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This Article Full Text Full Text (PDF) All Versions of this Article: 576/2/477    most recent jphysiol.2006.113068v1 Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, J. Articles by Ikeda, S. R. Search for Related Content PubMed PubMed Citation Articles by Guo, J. Articles by Ikeda, S. R. Related Collections Neuroscience

¹ Section on Transmitter Signalling, Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD 20892 USA

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, -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, ₂-adrenergic receptor-mediated I_Ca modulation, were observed following illumination of neurons expressing FL-BTX-labelled sodium channel ß2 subunits or ionotropic 5-HT₃ 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.

(Received 5 May 2006; accepted after revision 25 July 2006; first published online 27 July 2006)
Corresponding author S. R. Ikeda: Laboratory of Molecular Physiology, National Institute on Alcohol Abuse and Alcoholism, 5625 Fishers Lane, Room TS-06, MSC 9411, Bethesda, MD, 20892-9941 USA. Email: sikeda{at}mail.nih.gov

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