuated by FALI. These data challenge the assumption that the fluorophore-tagged protein is the sole target of FALI and provide evidence that collateral damage to proximal proteins occurs following fluorophore illumination.
authors.affiliation: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, 5625 Fishers Lane, Room TS-06, MSC 9411, Bethesda, MD, 20892-9941 USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Guo
authors.forename: Huanmian
authors.initials: H
authors.lastname: Chen
authors.forename: Henry L
authors.initials: HL
authors.lastname: Puhl
authors.forename: Stephen R
authors.initials: SR
authors.lastname: Ikeda
mesh_terms: D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000737:chemistry; D020864:Calcium Channels, N-Type / Q000502:physiology*; D002460:Cell Line; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D008027:Light; D008297:Male; D009474:Neurons / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D017208:Rats, Wistar; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism; D018094:Receptors, Metabotropic Glutamate / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013564:Sympathetic Nervous System / Q000378:metabolism*; D013997:Time Factors
chemical_list: D002038:Bungarotoxins; D020864:Calcium Channels, N-Type; D005456:Fluorescent Dyes; D043562:Receptors, G-Protein-Coupled; D018094:Receptors, Metabotropic Glutamate; C111435:metabotropic glutamate receptor 8; D019793:Fluorescein