Binding of local anesthetics to reconstituted acetylcholine receptors: effect of protein surface potential.

Nicotinic acetylcholine receptor isolated from Torpedo californica electric organ is reconstituted into lipid bilayers of zwitterionic dioleoylphosphatidylcholine. These membranes are labeled with a spin-labeled quaternary amine local anesthetic (C6SLMeI), which has been shown previously to be a noncompetitive blocker of acetylcholine receptor-ion channel function in the micromolar concentration range. The electron spin resonance spectral component corresponding to protein-immobilized anesthetic spin-label can be resolved from the composite data spectrum by using spectral subtraction of lipid components. This protein-immobilized component is shown to represent C6SLMeI bound to a finite number of sites on the receptor. We demonstrate that C6SLMeI binds to the receptor as a function of the surface potential on the protein and suggest that the acetylcholine receptor reconstituted into zwitterionic phospholipid, which has no surface potential of its own, provides an excellent model system with which to study effects of protein surface charge. We hypothesize that the primary pathway of interaction of C6SLMeI with the acetylcholine receptor is via the aqueous medium.