Binding of spin-labeled local anesthetics to lobster nerves

Abstract

Summary. The hyperfine coupling constant of spin-labeled local anesthetics, 2-(N-methyl N-(2,2,6,6-tetramethylpiperidinooxyl)) ethyl 4-alkoxybenzoates, showed these compounds to partition between the aqueous exterior and the hydrocarbon phase of the membrane. Increased partitioning into the hydrocarbon phase of the membrane was in the order: hexyloxy>butoxy>ethoxy. Since these compounds are known to have different durations of anesthesia in the same order, this suggests that durations of activity and ability to partition into the hydrocarbon region of the membrane are related.Local anesthetics block the action potential of nerve axons by preventing the transient increase in permeability of sodium to the axonal membrane [14,16]. The first step in the production of local anesthesia is believed to be an interaction of the drug with the lipid bilayer [15,19]. Since most local anesthetics are basic tertiary amines with pKa values of 7 to 9, at physiological pH they exist in both charged and uncharged forms. Narahashi [13] and others have shown that the charged form is responsible for nerve block activity and exerts its effect from inside of the membrane. However, whether these compounds are completely buried in the bilayer, partially or completely exposed to the outside, is unknown.A technique which is ideally suited to study these possibilities is that of spin labeling as developed by McConnell and colleagues [10] and Jost et aL [7]. It is based on line shape changes of the electron spin resonance spectrum of the nitroxide free radical. In situations where the radical can tumble unrestricted as in solution, three narrow symmetrical lines result. As the molecular motion of the radical decreases the lines become broad and asymmetrical. In addition, since the spectrum of the nitroxide radical is dependent on the dielectric constant of its solvent environment, it can give information concerning the polarity of its binding site. Although other techniques are more capable of giving quantitative data on binding, such as radioactive labeling, they cannot give information concerning the molecdlar properties of the binding sites.16"