Ric-3 Promotes Functional Expression of the Nicotinic Acetylcholine Receptor α7 Subunit in Mammalian Cells

Bill Burton,Anatoly Shcherbatko,Mark E. Williams,Jayashree Aiyar,Laura E. Chavez-Noriega,Charles J. Cohen,Arturo Urrutia

doi:10.1074/jbc.m410039200

Abstract

Expression of functional, recombinant alpha7 nicotinic acetylcholine receptors in several mammalian cell types, including HEK293 cells, has been problematic. We have isolated the recently described human ric-3 cDNA and co-expressed it in Xenopus oocytes and HEK293 cells with the human nicotinic acetylcholine receptor alpha7 subunit. In addition to confirming the previously reported effect on alpha7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional alpha7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface alpha-bungarotoxin binding. Upon application of 1 mm nicotine, currents were undetectable in HEK293 cells expressing only the alpha7 subunit. In contrast, co-expression of alpha7 and ric-3 cDNAs resulted in currents that averaged 42 pA/pF with kinetics similar to those observed in cells expressing endogenous alpha7 receptors. Immunoprecipitation studies demonstrate that alpha7 and ric-3 proteins co-associate. Additionally, cell surface labeling with biotin revealed the presence of alpha7 protein on the plasma membrane of cells lacking ric-3, but surface alpha-bungarotoxin staining was only observed in cells co-expressing ric-3. Thus, ric-3 appears to be necessary for proper folding and/or assembly of alpha7 receptors in HEK293 cells.

Highlights

Nicotinic acetylcholine receptors1 are members of the neurotransmitter-gated ion channel superfamily
In addition to confirming the previously reported effect on ␣7 receptor expression in Xenopus oocytes we demonstrate that ric-3 promotes the formation of functional ␣7 receptors in mammalian cells, as determined by whole cell patch clamp recording and surface ␣-bungarotoxin binding
Hric3 mRNA Is Present in Neuronal Cells and Absent in HEK293 Cells—We amplified and subcloned the hric3 coding sequence from human adult brain RNA using primers based on sequence in the GenBank data base

Summary

EXPERIMENTAL PROCEDURES

Isolation of Hric Coding Sequence—Sequences encoding the hric subunit were isolated by standard PCR techniques. For biochemical studies a hric cDNA construct containing the hemagglutinin (HA) tag sequence, YPYDVPDYAL, at its COOH terminus was generated by standard PCR techniques. Samples were washed 4 times with 1 ml of chilled PBS containing protease inhibitors, resuspended in SDS-PAGE sample buffer, and analyzed by Western blot as described above. Cell Surface Biotinylation—Cells on tissue culture dishes were washed 3 times with chilled PBS and incubated in the same buffer containing 0.5 mg/ml sulfo-NHS-LC-biotin (Pierce) for 20 min at 4 °C. Cells were washed with mammalian Ringer’s solution, incubated for ϳ10 min in a solution containing a 1/1000 dilution of M-450 CD4ϩ Dynabeads (Dynal Inc., Lake Success, NY) and rewashed with mammalian Ringer’s solution to remove excess beads. Oocytes were incubated at 16 –19 °C for 4 –7 days in Oocyte Ringer-3 medium containing 50% L-15 medium, 100 ␮g/ml gentamicin, 25 ␮g/ml tetracycline, 4 mM glutamine, and 30 mM Na-HEPES (all from Invitrogen), with pH ad-

Reverse primer

RESULTS

DISCUSSION