Toni Claudio

Acetylcholine receptor assembly: Subunit folding and oligomerization occur sequentially

The temperature sensitivity of nicotinic acetylcholine receptors (AChRs) from T. californica was used to identify steps in AChR subunit folding and oligomerization. Assembly intermediates were isolated by lowering to an assembly-permissive temperature. The earliest identifiable assembly intermediates, alpha beta gamma trimers, form minutes after subunit synthesis. alpha beta gamma delta tetramers are formed slowly by the addition of delta subunits to trimers, and finally a second alpha subunit is added to form alpha 2 beta gamma delta pentamers. Between these oligomerization steps, subunits fold as monitored by alpha-bungarotoxin-binding site formation, appearance of antigenic epitopes, changes in apparent molecular weight, and changes in detergent solubility. Subunit folding requires specific combinations of subunits and correlates in time with subunit additions, suggesting that these subunit folding events contribute to subunit recognition site formation during assembly.

Acetylcholine receptor assembly is stimulated by phosphorylation of its γ subunit

Abstract Different combinations of Torpedo acetylcholine receptor (AChR) subunits stably expressed in mouse fibroblasts were used to establish a role for phosphorylation in AChR biogenesis. When cell lines expressing fully functional AChR complexes ( α 2 βγδ ) were labeled with 32 P, only γ and δ subunits were phosphoryaated. Forskolin, which causes a 2- to 3-fold increase in AChR expression by stimulating subunit assembly, increased unassembled γ phosphorylation, but had little effect on unassembled δ. The forskolin effect on subunit phosphorylation was rapid, significantly preceding its effect on expression. The pivotal role of the γ subunit was established by treating αβγ and αβδ cell lines with forskolin and observing increased expression of only αβγ complexes. This effect was also observed in αγ, but not αδ cells. We conclude that the cAMP-induced increase in expression of cell surface AChRs is due to phosphorylation of unassembled γ subunits, which leads to increased efficiency of assembly of all four subunits.

Recombinant DNA technology in the study of ion channels

Abstract Molecular biological techniques have allowed more questions regarding the structure and function of the nicotinic acetylcholine receptor to be addressed. Toni Claudio describes the progress that has been made in characterizing the receptor-channel complex as regards subunit assembly and expression and explains how cDNA probes can be used to identify receptor subtypes and monitor receptor levels during development. Functional - biochemical and pharmacological - techniques are required to validate the information gained from cloning techniques; pharmacologists are far from being redundant!

Stable Expression of Multisubunit Protein Complexes in Mammalian Cells

There are advantages and disadvantages to each of the different protein expression systems available. Whereas ease, speed, and expense are important factors in determining which type of expression system to use, the complexity of the protein to be expressed and the kinds of questions addressed will often be the factors that ultimately determine which system is used. Our interest has been in expressing the nicotinic acetylcholine receptor (AChR). This protein is a heterologous multisubunit complex composed of four different subunits in the stoichiometry α2βγδ. In addition to the complex subunit composition, each subunit undergoes several posttranslational modifications and spans the membrane a multiple number of times. In order to reconstitute all of the functional properties of the AChR, the four subunits must be present and at least some of the posttranslational modifications must be executed correctly. Thus, whichever gene transfer technique one uses to express AChRs, the ability to introduce all four of the subunits into the same cell and to introduce them into a cell type that is capable of performing the necessary modifications, is of considerable importance. A mechanical technique such as microinjection would clearly be the easiest solution to the problem of multiple subunits.

Chapter 11 Establishing a Stable Expression System for Studies of Acetylcholine Receptors

Publisher Summary The chapter uses two methods for introducing the acetylcholine receptor (AChR) subunit cDNAs into cultured cells: DNA-mediated gene transfer using calcium phosphate precipitation and retroviral infection. These two methods were chosen because of the properties peculiar to, but not necessarily unique to, the AChR. One consideration was the need to stably introduce four different cDNAs into the same cell; another was the goal of introducing the cDNAs into muscle cell lines. Using DNA-mediated gene transfer, the chapter demonstrates that the cotransformation efficiency of introducing all four AChR cDNAs plus the selectable marker gene into the same Ltk - aprt - fibroblast cell was 80%. In addition, half of these cells took up approximately equal copy numbers of each cDNA. Thus, DNA-mediated gene transfer is a technique that will readily allow the introduction of multiple cDNAs into the genome of the same cell.

FORSKOLIN STABILIZES EPSILON SUBUNIT-CONTAINING ACETYLCHOLINE RECEPTORS

Fetal muscle-like acetylcholine receptors (AChRs) are composed of alpha, beta, gamma, delta subunits (gamma-AChRs) and have a rapid half life (t1/2), whereas adult muscle-like AChRs are composed of alpha, beta, epsilon, delta subunits (epsilon-AChRs) and have a slow t1/2. Two populations of AChRs, a slowly degrading population and a rapidly degrading population, have been shown to coexist in the postsynaptic membrane after denervation [In: Penn et al. (Eds.), Myasthenia Gravis and Related Disorders, Vol. 681, NY Acad. Sci., 1993, pp. 155-164]. Treatment of rat myotubes or mouse diaphragm muscle in organ culture with forskolin or cAMP analogues causes and increase in the t1/2 of the slowly degrading population of AChRs with no apparent effect on the rapidly degrading population of AChRs19. In this study, we have investigated the effect of forskolin on the cell surface half-lives of mouse gamma-AChRs, epsilon-AChRs and alpha beta delta complexes stably expressed in mouse fibroblasts. Forskolin had no significant effect on the t1/2 of gamma-AChRs or alpha beta delta complexes. The effect of forskolin on surface AChRs (alpha beta gamma delta) expressed in the C2 muscle cell line was similar to its effect on gamma-AChRs expressed in fibroblasts. In contrast, forskolin stabilized the epsilon-AChRs by approximately 2 fold. We show that the epsilon-subunit is phosphorylated in vivo, phosphorylation of epsilon increases with forskolin treatment, and the forskolin effect is reversible. Although the precise role of epsilon-subunit phosphorylation is yet to be determined, our results support the hypothesis that the slowly degrading population of AChRs consists of epsilon-AChRs and the rapidly degrading population of AChRs consists of gamma-AChRs.

Establishing a System for the Stable Expression of Torpedo Acetylcholine Receptors

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.

Acetylcholine Receptor-Expressing Fibroblasts

Two key and as yet unresolved questions concerning the autoimmune disease myasthenia gravis (MG) are: 1) what initiates the autoimmune response, and 2) what sustains it. It is known that autoantibodies to the acetylcholine receptor (AChR) can be detected in MG patient sera, that the disease symptoms can be passively transferred from humans to mice with the IgG fraction from MG patient sera (Drachman et al. 1976), that the AChR can induce experimental autoimmune myasthenia gravis (EAMG) in test animals (reviewed in Lindstrom 1985; Newsom-Davis 1986; Engel 1987), and that the AChR is the only protein at endplates which is capable of inducing EAMG (Claudio & Raftery 1980). The importance of the AChR in this disease cannot be disputed, but what role does it play in the initiation and/or maintenance of the disease. Are there specific epitopes on the AChR that are required for induction? It has been suggested that alterations of AChRs on myoid cells in the thymus might serve to break tolerance and initiate the autoimmune response directed against AChRs. Penicillamine has been shown to react with AChRs and to enhance EAMG (Bever et al. 1984). If alterations in the AChR can cause MG, do they occur by an exogenous or an endogenous mechanism? An endogenous mechanism acting directly on AChRs could have several sites of action. Various posttranslational modifications of the AChR are known to occur naturally, including disulfide-bond formation, glycosylation, phosphorylation, and fatty acylation.

Chapter 9 Biogenesis and Cell Surface Distribution of Acetylcholine Receptors Stably Expressed in Fibroblasts

Publisher Summary The ligand-gated family of receptor channels consists of the nicotinic acetylcholine receptors (AChRs). Several stable cell lines have been established that express functional cell surface Torpedo AChRs using two different gene transfer methods: calcium phosphate-DNA-mediated cotransfection and viral infection. Muscle and neuronal AChR subunit genes and complementary DNA (cDNAs) have been isolated from a number of species. The AChR isolated from Torpedo californica electric organ are chosen for work for several reasons: (1) it is the most extensively studied and best characterized AChR, (2) it is the one for which the most structural information is available, and (3) many tools are available for its analysis, including the libraries of monoclonal antibodies containing subunit- and conformation-specific antibodies. Using cotransfection, viral infection, or a combination of the two techniques, cell lines expressing one, two, three, or four different AChR subunits are established. Having cell lines expressing individual subunits has been of great use in analyzing biosynthesis, posttranslational processing, transport, and whether subunits form stable self-associations. In cell lines expressing all four subunits, fully functional, cell surface AChRs are expressed that can be easily analyzed using biochemical, pharmacological, immunological, or electrophysiological techniques.

Chapter 10 Expression of Acetylcholine Receptor Subunits in Saccharomyces cerevisiae (Yeast)

Publisher Summary The chapter presents yeast as an expression system for neuronal receptors: to use yeast to amplify the amounts of receptor proteins that normally exist in only small quantities in cells so that their structure and function can be studied. Because yeast can easily be grown in large quantities, it has obvious advantages over oocytes—an expression system in which each cell has to be injected to obtain relatively little protein. Therefore, with the development of the technology to produce large enough quantities of membrane proteins in yeast to investigate their structure and function, yeast cells can become a good source for the approximately 20 neuronal receptors that exist in only small quantities in cells and about which relatively little is known.