Two residues determine nicotinic acetylcholine receptor requirement for RIC‐3

Abstract

AbstractNicotinic acetylcholine receptors (N‐AChRs) mediate fast synaptic signaling and are members of the pentameric ligand‐gated ion channel (pLGIC) family. They rely on a network of accessory proteins in vivo for correct formation and transport to the cell surface. Resistance to cholinesterase 3 (RIC‐3) is an endoplasmic reticulum protein that physically interacts with nascent pLGIC subunits and promotes their oligomerization. It is not known why some N‐AChRs require RIC‐3 in heterologous expression systems, whereas others do not. Previously we reported that the ACR‐16 N‐AChR from the parasitic nematode Dracunculus medinensis does not require RIC‐3 in Xenopus laevis oocytes. This is unusual because all other nematode ACR‐16, like the closely related Ascaris suum ACR‐16, require RIC‐3. Their high sequence similarity limits the number of amino acids that may be responsible, and the goal of this study was to identify them. A series of chimeras and point mutations between A. suum and D. medinensis ACR‐16, followed by functional characterization with electrophysiology, identified two residues that account for a majority of the receptor requirement for RIC‐3. ACR‐16 with R/K159 in the cys‐loop and I504 in the C‐terminal tail did not require RIC‐3 for functional expression. Mutating either of these to R/K159E or I504T, residues found in other nematode ACR‐16, conferred a RIC‐3 requirement. Our results agree with previous studies showing that these regions interact and are involved in receptor synthesis. Although it is currently unclear what precise mechanism they regulate, these residues may be critical during specific subunit folding and/or assembly cascades that RIC‐3 may promote.