Reconstitution of a Functional Acetylcholine Receptor

Abstract

The 'functional' state of the acetylcholine receptor protein has been followed during reconstitution with the fluorescent agonist [l-(5-dimethylaminonaplithalene)-sulfonamido]-n-hexanoic acid-P-Ntrimethylammonium bromide ethyl ester (Dns-C6-Cho) and rapid-mixing techniques. Under appropriate conditions, a majority of the acetylcholine receptor sites can be recovered in a low-affinity state(s) for Dns-C6-Cho, similar to that found with the native membrane-bound receptor. This state can be slowly interconverted to a high-affinity state after rapid mixing with the agonist, and the noncompetitive channel blockers, like the local anesthetics, still regulate this transition in an allosteric manner.Several experimental conditions commonly used for the solubilization of the receptor and for its purification in the presence of sodium cholate result in the failure of reconstitution: the soluble receptor protein is stabilized in a low-affinity state which can no longer be interconverted to a high-affinity state in the presence of agonists or local anesthetics. On the other hand, it is demonstrated that if the concentration of lipids remains elevated in the presence of sodium cholate, a soluble (9 S) lowaffinity form of the receptor protein can be obtained which shows most of the characteristic properties of the membrane-bound receptor and in particular the slow interconversion to the highaffinity state and the effect of local anesthetics on this transition ; furthermore, in these conditions the soluble protein can be manipulated ad libitum and submitted, for instance, to column filtrations and sucrose gradient centrifugations in the presence of detergent, without losing its characteristic conformational and allosteric transitions. After elimination of the detergent this form yields a reconstituted receptor which presents binding properties identical to those of the native membranebound receptor and leads to the formation of vesicles which exhibit carbamylcholine-sensitive ion fluxes.A necessary and sufficient condition for functional reconstitution is therefore the conservation, in the presence of lipids, of the allosteric properties of the receptor protein in its detergent-soluble form.The aim of the reconstitution experiments presented in this paper and in the preceding one, is to identify the components of the subsynaptic membrane which are necessary and sufficient for the occurrence of the physiological response to acetylcholine. Since in vivo this response is characterized by a change of electrical conductance it appeared legitimate to [21,23 -25,28,29]. These investigations have lead to an altogether more precise and complete

Structural Sensitivity of a Prokaryotic Pentameric Ligand-gated Ion Channel to Its Membrane Environment

“…The underlying mechanisms by which lipids modulate nAChR function have been the subject of continuous debate for over 30 years (6). There has long been a consensus that both cholesterol and anionic lipids are required in a reconstituted membrane to obtain optimal nAChR function (7-10, 34 -36).…”

“…Early attempts at purifying and reconstituting the prototypic Cys-loop receptor, the Torpedo nicotinic acetylcholine receptor (nAChR), 2 in lipid bilayers revealed the importance of lipids in Cys-loop receptor function (6). In the presence of both cholesterol and anionic lipids, the nAChR is stabilized predominantly in an activatable resting conformation (7)(8)(9)(10).…”

Selective labelling by [3H]trimethisoquin azide of polypeptide chains present in acetylcholine receptor‐rich membranes from Torpedo marmorata

“…Finally, the labelling should disappear after dissolution of the ACh-receptor protein by high concentration of Na-cholate under conditions where the allosteric interaction between the ACh-receptor site and the site for non-competitive blockers is abolished [23,24]. We report here the labelling of two peptides of mol.…”

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

“…However, it is to be noted that these interactions are not conserved across the family (21). The strategic location of the ECD-TMD interface also makes this region a likely target for lipid-mediated effects on channel function (22)(23)(24). Interestingly, sequence analysis predicts the presence of a potential cholesterol-binding motif in GLIC at the pre-M1 region of the interface (25).…”