The MX-Helix of Muscle nAChR Subunits Regulates Receptor Assembly and Surface Trafficking

Abstract

Nicotinic acetylcholine receptors (AChRs) are pentameric channels that mediate fast transmission at the neuromuscular junction (NMJ) and defects in receptor expression underlie neuromuscular disorders such as myasthenia gravis and congenital myasthenic syndrome (CMS). Nicotinic receptor expression at the NMJ is tightly regulated and we previously identified novel Golgi-retention signals in the β and δ subunit cytoplasmic loops that regulate trafficking of the receptor to the cell surface. Here, we show that the Golgi retention motifs are localized in the MX-helix, a juxta-membrane alpha-helix present in the proximal cytoplasmic loop of receptor subunits, which was defined in recent crystal structures of cys-loop receptor family members. First, mutational analysis of CD4-MX-helix chimeric proteins showed that the Golgi retention signal was dependent on both the amphipathic nature of the MX-helix and on specific lysine residues (βK353 and δK351). Moreover, retention was associated with ubiquitination of the lysines, and βK353R and δK351R mutations reduced ubiquitination and increased surface expression of CD4-β and δ MX-helix chimeric proteins. Second, mutation of these lysines in intact β and δ subunits perturbed Golgi-based glycosylation and surface trafficking of the AChR. Notably, combined βK353R and δK351R mutations increased the amount of surface AChR with immature forms of glycosylation, consistent with decreased Golgi retention and processing. Third, we found that previously identified CMS mutations in the ε subunit MX-helix decreased receptor assembly and surface levels, as did an analogous mutation introduced into the β subunit MX-helix. Together, these findings indicate that the subunit MX-helix contributes to receptor assembly and is required for normal expression of the AChR and function of the NMJ. In addition, specific determinants in the β and δ subunit MX-helix contribute to quality control of AChR expression by intracellular retention and ubiquitination of unassembled subunits, and by facilitating the appropriate glycosylation of assembled surface AChR.

Speculation on How RIC-3 and Other Chaperones Facilitate α7 Nicotinic Receptor Folding and Assembly

“…If these two domains interact, it would be better if their interaction is before the MX helix is put in place ( Figure 7 C). The MX domains of muscle AChR (α1β1γδ) β and δ subunits are recently proposed to play a role in Golgi retention, ubiquitination and to act as a quality control site in muscle receptor folding [ 160 ]. No such role has yet been proposed for the α7 MX domain, but this possibility bears investigation, and as said above, the exact order of structural folding in α7-nAChR is not known.…”

Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR

“…εMet430 extends toward εMX into a hydrophobic pocket formed by residues on εM3, εM4, and εMX. εMX is implicated in the assembly/cell surface trafficking of the muscle nAChR, with mutations in εMX reducing cell surface expression leading to CMS ( 28 ). Residues in M4 that project toward MX may play a particularly important role in nAChR expression.…”

“…To identify interactions that are essential to channel function, we generated alanine mutations of every M4 residue in each subunit. Surprisingly, all the generated mutants expressed robustly in frog oocytes except for one, εM430A, which extends toward a structure, εMX, that has been implicated in assembly/cell surface trafficking ( 28 ). Of those that expressed, 54 of 155 mutations led to statistically significant changes in the measured EC 50 values and thus in channel function.…”

Pursuing High-Resolution Structures of Nicotinic Acetylcholine Receptors: Lessons Learned from Five Decades

“…The amino acid residues at the MX-helix of nAChR subunits have been implicated in molecular signals that regulate the assembly, trafficking, and expression of muscle nAChR [ 172 , 173 ]. Furthermore, the crystal structures of the human neuronal α4β2 and the cryo-map densities of α7 nAChR, Tc, and Tm have shown the presence of this secondary structure.…”