Local Anesthetic-membrane Interaction: a Multiequilibrium Model

We report the detection of electrostatic interactions between local anesthetics and membrane phospholipids and proteins. A spin-labeled local anesthetic was used to study how membrane-bound tertiary amine anesthetics interact with major molecular components in the membrane. The nitroxyl reporter group of this spin label is located at the polar end of the amphiphilic local anesthetic; it is therefore a uniquely suitable probe for detecting immobilization of the anesthetic due to binding interactions at the polar regions of the bilayer. The binding properties of this spin-labeled anesthetic to human erythrocyte membranes and to vesicles made from human erythrocyte lipids were studied. Lipid vesicle-bound spin labels give rise to a composite electron spin resonance spectrum from which two subcomponent spectra were resolved. Both components are membrane-bound; the first component has a narrower linewidth, indicating a greater mobility of the nitroxyl moiety of the anesthetic probe. The second component has a broader linewidth, indicating a population of constrained spin labels. We infer from the experimental results that electrostatic binding between cationic anesthetics and anionic phosphate of phospholipids produced the constrained component. In similar studies using erythrocyte ghost membranes, both a mobile (nonelectrostatic) component and a constrained (electrostatic) component were resolved from the composite spectrum. However, the constrained component in this case is much broader than the corresponding constrained component from the vesicles. We interpret this broad component in the erythrocyte membrane as an electrostatic interaction of cationic anesthetic probes with phospholipids and with membrane proteins. We conclude that membrane-bound tertiary amine anesthetics in cationic form do interact selectively with phospholipids and proteins.