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
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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 5

Endogenous IRE1α enhances α6β4 assembly.

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Endogenous IRE1α enhances α6β4 assembly. (A) HEK293T cells were transfec...
(A) HEK293T cells were transfected with α6β4 and treated with tunicamycin (Tm, 100 ng/mL) as indicated. Tm induced splicing of XBP1 (XBP1s) at 4 hours. XBP1u, unspliced XBP1. (B and C) Tm treatment for 4, but not 12 or 24 hours enhanced nicotine-induced (7.5 μM) α6β4 FLIPR responses. n = 6 for each condition except n = 5 for BARP + IRE1α. (D) [3H]epibatidine binding to α6β4 in HEK293T cells with indicated treatments. n = 8 each. (E) [3H]epibatidine binding to cortical neuron lysates transduced with α6β4. IRE1α inhibitor STF-083010 (20 μM) applied 30 minutes before Tm (100 ng/mL). n = 8 each. (F) Nicotine-evoked (50 μM) Ca2+ responses in cortical neurons transduced with α6β4 lentiviral particles. n = 5 for each condition. (G) Endogenous α4β2-mediated Ca2+ responses in cortical neurons. n = 10 for each condition. (H) Top: CRISPR/Cas9-mediated strategy for stop codon (*) insertion in IRE1α. Middle: Immunoblotting confirmed IRE1α protein knockout. Bottom: IRE1α activity in IRE1α-heterozygous (IRE1α-HET) and IRE1α-knockout (IRE1α-KO) lines. RT-PCR shows that the KO line lacks XBP1 splicing activity. (I) Nicotine-evoked (100 μM) FLIPR traces (left) and quantification (right) from IRE1α-WT and IRE1α-KO HEK293T cell lines transfected with α6β4 and BARP. n = 6 for each condition. (J) [3H]epibatidine binding in IRE1α-WT and IRE1α-KO cells transfected with α6β4. n = 8 each. (K) [3H]epibatidine binding to cells transfected with α6β4. Tm treatment increased binding in WT, but not IRE1α-KO cells, and cotransfection with IRE1α increased binding in both lines. n = 8 each. *P < 0.05; **P < 0.01; ***P < 0.001 by 1-way ANOVA with Dunnett’s post hoc test compared with α6β4 alone (C and D), unpaired t test (F–J), or 1-way ANOVA with Tukey’s post hoc test (E and K). C: F4,25 = 24.92. D: F3,28 = 22.29. E: F3,27 = 8.115. K: F2,21 = 41.93 for IRE1α-WT, F2,21 = 6.197 for IRE1α-KO. Graphs are the mean ± SEM and depict 1 experiment that was replicated with similar results.