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Updated: Oct 27, 2026

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time
Published on: March 11, 2021
Agonist binding and affinity state transitions in reconstituted nicotinic acetylcholine receptors revealed by single
1Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston 02114, USA. raines@etherdome.mgh.harvard.edu
Abstract:
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.
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