Isoflurane activates the type 1 ryanodine receptor to induce anesthesia in mice
Fig 6
Identification of RyR1 agonists that share the binding site with isoflurane.
(A) Scheme of the chemical screening. Following in silico screening, the agonistic actions of some of the hit compounds were tested with tetracycline-induced expressions of RyR1(WT) and RyR1(M4000F). (B) Results of the in vitro screening. As controls, stimulations by caffeine, isoflurane, and basal buffer without chemicals were included. 4-bromophenol (4-BP) is identified as a hit compound. (C) Putative binding formation of 4-BP. (D) Dose-dependent effects of 4-BP and the isomers 3-BP and 2-BP on RyR1(WT), RyR1(M4000F), RyR2, and RyR3. Data were fitted using logistic functions (equation 4 in Materials and methods). N = 4. (E, F) Effects of methyl phenols (MPs) (E) and ethyl phenols (EPs) (F) on RyR1. N = 5. (G) Effects of a series of 4-alkylphenols on RyR1, RyR2, and RyR3. N = 5. (H) Effects of 4-MP, 4-EP, and 4-PP on RyR1(WT) and RyR1(M4000F). The time-course reactions and the peak intensities are shown, respectively. N = 4. Data are presented as Mean ± SD. RyR1 and the M4000F mutant were expressed through tetracycline induction in the stable cell lines except for the data shown in panel H. RyR1, the type 1 ryanodine receptor; Fmax/F0, the maximum fluorescence intensity; 4-BP, 4-bromophenol; RyR2, the type 2 ryanodine receptor; RyR3, the type 3 ryanodine receptor; 4-BP, 4-bromophenol; 3-BP, 3-bromophenol; 2-BP, 2-bromophenol; 4-MP, 4-methylphenol; 3-MP, 3-methylphenol; 2-MP, 2-methylphenol; 4-EP, 4-ethylphenol; 3-EP, 3-ethylphenol; 2-EP, 2-ethylphenol; 4-PP, 4-propylphenol; Caf., Caffeine.