Given that the cellular labeling patterns of NRB-AF12 and ER-Tr are near identical, it could be proposed that the two dyes share a common cellular target/s. ER-Tr is a BODIPY derivative of glibenclamide, a widely used antidiabetic drug belonging to the sulfonylurea family. Glibenclamide is a non-selective inhibitor of ATP-sensitive potassium channels (Proks et al., 2002), a multimeric protein complex consisting of four pore-lining inward rectifier α subunits (Kir6.1/2) and four regulatory sulfonylurea-binding β subunits (SUR1/2A/2B) that belong to the ATP-binding cassette family (Yokoshiki et al., 1998; Zerangue et al., 1999; Shi et al., 2005; Ng et al., 2010). Co-assembly of different Kir and SUR subunits generates ion channel combinations with different channel properties, and with different cell compartment or tissue distribution (Yokoshiki et al., 1998), such as the SUR1/Kir6.2 pancreatic type, the SUR2A/Kir6.2 cardiac type and the SUR2B/Kir6.1 vascular smooth muscle type. The ER-Tr and NRB-AF12 fluorescence characteristics may therefore reflect the cellular distribution of KATP channels. Inhibition of plasma membrane ATP-sensitive potassium channels leads to an increased intracellular K+ concentration, plasma membrane depolarization, opening of voltage-dependent calcium channels, and increased intracellular Ca2+ concentration (Ko et al., 2008) that, in vascular smooth muscle, triggers the contractile process. It has been reported that vascular smooth muscle plasma membrane selectively expresses SUR2B subunits of KATP channels (Morrissey et al., 2005; Teramoto, 2006; Tinker et al., 2014). These data, together with the finding (this study) that NRB-AF12 localizes to the plasma membrane only in NRB-sensitive cells, might explain the species- and tissue-selective activity of NRB, by hypothesizing an inhibitory effect of the drug on rat-specific SUR2B-containing KATP channels expressed on the plasma membrane of the rat peripheral artery myocytes. In contrast to this hypothesis is the fact that neither unlabeled NRB nor glibenclamide are able to displace NRB-AF12 or ER-Tr from their binding site, which presents the prospect of a different/additional site of action from/to KATP channels. It also raises the question as to whether the fluorescently labeled compounds have the same binding characteristics of the corresponding unlabeled compounds. However, the lack of displacement is in itself not sufficient to rule out the possibility that NRB-AF12 does bind to KATP channels, with there being at least two plausible explanations: (1) NRB-AF12 and NRB could bind to different sites within the SUR2B moiety, thus allowing the attachment of both compounds; (2) the fluorophore group of NRB-AF12 may have multiple binding sites preventing its displacement by unlabeled NRB. The existence of more than one binding site, at least for NRB-AF12, is supported by the fact that this dye, in contrast to ER-Tr, was able to label both endosomes and lysosomes. As with SUR2-containing channels, NRB-AF12 appears to be similarly targeted to the endosomal/lysosomal pathway, supporting the idea that this compound can be recycled by endocytosis as has been shown for the KATP channel subunits (Bao et al., 2011).