Fluorophore‐assisted light inactivation produces both targeted and collateral effects on N‐type calcium channel modulation in rat sympathetic neurons

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, α‐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, α 2 ‐adrenergic receptor‐mediated I Ca modulation, were observed following illumination of neurons expressing FL‐BTX‐labelled sodium channel β2 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.