LIPID-PROTEIN INTERACTIONS AND PROTEIN DYNAMICS IN VESICLES CONTAINING THE NICOTINIC ACETYLCHOLINE-RECEPTOR - A STUDY WITH ETHANOL

Electron paramagnetic resonance (EPR) spectroscopy was used to study t he action of ethanol on the protein side chain motions of the nicotini c acetylcholine receptor (nAcChoR) in alkaline extracted membranes fro m Torpedo nobiliana. EPR spectra of the nAcChoR derivatized with malei mide spin label contain both strongly and weakly immobilized component s. The rotational correlation time of the strongly immobilized compone nt decreases by a factor of 2-3-fold with the addition of 1.6 M ethano l, while that of the weakly immobilized component is not significantly altered. EPR spectroscopy was also used to probe the lipid environmen t immediately surrounding the nAcChoR with stearic acid and phosphatid ylcholine spin labeled at the fourteenth acyl carbons (14-SASL and 14- PCSL, respectively), and the steroid spin label androstanol (ASL). EPR spectra of these probes reveal a component corresponding to lipids th at are motionally restricted by the receptor (annular lipids) in addit ion to a more fluid component arising from bulk lipid. Using spectral subtraction, the order of selectivity of these spin labels for the nAc ChoR was determined to be ASL greater than or equal to 14-SASL > 14-PC SL. The estimated rotational correlation times of the high affinity 14 -SASL and ASL probes ranged from approx. 20 to 35 ns. The correlation times of the lower affinity 14-PCSL were generally shorter than those for 14-SASL and ASL and ranged from about 10 to 25 ns. The addition of up to 0.9 M ethanol altered neither the affinity nor the mobility of the motionally restricted EPR component. This suggests that ethanol's actions on the nAcChoR are not mediated via changes at the lipid/prote in interface near the center of the bilayer.