Isoflurane activates the type 1 ryanodine receptor to induce anesthesia in mice
Fig 2
Identification of RyR1 residues responsible for the sensitivity to isoflurane.
(A) Transient transfection of RyR1, RyR2, and the chimeric receptors (ChR.1-3, ChR.1′-3′) into HEK 293T cells. The time-course reaction (F/F0) and the peak of the isoflurane response normalized by the caffeine response are shown. N = 4. (B) The RyR1-RyR2 chimeric receptor 10 (ChR.10) and ChR.10′. N = 4. (C) The chimeric receptors, ChR.10, ChR.13, ChR.13-6, and the amino acid substitution mutants. N = 8. (D) Normalized isoflurane responses of M4000 mutants with single amino acid substitutions. The amino acid substitution was color-coded as follows: cyan for basic amino acids; red for acidic amino acids; orange for amino acids with polar uncharged side chains; green for amino acids with alkyl chains; and purple for amino acids with aromatic rings. N = 4–12. (E) Time-course reaction and normalized isoflurane response of RyR1(M4000F). N = 4. (F) Stable cell lines that express RyR1(WT) and RyR1(M4000F). Basal buffer without pharmacological agents, 1.25 mM caffeine, or 0.63 mM anesthetics (isoflurane, sevoflurane, and halothane) were given. N = 4. Data are represented as Mean ± SD. ** Adjusted P-values (Adj. P) < 0.01 by the Steel’s test (multiple comparisons between RyR1 and other groups). For the calculation of the Normalized Iso. Response (equation 1 in Materials and methods), the peaks at 5.0 mM caffeine and the control (without pharmacological agents) were normalized to 1.0 and 0, respectively, in the transient expression experiment (A–E), while the peaks at 1.25 mM caffeine and the control were 1.0 and 0, respectively, in the experiments with stable cell lines (F). ChR., Chimeric receptor; RyR1, the type 1 ryanodine receptor; RyR2, the type 2 ryanodine receptor; Caf., Caffeine; Iso., Isoflurane; Sevo., Sevoflurane; Halo., Halothane.