Lipid-protein interactions and protein dynamics in vesicles containing the nicotinic acetylcholine receptor: a study with ethanol

Abstract

Electron paramagnetic resonance (EPR) spectroscopy was used to study the action of ethanol on the protein side chain motions of the nicotinic acetylcholine receptor (nAcChoR) in alkaline extracted membranes from Torpedo nobiliana. EPR spectra of the nAcChoR derivatized with maleimide spin label contain both strongly and weakly immobilized components. The rotational correlation time of the strongly immobilized component decreases by a factor of 2–3-fold with the addition of 1.6 M ethanol, while that of the weakly immobilized component is not significantly altered. EPR spectroscopy was also used to probe the lipid environment immediately surrounding the nAcChoR with stearic acid and phosphatidylcholine 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 that are motionally restricted by the receptor (annular lipids) in addition to a more fluid component arising from bulk lipid. Using spectral subtraction, the order of selectivity of these spin labels for the nAcChoR was determined to be ASL >- 14-SASL > 14-PCSL. 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/protein interface near the center of the bilayer.