Halothane and isoflurane alter the Ca2+ binding properties of calmodulin

Abstract

Background: Ca2+ plays an important role in signal transduction and anesthetic mechanisms. To date, no one has observed a direct effect of volatile anesthetics on a Ca2+-binding protein. We therefore examined the effects of halothane and isoflurane on the Ca2+-binding properties of bovine brain calmodulin. Methods: The fluorescence emission of calmodulin was obtained over a range of Ca2+ concentrations (10-7 - 10-4 M) in the presence and absence of halothane and isoflurane. The intrinsic tyrosine fluorescence of calmodulin was measured at an excitation wavelength of 280 nm and an emission wavelength of 320 nm. Fluorescence measurements were carried out in 50 mM hydroxyethylpiperazineethane sulfonic acid, 100 mM KCl, and 2 mM ethyleneglycol-bis-(β-aminoethyl ether) tetraacetic acid at pH 7.0 and 37°C. Experiments were performed in polytetrafluorethylene-sealed cuvettes so that the volatile anesthetic concentrations remained constant. The titration data were analyzed in two ways. The data were fit to the Hill equation by using nonlinear regression analysis to derive the Hill coefficient and the dissociation constant. The data were also analyzed by two-way analysis of variance with multiple comparisons to determine statistically significant effects. Volatile anesthetic concentrations were measured by gas chromatography. Results: The presence of volatile anesthetics altered the Ca2+-binding affinity of calmodulin in a dose-dependent fashion. At 0.57% (0.25 mM) halothane and 1.7% (0.66 mM) isoflurane, the affinity of calmodulin for Ca2+ relative to control was decreased. However, at higher concentrations of both anesthetics, the affinity for Ca2+ was increased. When the volatile anesthetics were allowed to evaporate from the experimental solutions, the observed rightward shift of the calmodulin- Ca2+-binding curve for Ca2+ at low concentrations of the anesthetics returned to the control position. The leftward shift seen at high concentrations of the anesthetics was irreversible after evaporation of 8.7% (3.3 mM) isoflurane and 5.7% (2.5 mM) halothane. Conclusions: These data demonstrate a complex interaction of two hydrophobic volatile anesthetics with calmodulin. A biphasic effect was observed both for halothane and for isoflurane. Calmodulin, an EF-hand Ca2+-binding protein, undergoes a conformational shift when binding Ca2+, exposing several hydrophobic residues. These residues may be sites at which the anesthetics act.