Incorporation of acetylcholine receptors and Cl- channels in Xenopus oocytes injected with Torpedo electroplaque membranes.

Abstract

A method was developed to transplant assembled nicotinic acetylcholine receptors (AcChoRs) and Cl1 channels from the electric organ of Torpedo to the membrane of Xenopus oocytes. Membrane vesicles from Torpedo electroplaques were injected into the oocytes and,within a few hours, the oocyte membrane acquired AcChoRs and Cl-channels.The mechanism of exprpssion of these receptors and channels is very different from that which follows the injection of mRNA, since the 4ppearance of receptors after membrane injection does not require'de novo protein synthesis or Nglycosylation. This, and other controls, indicate that the foreign receptor-bearing membranes fuse with the oocyte membrane and cause the appearance of functional receptors and channels. All this makes the Xenopus oocyte an even more powerful tool for studies of the structure and function of membrane proteins.Xenopus oocytes are a useful system to study the structure and function of many neurotransmitter receptors, ion channels, and other membrane proteins. In the first place, Xenopus oocytes and their enveloping follicular cells, which are electrically coupled to the oocyte, are endowed with a variety of native neurotransmitter and hormone receptors and voltageoperated channels (1-4). Moreover, after injection of appropriate mRNAs, Xenopus oocytes will express a large variety of foreign receptors and channels.' The oocytes translate the heterologous mRNA, process the products, and incorporate them into their plasma membrane where they form functional receptors and channels (5).The present experiments were done to see if the oocyte membrane could be made to acquire foreign receptors, already assembled in other cell membranes, without injecting the nucleic acids. If this could be achieved, the oocytes would become even more useful for studies of many receptors, channels, and signaling events. Our experiments, carried out early in 1990, showed that acetylcholine receptors (AcChoRs) and Cl-channels from Torpedo can be transplanted directly from the Torpedo electroplaques to the oocyte membrane. Some of the results have been presented in brief (6). MATERIALS AND METHODSTo determine whether foreign membranes carrying neurotransmitter receptors could be incorporated directly into the oocyte plasma membrane, Xenopus oocytes were injected with membranes isolated from the electric organ of Torpedo, because the electroplaque cells are very rich in AcChoRs and C1 channels (7-11). Membrane Preparation. Membranes were prepared as described (10, 11) with slight modifications. About 300 g of frozen electric organ from Torpedo californica was thawed in 1-2 liters of a hypoosmotic solution (1 mM EDTA, 10. mM Hepes buffer, pH 7.2). This solution was discarded and freshThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.solution was added to make 1 liter. The thawed, or sometimes fresh, tissue was homogenized first ...

Guanine Nucleotides, Including GMP, Antagonize Kainate Responses in Xenopus Oocytes Injected with Chick Cerebellar Membranes

“…As has been shown previously for fish and mammalian membranes (Marsal et al, 1995;Canals et al, 1996;Aleu et al, 1997), the chick cerebellar membrane fragments become integrated into the oocyte plasma membrane in a seamless way (Fig. 1).…”

“…First, the receptors to be studied are injected in a ready-to-work state so that their presence in the oocyte membrane can be detected as early as a few hours after injection (Marsal et al, 1995). Second, the foreign receptors retain their own membrane environment, presumably incorporating whichever protein subunits and lipid matrix are needed for full physiological function, including regulatory phenomena such as desensitization or facilitation, thus circumventing the need for the coordinated expression of a number of mRNAs, plus the appropriate posttranslational modification and assembly, and the association to the right lipidic environment.…”

“…Bath application of kainate to oocytes injected with just the vehicle solution did not elicit any responses. The amplitude of the current recorded for a given concentration of kainate, at a holding potential of -70 mV, was somewhat variable, probably depending on variations in the donor frogs as well as in the different membrane batches (Marsal et al, 1995).…”

“…This method has the advantage of incorporating foreign receptors into the oocyte membrane in their own natural molecular environment, with higher possibilities of their behaving in a thoroughly physiological way (Marsal et al, 1995;Canals et al, 1996).…”

Phosphatase inhibitors remove the run-down of γ-aminobutyric acid type A receptors in the human epileptic brain

“…Essentially, this method consists in injecting the oocytes with cell membranes prepared from foreign tissues. The oocyte plasma membrane incorporates the foreign membranes and efficiently acquires functional human neurotransmitter receptors and voltage-operated channels, which retain the properties they have in their native tissues (13)(14)(15).…”