Fluidity of the lipids next to the acetylcholine receptor protein of torpedo membrane fragments. Use of amphiphilic reversible spin-labels.
Choline esters of spin-labeled fatty acids (long-chain acylcholines) were used to probe the hydrophobic environment of the acetylcholine receptor protein in membrane fragments from Torpedo marmorata. These spin-labels competitively inhibit the binding of [3H]acetylcholine to the receptor site. Their inhibition constants (KI) were close to 200 nM. At the high membrane concentration required for electron spin resonance (ESR) experiments, the apparent inhibition constants (KIapp) differed from KI determined by using dilute membrane concentration. This is due to the amphiphilic character of long-chain acylcholine. For most spin-labels used, only difference ESR spectroscopy provided reliable spectra corresponding to receptor-bound spin-labeled acylcholines. Acetylcholine receptor agonists and antagonists displaced the acylcholine from the receptor sites, whereas choline had only a weak effect. This produced a modification in the ESR spectra of the bound acylcholines and provided evidence that the acylcholines bound to the receptor sites in a specific manner. The interpretation of the spectra of receptor-bound spin-labels favored a strong barrier to the motion of the probe when attached to the middle of the acyl chain. However, when the probe was close to the methyl terminal of a stearoylcholine molecule a much greater fluidity was found. Short-range spin-spin interactions were created between spin-labels bound to the receptor site and spin-labels in a fluid phase. This indicates that lipids next to the receptor protein are not completely immobilized in spite of the semicrystalline organization of the proteins in the postsynaptic region.
D000109:Acetylcholine / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000502:physiology / Q000648:ultrastructure; D004557:Electric Organ / Q000378:metabolism* / Q000648:ultrastructure; D004578:Electron Spin Resonance Spectroscopy; D005399:Fishes; D007700:Kinetics; D008433:Mathematics; D008563:Membrane Lipids / Q000502:physiology*; D011950:Receptors, Cholinergic / Q000378:metabolism; D013113:Spin Labels; D013329:Structure-Activity Relationship
D008563:Membrane Lipids; D011950:Receptors, Cholinergic; D013113:Spin Labels; D000109:Acetylcholine