Assembly of the Nicotinic Acetylcholine Receptor

Abstract

To investigate the mechanism of assembly of the mouse muscle acetylcholine receptor, we have expressed truncated N-terminal fragments of the ␣ and ␦ subunits in COS cells and have examined their ability to fold, to associate into heterodimers, and to form a ligand-binding site. Truncated fragments of the ␣ subunit that include all, part, or none of the first transmembrane domain (M1) folded to acquire ␣-bungarotoxin binding activity. Neither the full-length ␣ subunit nor any of the fragments were expressed on the cell surface, although the shortest folded fragment lacking a transmembrane domain was secreted into the medium. When coexpressed with the ␦ subunit, the ␣ subunit fragment possessing M1 formed a heterodimer containing a ligand-binding site, but shorter fragments, which lack transmembrane segments, did not associate with the ␦ subunit. N-terminal ␦ subunit fragments gave similar results. An N-terminal ␦ subunit fragment that contains M1 associated with the ␣ subunit to form a heterodimer, while a fragment lacking M1 did not. These results show that a complete M1 domain is necessary for association of truncated N-terminal ␣ and ␦ subunits into a heterodimer with high affinity ligand binding activity.The nicotinic acetylcholine receptor (AChR) 1 of mammalian muscle and the electrical organ of Torpedo is the best characterized member of a family of ligand-gated ion channels that mediate rapid synaptic transmission in the nervous system (1). All members of the family are thought to have a common pentameric structure in which highly homologous subunits surround a central aqueous pore whose opening and closing is regulated by binding of the neurotransmitter ligand (2-4). The muscle receptor consists of four different subunits in the ratio ␣ 2 :␤:␥:␦ (5, 6). Each of the subunits is separately synthesized and translocated into the endoplasmic reticulum, where AChR assembly occurs (7-9). Before assembly, the ␣ subunit undergoes a folding reaction whose product can be recognized by its ability to bind ␣-bungarotoxin (␣-BuTx) (10 -12). The other subunits also presumably undergo folding reactions before assembly, although the intermediates have not been characterized. Association of the folded subunits to form the assembled receptor occurs by a defined pathway in which the first step is the formation of the heterodimers ␣␦ and ␣␥ (13-16). The binding of the ␣ subunit to the ␦ and ␥ subunits results in the formation of a ligand-binding site within each heterodimer. Each site is characterized by distinctive properties that correspond to those in the intact AChR (6, 17). Several observations suggest that the binding site for competitive antagonists occurs at or near the interface between ␣ and ␦ subunits or ␣ and ␥ subunits, respectively (18 -20).In previous experiments, we have sought to define the domains of the AChR subunits that participate in the subunit interactions required for heterodimer formation. Chimeric subunits and dominant negative experiments were used to show that the N-terminal domains of the AChR me...

Subtype-specific Assembly of α-Amino-3-hydroxy-5-methyl-4-isoxazole Propionic Acid Receptor Subunits Is Mediated by Their N-terminal Domains

“…Additional chimeric proteins and fragments should in future experiments allow a more detailed analysis of this assembly domain. The key role of N-terminal domains for subunit assembly found here mirrors a similar importance of these regions for assembly of other ion channels, for example nicotinic acetylcholine, GABA A , and glycine receptors, as well as potassium channels (26,56,59,60,64,65). The advantages of such an arrangement are presently unclear.…”

“…They indicate that this half of the protein does not play an important role in subunit assembly. Deletion of the first transmembrane region strongly reduced association, as has been shown for the assembly of nicotinic acetylcholine receptors and potassium channels (26,59,60). The association of two fusion proteins in which the first transmembrane domain was replaced by the third showed that the sequence of this domain was not important for assembly.…”

“…A classical example for such a stereotypic receptor assembly pathway is the muscle nicotinic acetylcholine receptor. N-terminal domains of individual subunits of this neurotransmitter receptor initiate association of specific subunits in an ordered process (23)(24)(25)(26).…”

Extracellular Domain Nicotinic Acetylcholine Receptors Formed by α4 and β2 Subunits

“…M1, however, played an important role for expressing these extracellular domain nAChRs. These results, with the properties of extracellular domain ␣7 nAChRs (33,34) and evidence of oligomerization of extracellular domains from ␣1 and ␦ subunits (31,32), from extracellular domains of muscle-type subunits (35), from chimeric ␣7/AChBP subunits (26), from glycine receptor subunits (68,69), and from GABA A receptor subunits (70), suggest that producing extracellular domain receptors might be possible for many subunits throughout the nicotinoid family. Expression of extracellular domain nicotinoid receptors with high structural fidelity, however, might be more feasible with M1 included in the subunit design than without M1.…”

“…They have been expressed with several subunit combinations. Dimeric extracellular domain nAChRs were produced by extracellular domains of ␣1 and ␦ subunits that also contained M1 or another membrane tether, suggesting that membrane attachment of subunits might be important for forming these extracellular domain nAChRs (31,32). Extracellular domains of ␣7 subunits linked to a glycosylphosphatidylinositol moiety were transported to the surface of Xenopus oocytes and bound ␣-bungarotoxin (␣Bgt) and d-tubocurarine (33), implying the formation of multimers.…”