To the Editor:--Raines reports the effect of volatile anesthetics (enflurane and isoflurane) and ‘nonanesthetics’(2,3-dichlorooctafluorobutane and 1,2-dichlorohexafluorocyclobutane) on the desensitization kinetics of the Torpedo nicotinic acetylcholine receptor (nAChR). [1]The less pronounced effect of nonanesthetics on the desensitization kinetics of this membrane protein is interpreted as evidence that the system is a valid model of the volatile anesthetic site of action, based on a somewhat contentious criterion suggested by Eger and colleagues. [2–4]Indeed, these nonanesthetics were described recently as inducing at least one component of the anesthetic state. [5].
The nonanesthetics studied were chosen, in part, because they lack a bromine atom that may quench fluorescence. However, halogenated molecules that contain chlorine atoms also may quench fluorescence, [6]in some cases with an efficiency (the probability that an encounter between quencher and fluorophore will result in energy transfer) that exceeds that of brominated compounds. Therefore, using quenching of indole fluorescence in methanol (as a model for tryptophan residues in proteins), 1,2-dichlorohexafluorocyclobutane is found to be a superior quencher compared with halothane, whereas 2,3-dichlorooctafluorobutane is 84% as efficient as halothane (Figure 1). The effective fluorescence quenching exhibited by these bichlorinated compounds is presumably due to the electron withdrawing influence of the neighboring fluorines, which results in favorable conditions for electron transfer to the chlorine atoms. The lack of a bromine atom, therefore, is not a valid reason to exclude direct fluorescence quenching by the test compound.
![Figure 1. Stern-Volmer plots for indole (10 micro Meter) fluorescence quenching in methanol by (A) 1,2-dichlorohexafluorocyclobutane, (B) halothane, and (C) 2,3-dichlorooctafluorobutane. Excitation at 295 nm with emission peaks at 331 nm. Data plotted as Fo/F = 1 + Ksv*symbol*[Haloalkane], where Foand F are the fluorescence intensities in the absence and presence of added haloalkane, respectively, and Ksvis the Stern-Volmer quenching constant. Data points are the means of three to seven experiments, with error bars representing SEM.](https://asa2.silverchair-cdn.com/asa2/content_public/journal/anesthesiology/85/2/10.1097_00000542-199608000-00030/2/m_30ff1.png?Expires=1697143429&Signature=Yqkpa82L7kgqFA5VYBg4rl3cnQgn294YTydLcKI66EM1XQ1aQAbcpu7~xNOFojFqEj4M-BXBd3JTsfdqWKmiIvwzn5pouFEtK0danBelVCZXnozLYkOxkgTa4X90dPhltiFMB7Z87rrbRh95xrKgZC7wfFxyHtHU0eylmLDga~OUcPqPRUEv182cbh~7oiJvtPQX~px5UxZDjz2Pr24xpEMISl0xSbN5gZ5zcaHcwNl6dkZ1rkbwvo9ypLK~gThDO4VwbzT78sg6WaPJAC52tMj-W2vFRRFiL8mKzWTyzgWwp-a5QsV4JYpdPIJsKR~R6apfHzgglizrQ16rkHYfhg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Figure 1. Stern-Volmer plots for indole (10 micro Meter) fluorescence quenching in methanol by (A) 1,2-dichlorohexafluorocyclobutane, (B) halothane, and (C) 2,3-dichlorooctafluorobutane. Excitation at 295 nm with emission peaks at 331 nm. Data plotted as Fo/F = 1 + Ksv*symbol*[Haloalkane], where Foand F are the fluorescence intensities in the absence and presence of added haloalkane, respectively, and Ksvis the Stern-Volmer quenching constant. Data points are the means of three to seven experiments, with error bars representing SEM.
Figure 1. Stern-Volmer plots for indole (10 micro Meter) fluorescence quenching in methanol by (A) 1,2-dichlorohexafluorocyclobutane, (B) halothane, and (C) 2,3-dichlorooctafluorobutane. Excitation at 295 nm with emission peaks at 331 nm. Data plotted as Fo/F = 1 + Ksv*symbol*[Haloalkane], where Foand F are the fluorescence intensities in the absence and presence of added haloalkane, respectively, and Ksvis the Stern-Volmer quenching constant. Data points are the means of three to seven experiments, with error bars representing SEM.
An alternative interpretation of the results reported by Raines [1]is that quenching of a portion of the native nAChR tryptophan fluorescence by the nonanesthetics leads to decreased energy transfer to the fluorescent acetylcholine analog, which consequently emits less of a signal. Fluorescence energy transfer [7]from tryptophan to a dansyl group is estimated to occur efficiently over distances of up to 40–50 Angstrom (for comparison, the width of the phospholipid bilayer is 30–40 Angstrom), suggesting that multiple tryptophan residues in the nAChR are responsible for exciting the fluorescent acetylcholine analog. The observation that the fluorescence intensity of the added acetylcholine analog increases at higher concentrations of nonanesthetic might then reflect the balance between fluorescence quenching of selected tryptophan residues and nonanesthetic-induced conformational changes in the nAChR associated with desensitization. In support of this interpretation is the finding that the relative inability of the two nonanesthetics to promote nAChR desensitization (as shown in Figure 5 and Figure 6 of [1]) correlates with their fluorescence quenching efficiency, and not with their potency as predicted by oil-gas solubility. [2]It is open to question whether the approach used in this study, which relies on changes in the fluorescence intensity of an added ligand, that is in turn excited by energy transfer from multiple protein tryptophan residues, rules out an effect of non-anesthetics on nAChR desensitization kinetics.
Jonas S. Johansson, M.D., Ph.D.; Assistant Professor of Anesthesia, University of Pennsylvania, 3400 Spruce St., Philadelphia, Pennsylvania 19104–4238.
(Accepted for publication April 29, 1996.)
