Functional α6β4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties
Daniel Knowland, … , Anindya Bhattacharya, David S. Bredt
Daniel Knowland, … , Anindya Bhattacharya, David S. Bredt
Published October 19, 2020
Citation Information: J Clin Invest. 2020;130(11):6158-6170. https://doi.org/10.1172/JCI140311.
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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

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Figure 4

IRE1α RNase activity and XBP1 splicing mediate assembly of α6β4.

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IRE1α RNase activity and XBP1 splicing mediate assembly of α6β4. (A) Sch...
(A) Schematics of IRE1α kinase domain (K599A, KINmut), RNase domain (K907A, RNAmut), and conditional (I642G, CONDmut) mutants that were transfected into HEK293T cells. (B) RT-PCR shows that IRE1α mutants decrease XBP1 splicing (lower band) as compared with WT IRE1α. 1NM-PP1 rescues XBP1s in I642G CONDmut. u = full-length unspliced XBP1, s = spliced XBP1 (XBP1s), * = hybrid amplicon. (C) FLIPR traces (left) and quantification (right) of HEK293T cells transfected with WT IRE1α, K599A mutant, or K907A mutant. IRE1α and mutants were cotransfected with BARP. n = 6 for each group. (D) IRE1α mutants reduce [3H]epibatidine binding in HEK293T cell lysates compared with WT IRE1α. n = 8 for each condition. (E) FLIPR traces (left) and quantification (right) of I642G CONDmut without and with 5 μM 1NM-PP1, which rescued the FLIPR response. (F) CONDmut reduces [3H]epibatidine binding. RNase activation of CONDmut with 5 μM 1NM-PP1 increases α6β4 assembly. n = 8 for each condition. (G and H) FLIPR traces (G, left), quantification (G, right), and [3H]epibatidine binding (H) from HEK293T cells cotransfected with α6β4 and XBP1s as indicated. n = 12 for each condition in G, n = 8 for each group in H. ***P < 0.001 by 1-way ANOVA with Dunnett’s post hoc test to correct for multiple comparisons to WT IRE1α (C and D), 1-way ANOVA with Tukey’s multiple-comparisons post hoc test (E and F), unpaired t test (G), or 1-way ANOVA with Dunnett’s post hoc test to correct for multiple comparisons to vector (H). C: F3,20 = 702.6. D: F2,28 = 92.92. E: F3,20 = 136. F: F3,28 = 111.7. G: t = 5.86. H: F2,21 = 49.87. Graphs are the mean ± SEM and depict 1 experiment that was replicated with similar results.