Papke RL (2024), Functions and pharmacology of α2β2 nicotinic ac...

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Biochem Pharmacol 2024 Jul 10;225:116263. doi: 10.1016/j.bcp.2024.116263.

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Functions and pharmacology of α2β2 nicotinic acetylcholine receptors; in and out of the shadow of α4β2 nicotinic acetylcholine receptors.

Papke RL .

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Although α2 was the first neuronal nicotinic acetylcholine receptor (nAChR) receptor subunit to be cloned, due to its low level of expression in rodent brain, its study has largely been neglected. This study provides a comparison of the α2 and α4 structures and their functional similarities, especially in regard to the existence of low and high sensitivity forms based on subunit stoichiometry. We show that the pharmacological profiles of the low and high sensitivity forms of α2β2 and α4β2 receptors are very similar in their responses to nicotine, with high sensitivity receptors showing protracted responses. Sazetidine A, an agonist that is selective for the high sensitivity α4 receptors also selectively activates high sensitivity α2 receptors. Likewise, α2 receptors have similar responses as α4 receptors to the positive allosteric modulators (PAMs) desformylflustrabromine (dFBr) and NS9283. We show that the partial agonists for α4β2 receptors, cytisine and varenicline are also partial agonists for α2β2 receptors. Studies have shown that levels of α2 expression may be much higher in the brains of primates than those of rodents, suggesting a potential importance for human therapeutics. High-affinity nAChR have been studied in humans with PET ligands such as flubatine. We show that flubatine has similar activity with α2β2 and α4β2 receptors so that α2 receptors will also be detected in PET studies that have previously presumed to selectively detect α4β2 receptors. Therefore, α2 receptors need more consideration in the development of therapeutics to manage nicotine addiction and declining cholinergic function in age and disease.

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Species referenced: Xenopus laevis

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	Graphical abstract
	Fig. 1. Hydrophobicity profiles of α2, α4, and α3 nAChR subunits. Kyte-Doolittle plots were generated in DNA Strider 1.4f17 (CEA, France). The protein sequences are provided in Table 1 . The protein subdomains are as indicated for α2 (in black). There is a relatively hydrophilic extracellular domain, followed by three hydrophobic transmembrane (TM) domains, the intracellular domain, and a fourth TM domain. The question mark indicates the uniquely long unstructured amino terminal loop domain of α2. The hydrophobicity profile of α4 is shown in red. The two sequences are aligned below. For comparison, the hydrophobicity profile of α3 is shown in green. While it is similar to α2 in length, the fine structures of the profiles differ in several regions.
	Fig. 2. LS and HS α2β2 responses to nicotine compared to ACh control responses. A) The average normalized responses of cells expressing receptors with the configuration α2(2) β2(3) (n = 6) or α2(2) β2(3) (n = 7). See Table 2 for details. Peak-current responses were measured corresponding to the drug applications, as well as late currents, which were averaged over a 30 s interval, 150–180 s after the initial drug application, as indicated by the dotted box. Dot plots of single-cell responses are shown to the right. See Table 2 for statistical analyses. Averaged responses are indicated by lines. B) Hypothetical energy landscapes for the two types of α2β2 receptors to ACh and nicotine, illustrating a low barrier for the HS receptors to reopen repeatedly after desensitization by nicotine.
	Fig. 3. Sazetidine-A responses of LS and HS α2β2 nAChR. LS α2(3) β2(2) and HS α2(2) β2(3) forms of the receptor were generated by co-expressing the α2β2 concatamer with monomeric α2 or β2, respectively. Following two initial control applications of ACh (100 µM ACh for LS receptors and 10 µM ACh for HS receptors), 10 µM Sazetidine-A was applied and followed by a final control application of ACh. Data were normalized to the average of the two initial ACh control responses, and both peak currents (A) and net charge (B) responses were measured. See Table 3 statistical analyses.
	Fig. 4. Potentiation of LS and HS α2β2 responses by 10 µM dFBr compared to α4β2 receptors. A) LS α2(3) β2(2) and HS α2(2) β2(3) forms of the receptor were generated by co-expressing the α2- β2 concatamer with monomeric α2 or β2, respectively. Following two initial control applications of ACh (100 µM ACh for LS receptors and 10 µM ACh for HS receptors), 10 µM dFBr was co-applied with ACh, followed by a final control application of ACh. Peak-current data were normalized to the average of the two initial ACh control responses. B) LS α4(3) β2(2) and HS α4(2) β2(3) forms of the receptor were generated by co-expressing the α4β2 concatamer with monomeric α4 or β2, respectively. Following two initial control applications of ACh (100 µM ACh for LS receptors and 10 µM ACh for HS receptors), 10 µM dFBr was co-applied with ACh, followed by a final control application of ACh. Peak-current data were normalized to the average of the two initial ACh control responses. See Table 4 , Table 5 for statistical analyses.
	Fig. 5. Effects of NS9283 on responses of LS and HS α2β2. LS α2(3) β2(2) and HS α2(2) β2(3) forms of the receptor were generated by co-expressing the α2β2 concatamer with monomeric α2 or β2, respectively. Following two initial control applications of ACh (100 µM ACh for LS receptors and 10 µM ACh for HS receptors), 10 µM NS9283 was applied, followed by a final control application of ACh. Peak-current data were normalized to the average of the two initial ACh control responses. See Table 6 for statistical analyses.
	Fig. 6. Partial agonist responses of α4β2 (A) and α2β2 (B) nAChR. Shown are 30 µM ACh responses used for normalization and subsequent peak-current responses from the same cells to 30 µM cytisine, 30 µM varenicline, or 300 µM arecoline. Different sets of cells were used for each partial agonist.
	Fig. 7. Varenicline responses of α2β2 nAChR. A) Receptors were formed from the co-expression of subunit monomers and tested across a range of either ACh or varenicline concentrations. All data were normalized to 30 µM ACh control responses. The boxed-in area represents the 30 µM ACh and 30 µM varenicline responses, corresponding to the condition in Fig. 7 . B) LS α2(3) β2(2) and HS α2(2) β2(3) forms of the receptor were generated by co-expressing the α2β2 concatamer with monomeric α2 or β2, respectively and tested for their responses to varenicline across a range of concentrations. Peak-current data were initially normalized to the respective ACh controls and then adjusted to reflect the ratio between ACh control current and the ACh maximum responses as determined in separate experiments [22] .
	Fig. 8. Flubatine effects on HS receptors. A) Flubatine was applied to HS α4(2) β2(3) receptors with two different protocols. In the upper panel, following the two initial control applications of 10 µM ACh, 10 µM flubatine was applied using our normal 6 s drug application. Subsequently four additional ACh control applications were made. The data plotted are the peak current responses of seven cells. Data in the lower panel show the effects when 1 µM flubatine was applied to the bath and the cells were allowed to incubate in the flubatine solution for 15 min. B) The same experimental procedures described for panel A were applied to cells expressing HS α2(2) β2(3) receptors with essentially the same result (see Table 9 for statistical analyses).