Mutations of cytosolic loop residues impair assembly and maturation of α7 nicotinic acetylcholine receptors

Abstract

Mechanisms that regulate early events in the biogenesis of the α7 nicotinic acetylcholine receptor (α7 AChR) are not well understood. Data presented here show that single amino acid mutations in the cytoplasmic loop of the α7 AChR, between position 335 and 343, abolish or attenuate expression of mature pentameric α7 AChRs in both human embryonic kidney tsA201 (HEK) and neuronal SH‐SY5Y cells. Although the number of mature α7 AChRs is increased significantly in the presence of the chaperone protein resistant to inhibitors of cholineesterase‐3 in HEK cells, sucrose gradient sedimentation reveals that the vast majority of α7 subunits are aggregated or improperly assembled. Transfection of α7 AChRs in SH‐SY5Y cells, which endogenously express the α7 AChR, results in a much larger fraction of subunits assembled into mature AChRs. Thus, efficient assembly of α7 AChRs is influenced by several regions of the large cytoplasmic domain, as well perhaps by other parts of its structure, and requires as yet unknown factors not required by other AChR subtypes.

The Duplicated α7 Subunits Assemble and Form Functional Nicotinic Receptors with the Full-length α7

“…Further downstream, the M3-M4 intracellular loop also affects nAChR assembly (49,50). Assembly of dup␣7 and dup⌬␣7 with full-length ␣7 suggests that other regions of sequence may mediate the interaction between subunits and thus compensate for the lack of the N terminus.…”

Chemical Chaperones Exceed the Chaperone Effects of RIC-3 in Promoting Assembly of Functional α7 AChRs

“…It was cloned in the pcDNA3.1 vector [21]. Human RIC-3 was subcloned in the p3xFLAG–CMV vector between EcoRI and XbaI sites [22]. cDNAs were amplified and purified using the Qiagen Maxi Prep Kit.…”

Mutagenic Analysis of the Intracellular Portals of the Human 5-HT3A Receptor

“…Notably, the large intracellular loop is virtually absent from prokaryotic pLGIC subunits (9), and almost complete deletion of this region, among others, was necessary to optimize crystallization of GLC-1, once more suggesting it to be largely unstructured (10). This region of Cys-loop receptors is an established target for phosphorylation (19, 20), and it exerts important influences on, for example, the assembly, maturation, targeting, and gating kinetics of the nACh receptor (18, 21–26). The M3-M4 loop has also been shown to be a critical determinant of single-channel conductance (γ) (27).…”