Establishing a System for the Stable Expression of Torpedo Acetylcholine Receptors

Abstract

Several laboratories have shown that functional Torpedo acetylcholine receptors (AChRs) can be transiently expressed on the surface of Xenopus oocytes (1–3, Claudio & Sigworth, unpublished). Our laboratory is interested in studying several aspects of receptor biology. Many of the problems we wish to address can best be studied if AChR is expresssed continuously, in a stable fashion, rather than transiently. We are interested in the process of subunit assembly, the interactions of the nAChR with other components of nerve and muscle cells, and we would like to determine how certain regions of the molecule are folded and how they effect ion permeation. Our approach to beginning to study some of these processes is to introduce the 4 subunit cDNAs into the chromosomes of tissue culture cells and establish new lines that stably express functional Torpedo AChRs on their cell surface. There are several advantages of such a stable system over the currently popular system of transient expression of AChRs in Xenopus oocytes: 1) The expression of receptor is stable and continuous, not transient, therefore eliminating the need to establish expression every time one wishes to do an experiment. 2) Because new cell lines can be derived from a single transduced cell, all the cells are identical and the line only need be characterized once. 3) Large quantities of identical cells are easily obtained, making feasible, many types of biochemical and kinetic studies that would be difficult, tedious or impossible to perform on oocytes. 4) Several cell biological processes and protein-protein interactions can only be studied in a system where the proteins are continuously expressed for long periods of time. 5) Certain biophysical studies, such as single channel recordings using patch clamp techniques, are more easily performed on tissue culture cells than oocytes.