Incorporation of reconstituted acetylcholine receptors from Torpedo into the Xenopus oocyte membrane.

Abstract

Xenopus oocytes are a valuable aid for studying the molecular structure and function of ionic channels and neurotransmitter receptors. Their use has recently been extended by the demonstration that oocytes can incorporate foreign membranes carrying preassembled receptors and channels. Here we show that when reconstituted in an artificial lipid matrix and injected into Xenopus oocytes, purified nicotinic acetylcholine receptors are efficiently inserted into the plasma membrane, where they form "clusters" of receptors that retain their native properties. This constitutes an innovative approach that, besides allowing the analyses of membrane fusion processes, is also a powerful technique for studying the characteristics and regulation of many membrane proteins (with their native stoichiometry and configuration) upon reinsertion into the membrane of a very convenient host cell system.The functional properties of nicotinic acetylcholine receptors (nAcChoRs) have been elucidated by many approaches: for example, electrophysiological recordings from muscle (1), measurements of ion fluxes in membrane vesicles containing nAcChoRs (2), expression of mRNAs encoding nAcChoRs in Xenopus oocytes (3)(4)(5), and, more recently, by injecting oocytes with membranes from Torpedo electroplaques (6). The latter approach results in the incorporation of native Torpedo nAcChoRs and other proteins into the oocyte's plasma membrane (6). This procedure has some important advantages over the usual oocyte expression system, since it allows the study of receptors that have been fully processed and assembled, with their natural subunit stoichiometry, in the original cell membrane. Nevertheless, there are many reasons that make the incorporation of well-defined purified proteins into the host cell membranes highly desirable. Therefore, we set to find out whether purified nAcChoRs reconstituted in a lipid matrix could be incorporated in a host cellular system such as the Xenopus oocyte, in which the function and cellular regulation of the protein can be examined in detail. Preliminary results have been presented elsewhere (7,8). METHODSSolubilization and Reconstitution of nAcChoRs. nAcChoRrich membranes from the electric organ of Torpedo marmorata were used to purify nAcChoRs by affinity chromatography in the presence of asolectin lipids and with cholate as detergent (9-11) (Fig. 1). The specific activity of the purified nAcChoRs was 'z-8 nmol of a-bungarotoxin bound per mg of protein (10).Reconstitution of nAcChoRs in asolectin lipid vesicles was accomplished by a detergent dialysis method (10). Final concentrations in the reconstitution mixtures were as follows: nAcChoRs, 0.3-1.2 mg of protein per ml; asolectin lipids, -5 mg/ml; and sodium cholate, 1% (wt/vol). After dialysis, reconstituted nAcChoR samples were aliquoted and injected immediately into oocytes or stored in liquid nitrogen, either alone or in the presence of trehalose (5 mg/mg of protein) to prevent protein denaturation (10).Oocyte Preparation and Microinjection. Xenopus ...

Effects of Bis(7)-Tacrine on Spontaneous Synaptic Activity and on the Nicotinic ACh Receptor of Torpedo Electric Organ

“…Membrane transplantation preserves the activity of native receptors much better than the receptors translated in oocytes from mRNA (Marsal et al 1995;Morales et al 1995). The IC 50 for ACh-induced currents of bis(7)-tacrine (162 nM) was much lower than previously reported for tacrine (8.9 M) (Cantí et al 1998), which indicates that bis(7)-tacrine is a more potent inhibitor of nicotinic acetylcholine receptor than tacrine.…”

From Nuclear Transfer to Nuclear Reprogramming: The Reversal of Cell Differentiation

“…Since the work of Krieg & Melton (1984), the injection of in vitro-synthesized mRNA to eggs has become a very widely used procedure for analyzing gene function by overexpression. The Xenopus mRNA injection procedure was later to prove of great value in identifying genes that encode neural receptors (Morales et al 1995, Soloviev & Barnard 1997. The wider use of injected living eggs of Xenopus was followed by the work of my students Alan Colman and Charles Lane, the son of Dame Miriam Rothschild, famous for flea taxonomy and nature conservation, showing secretion and other gene expression steps that injected eggs perform (Drummond et al 1985;Gurdon & Melton 1981;Lane et al 1971Lane et al , 1980.…”

Expression of functional neurotransmitter receptors in Xenopus oocytes after injection of human brain membranes

“…Significantly, many steps between foreign mRNA injection into the oocyte and the insertion of AcChoRs into the oocyte's plasma membrane were avoided by injecting the oocytes with membrane vesicles from the electroplaques. With this method the oocyte membrane efficiently acquired functional AcChoRs and Cl Ϫ channels, still embedded in their native membrane (6,7).…”