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 6

BARP promotes α6β4 function in vivo and mediates antiallodynia.

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BARP promotes α6β4 function in vivo and mediates antiallodynia. (A) Immu...
(A) Immunoblotting identified BARP protein in cerebral cortex and dorsal root ganglia (DRG) from WT but not BARP-KO mice. BARP-transfected HEK293T cells and β-actin served as controls. (B and C) Imaging (B) and quantification (C) of surface α6 in DRG neurons from WT and BARP-KO mice. Neurons were transduced with lentivirus expressing α6-V5 and β4 subunits and were stained with anti-V5 antibody. Surface staining for α6 is reduced in BARP-KO neurons, whereas permeabilized (Perm) neurons show similar total levels of receptor. Scale bar: 10 μm. n = 23 and 17 neurons for WT and BARP-KO, respectively. (D) FLIPR responses in transfected HEK293T cells stimulated with indicated compounds. Cells transfected with α6β4 were cotransfected with BARP, IRE1α, and SULT2B1 (for values see Supplemental Tables 1 and 2). Shown are concentration-response curves for α6β4 and α4β2 and a single high concentration for α3β4. (E) Quantification of maximal compound efficacy on α6β4 vs. α4β2 (left) or α3β4 (right). (F and G) Mechanical allodynia was assessed in WT and BARP-KO mice before (Pre) and following spared nerve injury (SNI) surgery. WT, but not BARP-KO mice exhibited significant (F) nicotine-mediated and (G) ABT-594–mediated antiallodynia. n = 10 and 12 for WT and BARP-KO, respectively in F. n = 11 for each group for G. *P < 0.05; ***P < 0.001 by Mann-Whitney test (C) or linear mixed-effects model for repeated measures comparing SNI-baseline to SNI-treatment time point (F and G). C: U = 57. F: P = 0.05 for WT, SNI vs. SNI + nicotine; P = 0.35 for BARP-KO. G: P < 0.001 for WT, SNI vs. SNI + nicotine; P = 0.4 for BARP-KO. Graphs are the mean ± SEM. Graphs in C–E are representative of 1 experiment and were replicated with similar results.