Free
Correspondence  |   August 1996
In Reply: Nonanesthetic Haloalkanes and Nicotinic Acetylcholine Receptor Desensitization Kinetics
Author Notes
  • Assistant Professor of Anesthesia, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114–2696.
Article Information
Correspondence
Correspondence   |   August 1996
In Reply: Nonanesthetic Haloalkanes and Nicotinic Acetylcholine Receptor Desensitization Kinetics
Anesthesiology 8 1996, Vol.85, 431-432. doi:
Anesthesiology 8 1996, Vol.85, 431-432. doi:
In Reply:--The premise of Johansson's alternative interpretation is that nonanesthetics decrease energy transfer and reduce the fluorescence emission of the acetylcholine analog, Dns-C6-Chol. However, this premise is not supported by experimental data; titrations using increasingly higher nonanesthetic concentrations under conditions of energy transfer from nAcChoRs revealed no reduction in Dns-C6-chol fluorescence emission at equilibrium (Figure 1(A)). Therefore, the nonanesthetics 1,2-dichlorohexafluorocyclobutane and 2,3-dichlorooctafluorobutane do not significantly disrupt energy transfer from nAcChoR tryptophan residues to Dns-C6-Chol.
Figure 1. (A) Equilibrium fluorescence intensity of Dns-C6-Chol under conditions of energy transfer from nAcChoR tryptophan residues in the presence of nonanesthetics. The excitation wavelength was 290 nm, and emission was recorded above 560 nm. The predicted EC50s for 1,2-dichlorohexafluorocyclobutane and and 2,3-dichlorooctafluorobutane are 16 micro Meter and 4.5 micro Meter, respectively. Data are the means (+/-SD) of at least three determinations. (B) Stern-Volmer plots for nAcChoR-rich membrane intrinsic fluorescence quenching by halothane. Data are the means (+/-SD) of at least three determinations.
Figure 1. (A) Equilibrium fluorescence intensity of Dns-C6-Chol under conditions of energy transfer from nAcChoR tryptophan residues in the presence of nonanesthetics. The excitation wavelength was 290 nm, and emission was recorded above 560 nm. The predicted EC50s for 1,2-dichlorohexafluorocyclobutane and and 2,3-dichlorooctafluorobutane are 16 micro Meter and 4.5 micro Meter, respectively. Data are the means (+/-SD) of at least three determinations. (B) Stern-Volmer plots for nAcChoR-rich membrane intrinsic fluorescence quenching by halothane. Data are the means (+/-SD) of at least three determinations.
Figure 1. (A) Equilibrium fluorescence intensity of Dns-C6-Chol under conditions of energy transfer from nAcChoR tryptophan residues in the presence of nonanesthetics. The excitation wavelength was 290 nm, and emission was recorded above 560 nm. The predicted EC50s for 1,2-dichlorohexafluorocyclobutane and and 2,3-dichlorooctafluorobutane are 16 micro Meter and 4.5 micro Meter, respectively. Data are the means (+/-SD) of at least three determinations. (B) Stern-Volmer plots for nAcChoR-rich membrane intrinsic fluorescence quenching by halothane. Data are the means (+/-SD) of at least three determinations.
×
Johansson assumes that indole in methanol is an adequate experimental model for tryptophan residues in intrinsic membrane proteins, and he bases his premise on the observation that anesthetic and nonanesthetic compounds quench essentially all of the fluorescence in this model system. However, a Stern-Volmer plot of halothane quenching of nAcChoR-rich membrane fluorescence is quite different from that of halothane quenching of indole fluorescence (Figure 1(B)). It is nonlinear, with curvature toward the concentration axis, indicating that a significant fraction of protein tryptophans are either poorly quenched or not quenched at all. [1–3] .
Finally, none of the kinetic parameters reported in my study are even derived from absolute amplitudes. They are derived from amplitudes that are normalized to their final intensities (at equilibrium) or from observed rates. [4,5] These values are affected little, if at all, by quenching. However, because quenching could have reduced the signal:noise ratio and made the analysis more difficult, reason dictated avoiding compounds that seemed to offer the greatest probability of quenching.
Douglas E. Raines, MD; Assistant Professor of Anesthesia, Massachusetts General Hospital, 32 Fruit Street, Boston, Massachusetts 02114–2696.
(Accepted for publication April 29, 1996.)
REFERENCES
Eftink MR, Ghiron CA: Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies. Biochemistry 1976; 15:672-80.
Calhoun DB, Vanderkooi JM, Englander SW: Penetration of small molecules into proteins studied by quenching of phosphorescence and fluorescence. Biochemistry 1983; 22:1533-9.
Eftink MR: Fluorescence quenching: Theory and applications, Topics in Fluorescence Spectroscopy. Edited by Lakowicz A. New York, Plenum Press, 1991, pp 53-126.
Heidmann T, Changeux JP: Fast kinetic studies on the interaction of a fluorescent agonist with the membrane-bound acetylcholine receptor from Torpedo marmorata. Eur J Biochem 1979; 94:255-79.
Raines DE, Rankin SE, Miller KW: General anesthetics modify the kinetics of nicotinic acetylcholine receptor desensitization at clinically relevant concentrations. ANESTHESIOLOGY 1995; 82:276-87.
Figure 1. (A) Equilibrium fluorescence intensity of Dns-C6-Chol under conditions of energy transfer from nAcChoR tryptophan residues in the presence of nonanesthetics. The excitation wavelength was 290 nm, and emission was recorded above 560 nm. The predicted EC50s for 1,2-dichlorohexafluorocyclobutane and and 2,3-dichlorooctafluorobutane are 16 micro Meter and 4.5 micro Meter, respectively. Data are the means (+/-SD) of at least three determinations. (B) Stern-Volmer plots for nAcChoR-rich membrane intrinsic fluorescence quenching by halothane. Data are the means (+/-SD) of at least three determinations.
Figure 1. (A) Equilibrium fluorescence intensity of Dns-C6-Chol under conditions of energy transfer from nAcChoR tryptophan residues in the presence of nonanesthetics. The excitation wavelength was 290 nm, and emission was recorded above 560 nm. The predicted EC50s for 1,2-dichlorohexafluorocyclobutane and and 2,3-dichlorooctafluorobutane are 16 micro Meter and 4.5 micro Meter, respectively. Data are the means (+/-SD) of at least three determinations. (B) Stern-Volmer plots for nAcChoR-rich membrane intrinsic fluorescence quenching by halothane. Data are the means (+/-SD) of at least three determinations.
Figure 1. (A) Equilibrium fluorescence intensity of Dns-C6-Chol under conditions of energy transfer from nAcChoR tryptophan residues in the presence of nonanesthetics. The excitation wavelength was 290 nm, and emission was recorded above 560 nm. The predicted EC50s for 1,2-dichlorohexafluorocyclobutane and and 2,3-dichlorooctafluorobutane are 16 micro Meter and 4.5 micro Meter, respectively. Data are the means (+/-SD) of at least three determinations. (B) Stern-Volmer plots for nAcChoR-rich membrane intrinsic fluorescence quenching by halothane. Data are the means (+/-SD) of at least three determinations.
×