Recovery of Some Functional Properties of the Detergent-Extracted Cholinergic Receptor Protein from Torpedo marmorata after Reintegration into a Membrane Environment

Abstract

The change of affinity of the acetylcholine receptor for agonists and the influence of local anaesthetics has been studied in detail in receptor-rich membranes. These properties are changed after solubilisation by ionic detergents.A method for reproducibly reintegrating the receptor protein into a lipid environment is described. Reintegration of the receptor results in partial recovery of the binding and fluorescence properties of the membrane-bound receptor protein. In particular, the slow affinity change caused by agonists can be recovered but not the effect of local anaesthetics on this change. The fluorescence response to cholinergic ligands of the reintegrated receptor protein labelled with quinacrine does not appear identical to that found with the native receptor-rich membranes. It is suggested that the failure to recover the sensitivity to local anaesthetics is at the origin of the difficulties to regain functional reconstitution.Many attempts to reconstitute a functional 'excitable' membrane from preparations of purified nicotinic receptor protein from fish electric organ have been reported in the literature (for review see [l] and [2]). Crude detergent extracts of membrane preparations, rich in acetylcholine receptor protein [3], receptor protein purified by affinity chromatography in the presence of detergent [4,5] and even proteolipid fractions prepared directly from the electric organ [6,7] have been used and in several instances recovery of a permeability response to cholinergic agonists was found [3,4,8] but the reproducibility of these results is often questionable [1,2].As a first step to understanding the several parameters involved, our approach has been to study 'the binding properties of the receptor protein for cholinergic ligands in the membrane-bound and detergentsolubilised states and reintegrated into a membrane environment. Conditions have been previously defined under which a reversible transition between highaffinity and low-affinity states of the receptor protein takes place by varying the concentration of detergent [9]. Subsequently, it was shown that such transitions between affinity states exist in 'native' receptor-rich membrane fragments [lo] and are also regulated by cholinergic agonists. In the course of these studies it was also noticed that, in the same membrane fragments, local anaesthetics stabilize, at equilibrium, the receptor in its high-affinity state.In this paper we describe the reintegration of the receptor protein from Torpedo miirmorata electric organ into a lipid environment and demonstrate that, under these conditions, the agonist-binding characteristics of the membrane-bound receptor and the agonist-dependent affinity change can be recovered. The characteristic effect of local anaesthetics on this transition, however, cannot be demonstrated with the reintegrated receptor protein. The failure to recover the sensitivity to local anaesthetics might be at the origin of the difficulty to regain the full permeability response. MATERIALS AND METHODS Preparation ...

Regulation of receptor function by cholesterol

“…The interaction of the AChR with the surrounding lipids of the membrane has been studied for more than 20 years. In the late 1970s, delipidation of the AChR was found to modify the affinity of the AChR for agonists [28,29]. In addition, solubilization of the AChR in detergents without adequate lipids irreversibly stabilized the AChR in a low-affinity state [30].…”

Reconstitution of functional membrane-bound acetylcholine receptor from isolated Torpedo californica receptor protein and electroplax lipids.

“…Investigating these events, various groups have purified the AcChoR in solubilized and membrane-bound forms (1). Studies (5)(6)(7)(8)(9) or AcChoR purified by affinity chromatography (2)(3)(4) (Fig. 3).…”

“…Furthermore, purified AcChoR could be irreversibly altered by conventional purification methods (10), and thus could prove unsuitable for functional reconstitution. Alternatively, attempts at reconstitution with total solubilized protein from essentially intact membrane fragments generally yielded incompletely characterized membranes of low specific activity (i.e., a-neurotoxin binding) (5)(6)(7)(8)(9).…”

Identification of the polypeptide chains in Torpedo californica electroplax membranes that interact with a local anesthetic analog.

“…4). Because (20,32), detergents (32,45), caeruleotoxin (9,10), and, in some tissues (46) but not others (8), HTX …”

“…2B) and since local anesthetics have been reported to increase the rate of the Carb-induced transition in receptor affinity at concentrations below those that directly inhibit a-BuTx binding (20,32), the effects of PAA on the rate of this process were examined. Up to concentrations of 50 ,uM PAA, the rate of the transition of the receptor from low to high affinity caused by 1 (Fig.…”