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.

Keywords: Ion channels; Neuroscience; Pain.

Figure 1. Genomic screening identifies chaperones for functional α6β4 reconstitution.

(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 (GABA_BR) 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 GABA_BR 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: F_6,35 = 511.4. D: α7, F_2,15 = 18.08; α4β2, F_2,15 = 86.27; α3β2, F_2,15 = 1171; α6β2β3*, F_2,15 = 143.9; GluA1, F_2,15 = 21.46; GABA_BR, F_2,15 = 12.85. F: F_2,40 = 106.7. Graphs are the mean ± SEM and depict 1 experiment that was replicated with similar results.

Figure 2. BARP and IRE1α promote α6β4 function through distinct mechanisms.

(A) Confocal images of α6β4 and α3β4 surface staining in transfected HEK293T cells. The β4 subunit C-terminus contained an HA tag, which was visualized with an anti-HA antibody. (B) Quantification shows that BARP, but not IRE1α, enhances α6β4 surface staining. Neither has effects on α3β4. n = 10 for each group. Scale bar: 50 μm. (C) Quantification of [³H]epibatidine binding to HEK293T lysates transfected as indicated. n = 8 for each condition. (D) Imm unoprecipitation with anti-V5 of solubilized HEK293T cells transfected as indicated. Immunoblotting shows that V5-tagged α6 associates with β4 and BARP, but not with IRE1α. ***P < 0.001 by 1-way ANOVA with Dunnett’s post hoc correction for multiple comparisons to vector (B and C). B: F_3,36 = 515.9. C: F_3,28 = 106.7. Graphs are the mean ± SEM and depict 1 experiment that was replicated with similar results.

Figure 3. Transmembrane, but not N-terminal region, of α6 is critical for BARP effect.

(A) Schematics of α6/α4 chimeras. (B) FLIPR traces and quantification of nicotine-evoked (50 μM) Ca²⁺ responses. n = 6 for each group. BARP enhances FLIPR responses only in α6- and α4N/6-containing receptors cotransfected with β4. (C) Representative confocal images for β4^HA surface staining in cotransfected HEK293T cells with (bottom) and without (top) BARP. Scale bar: 50 μm. (D) Quantification of surface receptor intensity in C. n = 10 for each group. All chimeric α6/α4 nAChRs were cotransfected with β4 into HEK293T cells. ***P < 0.0001 by unpaired t test (B and D). B: α6β4, t = 29.95; α4N/6, t = 40.58. D: α6β4, t = 24.04; α4N/6, t = 18.19. Graphs are the mean ± SEM and depict 1 experiment that was replicated with similar results.

Figure 4. IRE1α RNase activity and XBP1 splicing mediate assembly of α6β4.

(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: F_3,20 = 702.6. D: F_2,28 = 92.92. E: F_3,20 = 136. F: F_3,28 = 111.7. G: t = 5.86. H: F_2,21 = 49.87. Graphs are the mean ± SEM and depict 1 experiment that was replicated with similar results.

Figure 5. Endogenous IRE1α enhances α6β4 assembly.

(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: F_4,25 = 24.92. D: F_3,28 = 22.29. E: F_3,27 = 8.115. K: F_2,21 = 41.93 for IRE1α-WT, F_2,21 = 6.197 for IRE1α-KO. Graphs are the mean ± SEM and depict 1 experiment that was replicated with similar results.

Figure 6. BARP promotes α6β4 function in vivo and mediates antiallodynia.

(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.