ELIC-α7 Nicotinic acetylcholine receptor (α7nAChR) chimeras reveal a prominent role of the extracellular-transmembrane domain interface in allosteric modulation

Abstract

The native α7 nicotinic acetylcholine receptor (α7nAChR) is a homopentameric ligand-gated ion channel mediating fast synaptic transmission and is of pharmaceutical interest for treatment of numerous disorders. The transmembrane domain (TMD) of α7nAChR has been identified as a target for positive allosteric modulators (PAMs), but it is unclear whether modulation occurs through changes entirely within the TMD or changes involving both the TMD and the extracellular domain (ECD)-TMD interface. In this study, we constructed multiple chimeras using the TMD of human α7nAChR and the ECD of a prokaryotic homolog, ELIC, which is not sensitive to these modulators, and for which a high resolution structure has been solved. Functional ELIC-α7nAChR (EA) chimeras were obtained when their ECD-TMD interfaces were modified to resemble either the ELIC interface (EAELIC) or α7nAChR interface (EAα7). Both EAα7 and EAELIC show similar activation response and desensitization characteristics, but only EAα7 retained the unique pharmacology of α7nAChR evoked by PAMs, including potentiation by ivermectin, PNU-120596, and TQS, as well as activation by 4BP-TQS. This study suggests that PAM modulation through the TMD has a more stringent requirement at the ECD-TMD interface than agonist activation.

Keywords: Allosteric Regulation; ELIC; Ion Channels; Neurotransmitter Receptors; Nicotinic Acetylcholine Receptors; Pentameric Ligand-gated Ion Channels; Protein Chimeras; pLGICs; α7nAChR.

FIGURE 1.

Homology model of ELIC-α7nAChR highlighting the interface between domains. The EA chimera was modeled to the ECD of ELIC (PDB ID code 3RQW) and the TMD of the α7nAChR (PDB ID code 2MAW). Colors depict the ELIC ECD (cyan) and the α7nAChR TMD (yellow). Residues predicted to comprise the ECD-TMD interface are colored purple in the ECD and green in the TMD. For clarity, the TMD helices of one subunit are labeled and depicted as cylinders.

FIGURE 2.

Activation of EA chimeras by cysteamine. a and b, representative current traces for EA_ELIC (a) and EA_α7 (b). Bars over the trace indicate the length of application and cysteamine concentrations. Horizontal and vertical scale bars indicate 1 min and 0.1 μA current, respectively. c, cysteamine response curves for the functional EA chimeras. Current is expressed as a fraction of maximal current, n ≥ 5 oocytes. Error bars indicate S.D. Data are fit to Hill equations with the following parameters: ■, EA_ELIC, EC₅₀ = 1.0 ± 0.02 mm; ●, EA_α7, EC₅₀ = 1.3 ± 0.1 mm; ▵, EA_ELIC⁵, EC₅₀ = 0.4 ± 0.02 mm; □, EA _α7¹, EC₅₀ = 2.2 ± 0.1 mm; ○, EA_α7², EC₅₀ = 2.6 ± 0.1 mm. The corresponding Hill coefficients are 1.7 ± 0.1, 1.3 ± 0.1, 1.5 ± 0.1, 1.9 ± 0.1, and 2.4 ± 0.2, respectively.

FIGURE 3.

EA chimeras desensitize much more slowly than α7nAChR. Representative current traces show a 1.5-min agonist application to Xenopus oocytes injected with the indicated constructs. Acetylcholine at 100 μm was applied to α7nAChR; 30 mm cysteamine was applied to ELIC and EA chimeras. Rate constants for the initial rate of desensitization were calculated (n = 3): α7nAChR = 0.18 ± 0.05; ELIC = 2.4 ± 0.2; EA_ELIC = 1.6 ± 0.2; and EA_α7 = 1.7 ± 0.2 min.

FIGURE 4.

EA_α7 resembles α7nAChR pharmacology. Representative traces of ELIC, α7nAChR, and EA_α7 show responses to the indicated allosteric modulators. Ivermectin (30 μm), TQS (100 μm), and propofol (100 μm) were applied with agonist cysteamine (ELIC, EA_α7) or acetylcholine (α7nAChR) at the EC₂₀ for each construct; PNU-120596 (30 μm) was applied with agonist at the EC₇₀ for each construct. Bars over the traces indicate the length of application. Horizontal and vertical scale bars indicate 1 min and 0.1 μA current, respectively.

FIGURE 5.

EA_α7 is activated and potentiated by 4BP-TQS. Representative traces of ELIC and EA_α7 show responses to cysteamine and 4BP-TQS. a, ELIC is insensitive to 4BP-TQS, but EA_α7 is activated (b) and potentiated (c) by 4BP-TQS (100 μm). Cysteamine concentrations are at the EC₂₀ for each construct. Horizontal and vertical scale bars indicate 1 min and 0.1 μA current, respectively.

FIGURE 6.

EA_ELIC is insensitive to allosteric modulators acting through the TMD. Representative traces of EA_ELIC (a) and EA_ELIC⁵ (b) show responses to the indicated allosteric modulators. Ivermectin (30 μm), TQS (100 μm), and propofol (100 μm) were applied with cysteamine at the EC₂₀; PNU-120596 (30 μm) was applied with cysteamine at the EC₇₀. Bars over the traces indicate length of application. Horizontal and vertical scale bars indicate 1 min and 0.1 μA current, respectively.