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Int J Mol Sci
2025 Sep 28;2619:. doi: 10.3390/ijms26199506.
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The Human Alpha3 Beta2 Neuronal Nicotinic Acetylcholine Receptor Can Form Two Distinguishable Subtypes.
Jackson DC
,
Hall MK
,
Sudweeks SN
.
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Diverse neuronal nicotinic acetylcholine receptor (nAChR) subtypes are expressed in hippocampal interneurons. Single-cell analysis of mRNA expression previously revealed prominent co-expression of the α3 and β2 subunits within rat interneurons in the CA1 region. Although the α3 subunit (traditionally expressed together with β4) is usually associated with the peripheral nervous system, its significant co-expression with the β2 subunit in hippocampal interneurons suggests a distinct, potentially novel central nervous system nAChR subtype. We demonstrate that the human α3 and β2 subunits injected into Xenopus laevis oocytes can assemble into at least two functionally distinct subtypes of nAChRs based on different subunit stoichiometries. These subtypes exhibit similar reversal potentials but differ significantly in their desensitization kinetics and acetylcholine (ACh) affinities. The response obtained from a 1:5 α3:β2 mRNA injection ratio shows a higher affinity for ACh and significantly greater desensitization during prolonged ACh application compared to the response obtained from a 5:1 α3:β2 mRNA injection ratio. The identification of distinct functional α3β2 subtypes, characterized by differential desensitization kinetics and ACh affinity, could represent novel targets for the potential development of highly selective cognitive therapeutics for conditions such as Alzheimer's disease, autism spectrum disorder, and attention deficit hyperactivity disorder, where hippocampal nAChRs are implicated.
Figure 1. Relative levels of α3 and β2 nicotinic acetylcholine receptor (nAChR) mRNA expression in rat hippocampal interneurons. The most commonly co-expressed α and β subunits from Jackson et al. 2024 [6] were the α3 and β2 subunits (n = 36 neurons). These were analyzed further to identify two populations, depending on which subunit was expressed higher. (a) A total of 56% of the α3β2 expressing neurons had more α3, with a 3.45-fold α3:β2 mRNA ratio (n = 20 for (a)). (b) A total of 44% of the α3β2 expressing neurons had more β2 with a 2.27-fold α3:β2 mRNA ratio (n = 16 for (b)).
Figure 2. Sample traces and likely stoichiometries. Injection of hα3 and hβ2 nicotinic acetylcholine receptor (nAChR) mRNA into Xenopus laevis oocytes at ratios of (a) 1:5 and (b) 5:1 formed functional and kinetically distinguishable receptors. The respective oocytes were perfused with 333 µM and 10 mM acetylcholine (ACh) (EC85) for up to 60 s to characterize desensitization. The percentage of the peak currents remaining after 30 s of sustained ACh application (30 s mark indicated by *) were compared and showed a significant difference between the two injected α3β2 ratios (see also Figure 1). Graphical representations of the likely potential stoichiometries are shown below each respective electrophysiological trace (i.e., either α3(2) + β2(3) or α3(3) + β2(2)), modeled on what is already published about stoichiometries for α4β2 and the α2β2 nAChRs [16,33,34,35].
Figure 3. IV plot. The IV plot obtained from the α3:β2 1:5 mRNA-injected oocytes fit a linear trendline of y = 0.0356x + 1.033 (R2 = 0.9892) (dashed line, n = 5). The IV plot obtained from the α3:β2 5:1 mRNA-injected oocytes fit a linear trendline of y = 0.0537x + 1.7478 (R2 = 0.9362) (solid line, n = 8) when fitting the points between −90 mV and −30 mV (the linear part of the rectifying plot). The reversal potential for both is approximately −30 mV, with a strong inward rectification. ANOVA testing showed no significant difference between these IV responses.
Figure 4. Acetylcholine (ACh) Dose–Response Curves: 1:5 (α3:β2) EC50 = 12.2 ± 1.7 μM, nH = 0.49 ± 0.13 (R2 = 0.74) (n = 14 oocytes, replicates of 4, 1 individual data point identified as an outlier and removed), 5:1 (α3:β2) EC50 = 263.8 ± 1.6 μM, nH = 0.55 ± 0.15 (R2 = 0.77) (n = 12 oocytes, replicates of 4, 2 individual data points outliers). Grubb’s outlier tests were used for outliers (alpha = 0.05). The 1:5 injected ratio has a minimum response at ~100 nM and an Emax at ~1000 µM ACh. The 5:1 injected ratio has a minimum response at ~10 µM and an Emax at ~33 mM ACh (ANOVA, F[15,258] = 54.644, *** p < 0.001).
Figure 5. Comparison of rise times and half-widths. Comparisons were made at the EC50 (10 µM, 333 µM acetylcholine) and the EC85 (333 µM, 10 mM) for the 1:5 and 5:1 α3:β2 mRNA-injected ratios, respectively, using t-tests for significance. (a) Comparison of 10% to 90% rise time: 1:5 10 µM (x¯ = mean ± SEM) x¯ = 1166 ± 237 ms (n = 6, replicates of 3), 333 µM x¯ = 169 ± 9 ms (n = 21, replicates of 4, 3 replicate outliers removed), 5:1 333 µM x¯ = 821 ± 123 ms (n = 13, replicates of 4), 10 mM x¯ = 1424 ± 171 ms (n = 25, replicates of 4, 3 replicate outliers removed). (b) Comparison of half-width: 1:5 10 µM x¯ = 3084 ± 130 ms (n = 5, replicates of 3, 3 replicate outliers removed), 333 µM x¯ = 3522 ± 654 ms (n = 11, replicates of 4), 5:1 333 µM x¯ = 3988 ± 163 ms (n = 13, replicates of 4, 2 replicate outliers removed), 10 mM x¯ = 3003 ± 297 ms (n = 21, replicates of 4) (** p < 0.01, *** p < 0.001). Grubb’s outlier tests were used to identify significant outliers (alpha = 0.05).
Figure 6. Desensitization. Comparisons were made after 30 s of continuous acetylcholine (ACh) application at the EC85 for each receptor subtype. The 1:5 α3:β2-injected oocytes (n = 7 oocytes, performed in replicates of 3 traces, 1 replicate outlier removed) were much more desensitized, withless current remaining compared to the original peak, while the 5:1 α3:β2-injected oocytes (n = 3 oocytes, performed in replicates of 3 traces, 1 replicate outlier removed) were only desensitized minimally with much of the current remaining at 30 s compared to the original peak. Outliers were identified using Grubb’s outlier test (alpha = 0.05). A t-test was used to test for the significance of the amount of peak current remaining after 30 s of ACh administration (*** p < 0.001).
