Agonist binding and affinity state transitions in reconstituted nicotinic acetylcholine receptors revealed by single and sequential mixing stopped-flow fluorescence spectroscopies.
The affinity state of nicotinic acetylcholine receptors (nAcChoRs) reconstituted into either dioleoylphosphatidylcholine (DOPC) or a mixture of dioleoylphosphatidylcholine, dioleoylphosphatidic acid, and cholesterol (DOPC/DOPA/cholesterol) has been determined using single and sequential mixing stopped-flow fluorescence spectroscopies. These techniques have millisecond temporal resolution, permitting low- and high-affinity conformational states of the nAcChoR to be resolved following mixing with the fluorescent partial agonist Dns-C6-Cho from their characteristic Dns-C6-Cho dissociation rates. Our studies reveal that prior to agonist-induced affinity state conversion, nAcChoRs reconstituted into either DOPC or DOPC/DOPA/cholesterol are predominantly in a conformational state that has a low affinity for agonist. Prolonged exposure to Dns-C6-Cho converts nearly all DOPC/DOPA/cholesterol-reconstituted nAcChoRs to the high-affinity state. In contrast, Dns-C6-Cho converts only half of all DOPC-reconstituted nAcChoRs to the high-affinity state. The other half persists in a low-affinity state characterized by a Kd for Dns-C6-Cho of 0.61+/-0.07 microM. This Kd is similar to that previously reported for Dns-C6-Cho binding to low-affinity, resting-state nAcChoRs in native membranes. However, affinity state conversion of DOPC-reconstituted nAcChoRs may be facilitated by re-reconstituting them into bilayers composed of DOPC/DOPA/cholesterol. These results indicate that the lipid bilayer composition modulates nAcChoR agonist-induced affinity state transitions.
Raines
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Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA. raines@etherdome.mgh.harvard.edu
Krishnan
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D000818:Animals; D001665:Binding Sites; D002784:Cholesterol / Q000737:chemistry; D004557:Electric Organ / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry; D018722:Nicotinic Agonists / Q000378:metabolism*; D010712:Phosphatidic Acids / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014101:Torpedo / Q000378:metabolism
D008051:Lipid Bilayers; D018722:Nicotinic Agonists; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D011978:Receptors, Nicotinic; C037657:dioleoylphosphatidic acid; D002784:Cholesterol; C017251:1,2-oleoylphosphatidylcholine