title: A fluorescent calmodulin that reports the binding of hydrophobic inhibitory ligands.
abstract: Ca2+ binding to calmodulin in the pCa range 5.5-7.0 exposes hydrophobic sites that bind hydrophobic inhibitory ligands, including calmodulin antagonists, some Ca2+-antagonists and calmodulin-binding proteins. The binding of these hydrophobic ligands to calmodulin can be followed by the approx. 80% fluorescence increase they produce in dansylated (5-dimethylaminonaphthalene-1-sulphonylated) calmodulin (CDRDANS). In the presence of Ca2+, calmodulin binds the calmodulin inhibitor, R24571, with an affinity of approx. 2-3 nM and hydrophobic ligands, including trifluoperazine (TFP), W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide], fendiline, felodipine and prenylamine, with affinities in the micromolar range. This binding is strongly Ca2+-dependent and Mg2+-independent. Calmodulin shows a reasonably high degree of specificity in its binding of these ligands over other ligands tested. CDRDANS, therefore, provides a convenient and simple means of monitoring the interaction of a variety of hydrophobic ligands with the Ca2+-dependent regulatory protein, calmodulin. CDRDANS binds to phospholipid vesicles made of (dimyristoyl)phosphatidylcholine (DMPC) or (dipalmitoyl)phosphatidylcholine (DPPC) and produces fluorescence increases only in the presence of Ca2+ and at temperatures above their gel-to-liquid crystalline phase transition. Although the fluorescence changes in CDRDANS accurately report phase transitions in these liposomes, its binding to these vesicles is weak. Calmodulin probably requires a high-affinity lipid-bound receptor protein for its high-affinity binding to natural membranes.
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015736:Felodipine; D005456:Fluorescent Dyes / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D008024:Ligands; D008081:Liposomes / Q000378:metabolism; D009543:Nifedipine / Q000031:analogs & derivatives / Q000494:pharmacology; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D014268:Trifluoperazine / Q000494:pharmacology
chemical_list: C035684:5-dimethylaminonaphthalene-1-sulfonyl-calmodulin; D000817:Anilino Naphthalenesulfonates; D002135:Calcium-Binding Proteins; D002147:Calmodulin; D005456:Fluorescent Dyes; D007093:Imidazoles; D008024:Ligands; D008081:Liposomes; D014268:Trifluoperazine; C031938:calmidazolium; C027132:1-anilino-8-naphthalenesulfonate; D009543:Nifedipine; D015736:Felodipine; D002118:Calcium