ABSTRACT
Nicotinic acetylcholine receptors (nAChRs) belong to the pentameric ligand-gated ion channel superfamily (pLGICs). Among them, the neuronal homomeric α7 nAChR is highly permeable to calcium and plays critical roles in synaptic transmission, cell signaling, and inflammation modulation. The biogenesis of α7 nAChRs is enhanced by the chaperone proteins RIC-3 and NACHO. Previously, we reported a motif in the 5-HT 3A receptor, another pLGIC, involved in RIC-3 modulation. Residues in this motif are conserved and also found within the L1-MX segment of the α7 nACh subunit. We therefore explored the regulatory roles of these conserved residues in the biogenesis of α7 nAChRs using multiple approaches, including heterologous expression in Xenopus laevis oocytes, mutagenesis, pull-down assays, cell-surface labeling, and two-electrode voltage-clamp (TEVC) recordings. We find that synthetic α7 L1-MX peptide interacts with both RIC-3 and NACHO. In particular, conserved residues W330, R332, and L336 in the L1-MX positively regulates the assembly of α7 oligomers and the biogenesis of α7nAChR. In presence of residues W330, R332, and L336, NACHO promotes an assembly of an α7 pentamer which is resistant to strong denaturing conditions. NACHO-promoted α7 pentamer is also resistant to Endo H enzyme. Sensitivity of the pentamer to moderate temperatures (37 °C, 45 °C, and 50 °C) suggests that NACHO stabilizes the pentamer via non-covalent interactions. In contrast, Ala replacements at these residues disrupt the biogenesis and abolish α7 current. NACHO and RIC-3 co-expression yields partial rescue of functional expression for some Ala replacement constructs.
SUMMARY
This work identifies regulatory roles of conserved residues W330, R332, and L336 in the biogenesis of α7 nAChR. This discovery positions MX subdomain as a promising target for future drug development that can minimize adverse effects.
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