Tekarli B et al. (2025), Human α10 nicotinic acetylcholine receptor subu...

XB-ART-61176

J Biol Chem 2025 Feb 10;3012:108182. doi: 10.1016/j.jbc.2025.108182.

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Human α10 nicotinic acetylcholine receptor subunits assemble to form functional receptors.

Tekarli B , Azam L , Hone AJ , McIntosh JM .

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Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels. In mammals, there are 16 individual nAChR subunits allowing for numerous possible heteromeric compositions. nAChRs assembled from α7 or α9 subunits will form homopentamers. In contrast, the structurally related α10 nAChR subunit has historically been thought to require α9 subunits for function. Recently, however, strychnine was shown to enable the expression of human α10 nAChRs in Xenopus laevis oocytes or mammalian cells, prompting a re-examination of whether the human α10 subunit can self-assemble in the absence of strychnine. In the present study, acetylcholine-evoked ionic currents were obtained by co-expression of human α10 nAChR subunits with the transmembrane protein resistance to inhibitors of cholinesterase-3 (RIC-3) in Xenopus oocytes. Furthermore, the creation of a gain-of-function reporter mutation, V13'T, in the second transmembrane domain demonstrated that α10 subunits can self-assemble in the presence or absence of RIC-3. The antagonist sensitivity of the homomeric α10 nAChR is distinct from that of the closely related α7 and α9α10 subtypes. α10 homomers were blocked by α-bungarotoxin but were insensitive to α-conotoxin [V11L;V16D]ArIB and RgIA-5474, which potently block α7 nAChRs and α9α10 nAChRs, respectively. These studies yield insight into the assembly of functional human α10 homomers and provide tools for the development of α10 -nAChR-selective ligands.

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Species referenced: Xenopus laevis
GO keywords: ion channel activity

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	Figure 1. RIC-3 enables α10 nAChR expression.Xenopus laevis oocytes were injected with cRNA encoding the α10 nAChR subunit then incubated at 16 °C for 3 days. Oocytes from both conditions were voltage-clamped at −70 mV and pulsed with ACh (1 mM) for 1 s. A, oocytes injected with both α10 nAChR and RIC-3 cRNA had significantly larger responses to ACh compared to oocytes injected with only α10 with mean responses of −17 ± 8 (n = 20) and −9 ± 12 (n = 14) respectively; ∗∗p < 0.01. Statistical significance was assessed by the Mann–Whitney test. B, the current trace of α10 and RIC-3 containing oocyte in response to 1 mM ACh. The error bars and “±” indicate SD. The horizontal bar indicates the application of a 1 s pulse of ACh into the oocyte chamber.
	Figure 2. α10 nAChRs are inhibited by α-bungarotoxin but not by RgIA-5474.Xenopus laevis oocytes were injected with cRNA encoding for α10 nAChRs and RIC-3, then incubated at 16 °C for 3 days. ACh-evoked current amplitudes were voltage-clamped at −70 mV and pulsed with ACh (1 mM) for 1 s. Currents were assessed in saline, and then compared to currents after exposure to α-bungarotoxin or RgIA-5474. A, Scatter plot of percent response to ACh after application of antagonist. B, ACh-evoked currents mediated by α10 nAChRs before and after a 5-min static bath of α-bungarotoxin (10 μM), representative of 5 trials. C, traces of α10 nAChRs before and after 5-min of acute perfusion of RgIA-5474 (100 nM), representative of 4 trials. The horizontal bar indicates the application of a 1 s pulse of ACh into the oocyte chamber.
	Figure 3. Human [V13′T]α10 nAChR subunits form functional receptors. Xenopus oocytes were injected in various combinations of cRNAs encoding for the RIC-3 chaperone, α10 nAChRs, or [V13′T]α10 nAChRs. As described in Experimental Procedures, responses to 1-s pulses of 100 μM ACh were assessed by voltage clamp electrophysiology at −70 mV. A, oocytes were injected with cRNA encoding for [V13′T]α10 nAChR subunits, RIC-3, or both. [V13′T]α10 nAChRs demonstrated pronounced currents with a mean response of −38 ± 20 nA. These nAChR current amplitudes were enhanced to −208 ± 227 nA when co-injected with RIC-3 cRNA. Uninjected oocytes (n = 10) and oocytes injected with RIC-3 alone (n = 20) did not produce currents. B, oocytes were injected with cRNA for either α10 or [V13′T]α10 subunits in combination with RIC-3. Oocytes injected with α10 nAChR subunits had a mean ACh-induced response of −7 ± 6 nA and a range of −2 to −24 nA (n = 12); oocytes injected with human [V13′T]α10 cRNA had a mean response of −456 ± 294 nA with a range of −117 to −1120 nA (n = 12); ∗∗∗∗p < 0.0001. Statistically significant measurements were assessed using the Mann–Whitney test. The error bars and “±” indicate SD. C, the current trace of uninjected oocytes. D–F, current traces of oocytes containing [V13′T]α10 nAChRs, RIC-3 chaperones, or both. The horizontal bar indicates the application of a 1 s pulse of ACh into the oocyte chamber.
	Figure 4. ACh evoked currents in [V13′T]α10 nAChR expressing oocytes persist in the absence of Ca2+and when oocytes are incubated in actinomycin D. cRNA encoding for [V13′T]α10 nAChRs was injected into Xenopus laevis oocytes. Oocytes were voltage-clamped at −70 mV and pulsed with ACh (100 μM) for 1 s. A, oocytes were perfused with saline containing either Ca2+ or Ba2+. [V13′T]α10-expressing oocytes had a mean ACh-induced response of −437 ± 508 nA in the presence of calcium and −587 ± 539 nA in the presence of barium. (n = 12), p > 0.05. B, immediately after injection of [V13′T]α10 nAChR cRNA, oocytes were incubated in an actinomycin D solution (1 mM) or saline for 2 days. The mean response was not significantly different between oocytes incubated with the presence of actinomycin D (−233 ± 219 nA) and without actinomycin D (−208 ± 227 nA); n = 20, p > 0.05. Values shown are means ± SD. Statistical significance was assessed by the Mann–Whitney test. The error bars and “±” indicate SD.
	Figure 5. Acetylcholine (ACh), choline, and nicotine induce currents in [V13′T]α10 nAChR expressing oocytes. Oocytes were assessed by voltage-clamp electrophysiology for agonist-induced responses A, concentration-response curves of [V13′T]α10 nAChRs. ACh had an EC50 of 0.27 mM (0.21–0.33) with a Hill slope of 0.82 (0.67–1.01) (n = 7). Choline and nicotine were partial agonists with EC50s of 0.20 mM (0.15–0.28) and 0.35 mM (0.28–0.46), with Hill slopes of 0.49 (0.41–0.60), 0.59 (0.49–0.69) respectively (n = 6). B–D, current traces of [V13′T]α10 nAChRs activated by 1 mM ACh, choline, and nicotine. The error bars in (A) indicate SD and the values in parenthesis indicate 95% CI. Acetylcholine-induced currents were significantly larger than those induced by either choline or nicotine at all concentrations tested (see Table 1). The horizontal bar indicates the application of a 1 s pulse of ACh into the oocyte chamber.
	Figure 6. [V13′T]α10 nAChRs are resistant to block by RgIA-5474.Xenopus laevis oocytes were injected with cRNA encoding nAChR subunits α9, α9, and α10, or [V13′T]α10. Oocytes were voltage-clamped at −70 mV and pulsed with 1-s of 100 μM ACh in the absence of and then in the presence of RgIA-5474. Currents evoked by ACh on α9 (A) and α9α10 (B) nAChRs were potently blocked by RgIA-5474, whereas RgIA-5474 failed to block [V13′T]α10 (C). Note that block by RgIA-5474 of α9 and α9α10 nAChRs is only slowly reversed upon washout of RgIA-5474 (n = 5). The horizontal black bar represents the period of RgIA-5474 perfusion and arrows indicate ACh pulses.
	Figure 7. α-Bungarotoxin potently inhibits [V13′T]α10 nAChRs.Xenopus laevis oocytes were injected with cRNA for [V13′T]α10 and voltage clamped at −70 mV; oocytes were pulsed with 1 mM ACh for 1 s. A, concentration-response curve for α-bungarotoxin induced block of ACh-evoked currents. The IC50 was 331 (273–400) nM with a Hill slope of −1.17 (−1.36 to −1.00) (n = 4). The error bars indicate SD and the values in parenthesis indicate 95% CI. B, superimposed ACh-induced currents when exposed to the indicated concentrations of α-bungarotoxin. The red trace indicates the response measured before α-bungarotoxin exposure.
	Figure 8. ACh-induced currents are potentiated by atropine and strychnine. [V13′T]α10 expressing oocytes were exposed to ACh in the presence and absence of strychnine (STR) (1 μM) or other indicated ligands (3 μM) for 10 min. A, oocytes were exposed to 1-s pulses of ACh (100 μM) at 90-s intervals. Responses to the indicated ligands are indicated as a percent of the ACh response obtained at baseline. ACh-evoked responses were potentiated to 230 ± 44% (n = 6) when exposed to atropine and 572 ± 131% (n = 7) when exposed to strychnine. The other compounds listed showed no significant activity. The concentration of RgIA-4, RgIA-5474, and dihydro-β-erythroidine (DHβE) was 3 μM and [V11L,V16D]ArIB was 1 μM (n = 5). Statistical significance was assessed by the Students t-test as described in the Experimental Procedures. B, atropine effects have a rapid onset and reversal. A baseline response to ACh pulses was established, followed by a 10-min perfusion of atropine (3 μM). Atropine was then washed out.
	Figure 9. Concentration-dependent effects of strychnine.Xenopus laevis expressing [V13′T]α10 nAChRs were voltage clamped at −70 mV, and the responses to ACh in the presence and absence of strychnine were measured. Oocytes were exposed to 1-s pulses of ACh (100 μM) for ∼ 20 min until a stable baseline response was achieved. Strychnine was then perfused for 10 min at the indicated concentrations. A, The responses to 10 nM to 100 μM strychnine were 98.8 ± 9.6% (10 nM), 210 ± 23.2% (100 nM), 572 ± 131% (1 μM), 273 ± 146% (10 μM), and 4.2 ± 1.8% (n = 5–7). The error bars and “±” indicate SD. B, representative current traces of [V13′T]α10 nAChRs during acute perfusion of increasing concentrations of strychnine.