title: Fluorescent probing with felodipine of the dihydropyridine receptor and its interaction with the ryanodine receptor calcium release channel.
abstract: The intensity of the fluorescence emission of the fluorescent 1,4-dihydropyridine (DHP) derivative felodipine increased upon binding to both isolated cardiac sarcolemma (SLM) and skeletal muscle sarcoplasmic reticulum (SR) preparations, the latter containing SR-transversal tubule junctional diads and triads. The fluorescence enhancement was due to the binding of felodipine to high-affinity (Kd's of 0.35 and 1.25 nM in cardiac SLM and skeletal SR, respectively) 1,4-dihydropyridine sites of the dihydropyridine receptor (DHPR), as evidenced in competition experiments with the DHP analog isradipine. In both cardiac SLM and SR, the felodipine fluorescence was sensitive to conformational changes of the DHPR, as diltiazem that binds to DHPR at a separate site altered the values of both the Kd and the Hill coefficient characteristic for felodipine binding. In skeletal muscle membranes containing intact TT-SR junctions, ryanodine, a specific ligand of the ryanodine receptor calcium release channel (RyRC), also induced changes in felodipine fluorescence, which was eliminated by detergent and high-salt treatment to solubilize the RyRC. These results suggest that i) felodipine fluorescence is useful to probe conformational changes of the DHPR and ii) coupled conformational changes between the DHPR and the RyRC in skeletal muscle indeed occur and could be monitored by measuring felodipine fluorescence.
authors.affiliation: Department of Physiology and Endocrinology, Medical College of Georgia, Augusta 30912, USA.
authors.forename: I
authors.initials: I
authors.lastname: Minarovic
authors.forename: L G
authors.initials: LG
authors.lastname: Mészáros
mesh_terms: D000818:Animals; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels / Q000737:chemistry / Q000378:metabolism*; D020746:Calcium Channels, L-Type; D004110:Diltiazem / Q000378:metabolism; D004285:Dogs; D015736:Felodipine / Q000378:metabolism*; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D017275:Isradipine / Q000378:metabolism; D007700:Kinetics; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism*; D012508:Sarcolemma / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D013050:Spectrometry, Fluorescence
chemical_list: D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D005456:Fluorescent Dyes; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D004110:Diltiazem; D015736:Felodipine; D017275:Isradipine