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.
Highlights
The muscle nicotinic acetylcholine receptor (AChR) is a ligandgated ion channel belonging to the cys-loop superfamily of receptors that includes the neuronal ACh, GABAA, glycine and 5HT3 receptors
AChR function at the neuromuscular junction (NMJ) depends on its appropriate assembly, processing and trafficking to the cell surface, with each step in receptor biogenesis being regulated by specific molecular determinants in the receptor subunits
We identify important regulatory signals localized in the MX-helix, which is a juxta-membrane α-helix present in the proximal, FIGURE 5 | congenital myasthenic syndrome (CMS)-linked mutations in the MX-helix impair AChR assembly and expression. (A) Sequence alignment showing the initial portion of the beta and epsilon cytoplasmic (TM3-TM4) loops, with the MX-helix shown by the gray box
Summary
The muscle nicotinic acetylcholine receptor (AChR) is a ligandgated ion channel belonging to the cys-loop superfamily of receptors that includes the neuronal ACh, GABAA, glycine and 5HT3 receptors. One region that is only partially resolved in these structures, is the subunit large cytoplasmic loop domain, which contains critical determinants for trafficking and targeting of the receptor (Millar and Harkness, 2008; Albuquerque et al, 2009). This region begins with the short MX helix located just after TM3 and ends with the long MA helix that is continuous with TM4, but no structure is available for the intervening sequence (Wu et al, 2015)
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