Functional α6β4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties
Daniel Knowland, Shenyan Gu, William A. Eckert III, G. Brent Dawe, Jose A. Matta, James Limberis, Alan D. Wickenden, Anindya Bhattacharya, David S. Bredt
Daniel Knowland, Shenyan Gu, William A. Eckert III, G. Brent Dawe, Jose A. Matta, James Limberis, Alan D. Wickenden, Anindya Bhattacharya, David S. Bredt
View: Text | PDF
Research Article Neuroscience

Functional α6β4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties

Abstract

The α6β4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6β4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6β4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6β4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6β4 receptor assembly. Effects on α6β4 involve BARP’s N-terminal region and IRE1α’s splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6β4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6β4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6β4 receptor.

Authors

Daniel Knowland, Shenyan Gu, William A. Eckert III, G. Brent Dawe, Jose A. Matta, James Limberis, Alan D. Wickenden, Anindya Bhattacharya, David S. Bredt

×

Figure 1

Genomic screening identifies chaperones for functional α6β4 reconstitution.

Options: View larger image (or click on image) Download as PowerPoint
Genomic screening identifies chaperones for functional α6β4 reconstituti...
(A) Schematic of genomic screening. HEK293T cells were cotransfected with cDNAs encoding α6 and β4 and individual plasmids from a genome-wide expression library. Ca2+ signals were measured in a fluorescence imaging plate reader (FLIPR). (B) Exemplary FLIPR traces showing nicotine-induced Ca2+ responses for indicated transfections. (C) Quantification of FLIPR signals. Activity of α6β4 is enhanced by BARP, IRE1α, and SULT2B1. n = 6 for each group. B, BARP; I, IRE1α; S, SULT2B1. (D) Quantification of FLIPR Ca2+ response from other nAChRs and other ion channels (5-HT3A and GluA1), and G protein–coupled receptor (GABABR) cotransfected with either BARP or IRE1α. nAChRs were stimulated with 50 μM nicotine, 5-HT3A with 100 μM serotonin, GluA1 with 100 μM glutamate plus 100 μM cyclothiazide, and GABABR with 100 μM GABA. n = 6 each. Responses normalized to that of vector-transfected cells (100%). (E) Current traces from Xenopus oocytes injected with indicated cRNAs and stimulated with a 2-second pulse of 250 μM ACh. (F) Quantification of current amplitude (absolute value) responses in E: n = 14, 13, and 16 oocytes for α6β4, α6b4 plus IRE1α, and α6β4 plus BARP, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 by 1-way ANOVA with Dunnett’s post hoc correction for multiple comparisons to vector (C, D, and F). C: F6,35 = 511.4. D: α7, F2,15 = 18.08; α4β2, F2,15 = 86.27; α3β2, F2,15 = 1171; α6β2β3*, F2,15 = 143.9; GluA1, F2,15 = 21.46; GABABR, F2,15 = 12.85. F: F2,40 = 106.7. Graphs are the mean ± SEM and depict 1 experiment that was replicated with similar results.