Ric-3 chaperone-mediated stable cell-surface expression of the neuronal α7 nicotinic acetylcholine receptor in mammalian cells

Abstract

Aim: Studies of the α7-type neuronal nicotinic acetylcholine receptor (AChR), one of the receptor forms involved in many physiologically relevant processes in the central nervous system, have been hampered by the inability of this homomeric protein to assemble in most heterologous expression systems. In a recent study, it was shown that the chaperone Ric-3 is necessary for the maturation and functional expression of α7-type AChRs [1] . The current work aims at obtaining and characterizing a cell line with high functional expression of the human α7 AChR. Methods: Ric-3 cDNA was incorporated into SHE-P1-hα7 cells expressing the α7-type AChR. Functional studies were undertaken using single-channel patch-clamp recordings. Equilibrium and kinetic [ 125 I]α-bungarotoxin binding assays, as well as fluorescence microscopy using fluorescent α-bungarotoxin, anti-α7 antibody, and GFP-α7 were performed on the new clone. Results: The human α7-type AChR was stably expressed in a new cell line, which we coined SHE-P1-hα7-Ric-3, by co-expression of the chaperone Ric-3. Cell-surface AChRs exhibited [ 125 I]αBTX saturable binding with an apparent K D of about 55 nmol/L. Fluorescence microscopy revealed dispersed and micro-clustered AChR aggregates at the surface of SHE-P1-hα7-Ric-3 cells. Larger micron-sized clusters were observed in the absence of receptor-clustering proteins or upon aggregation with anti-α7 antibodies. In contrast, chaperone-less SHE-P1-hα7 cells expressed only intracellular α7 AChRs and failed to produce detectable single-channel currents. Conclusion:The production of a stable and functional cell line of neuroepithelial lineage with robust cell-surface expression of neuronal α7-type AChR, as reported here, constitutes an important advance in the study of homomeric receptors in mammalian cells.

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

“…Human α7 AChRs have been expressed alone or with RIC-3 in several different cell lines [31], [32], [33], [34], [35]. These have provided varying degrees of success at detecting function.…”

Activation of α7 Nicotinic Receptors by Orthosteric and Allosteric Agonists: Influence on Single-Channel Kinetics and Conductance

“…Studies of native rat hippocampal ␣7 nAChRs reported a mean single-channel conductance of 91.5 pS, and although a single Gaussian function did not provide a good fit of the amplitude distribution, it was not possible to identify unambiguously multiple conductance levels (Mike et al, 2000). In addition, studies of human recombinant ␣7 nAChRs expressed in SHEP1 cells have identified "two to three different levels of channel amplitudes" (Vallés et al, 2009). Likewise, studies of native chick ␣7 nAChRs identified multiple conductance levels (Nai et al, 2003).…”

Functional Expression of Human α9* Nicotinic Acetylcholine Receptors in X. laevis Oocytes Is Dependent on the α9 Subunit 5′ UTR

“…Nicotine exposure causes an upregulation of nicotinic AChRs in brain as well as in vitro , and a possible explanation of this effect is through the chaperoning by nicotine [90], [91], [92]. The RIC-3 is a chaperone that upregulates the expression of α7 nAChRs in oocytes [93], [94], [95], [96]. Interestingly, RIC-3 has no effect on the expression of α9 receptors [40], [97].…”