Role of the Endoplasmic Reticulum Chaperone Calnexin in Subunit Folding and Assembly of Nicotinic Acetylcholine Receptors

Abstract

The nicotinic acetylcholine receptor (AChR) is a pentameric complex assembled from four different gene products by mechanisms that are inadequately understood. In this study we investigated the role of the endoplasmic reticulum (ER)-resident molecular chaperone calnexin in AChR subunit folding and assembly. We have shown that calnexin interacts with nascent AChR alpha-subunits (AChR-alpha) in muscle cell cultures and in COS cells transfected with mouse AChR-alpha. In chick muscle cells maximal association of labeled alpha-subunits with calnexin was observed immediately after a 15-min pulse with [35S]methionine/cysteine and subsequently declined with a t1/2 of approximately 20 min. The decrease in association with calnexin was concomitant with the folding of the alpha-subunit to achieve conformational maturation shortly before assembly. Brefeldin A did not inhibit AChR subunit assembly or the dissociation of calnexin from the assembling subunits, confirming that the ER is the site of AChR assembly and that calnexin dissociation is not affected under conditions in which the exit of assembled AChR from the ER is blocked. These results indicate that calnexin participates directly in the molecular events that lead to AChR assembly.

Arrest of Subunit Folding and Assembly of Nicotinic Acetylcholine Receptors in Cultured Muscle Cells by Dithiothreitol

“…found that nascent AChR ␣-subunit binds to the molecular chaperone calnexin immediately after ␣-subunit synthesis (20). The time course of ␣-subunit-calnexin dissociation was observed to coincide with the conformational maturation of ␣-subunit, consistent with a role for calnexin in mediating ␣-subunit folding.…”

“…2 (left panel) shows the time course of AChR assembly under control conditions, as monitored by the accumulation of ␣-subunit immunoprecipitated with anti-␦-subunit antibody with increasing chase times. The ␦-subunit itself is not visible in [ 35 S]methionine-labeled preparations due to its diffuse migration as a heterogeneous band, high susceptibility to proteolysis, and nonspecific backgrounds in this region of the gel, as noted previously by ourselves (18,20) and others (43). However, the ␦-subunit is phosphorylated and is clearly visible in immunoprecipitates from cultures labeled with 32 P i (18).…”

“…In the experiment shown in The immunoprecipitated calnexin-substrate complexes were eluted from the pellet and reprecipitated with anti-␣-subunit antibody. The rate of ␣-subunit dissociation from calnexin in DTT-treated cells, with a half-time of 18 min, is indistinguishable from that observed in the absence of DTT (20). Thus, no difference is seen in the amounts of ␣ subunit bound to calnexin after 15 min of chase in DTT-treated and untreated cultures (Fig.…”

“…Clarification of the extracts was achieved by centrifugation for 15 min in the microcentrifuge at 4°C. The clarified supernatants were incubated at 4°C with the specified antiserum for 3 h. Then protein A-Sepharose beads were added, and incubation at 4°C was continued for a further 1 h. Where indicated, sequential immunoprecipitations were carried out essentially as described before (20,38). Briefly, cells were extracted in HBS buffer (50 mM HEPES, pH 7.5, 200 mM NaCl, 1 mM CaCl 2 , 10% glycerol, 1% aprotinin, 10 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride) supplemented with 2% sodium cholate.…”

A review of experimental techniques used for the heterologous expression of nicotinic acetylcholine receptors

“…Studies in transfected cells have helped in characterizing the interaction of nAChRs with chaperone proteins such as BiP [277,278] and calnexin [279][280][281]. Such studies have also helped to reveal the role of nAChR-interacting proteins such as 14-3-3 [282,283] and VILIP-1 [284] in regulating cell-surface expression of α4β2 nAChRs.…”