Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)₂α5 AChR function - PubMed (original) (raw)

Comparative Study

Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)₂α5 AChR function

Alexander Kuryatov et al. Mol Pharmacol. 2011 Jan.

Abstract

Genomic studies have identified a D398N variation in the α5 subunit of nicotinic acetylcholine receptors (AChRs) that increases risk of nicotine dependence and lung cancer. (α4β2)₂α5 AChRs are a significant brain presynaptic subtype in brain. Their high sensitivity to activation by nicotine and high Ca²+ permeability give them substantial functional impact. α3β4* and α3β2* AChRs are predominant postsynaptic AChRs in the autonomic nervous system, but rare in brain. The amino acid 398 of α5 is located in the large cytoplasmic domain near the amphipathic α helix preceding the M4 transmembrane domain. These helices have been shown to influence AChR conductance by forming portals to the central channel. We report that α5 Asn 398 lowers Ca²+ permeability and increases short-term desensitization in (α4β2)₂α5 but not in (α3β4)₂α5 or (α3β2)₂α5 AChRs. This suggests that a positive allosteric modulator would augment nicotine replacement therapy for those with this risk variant. α5 D398N variation does not alter sensitivity to activation. The high sensitivity to activation and desensitization of (α4β2)₂α5 AChRs by nicotine results in a narrow concentration range in which activation and desensitization curves overlap. This region centers on 0.2 μM nicotine, a concentration typically sustained in smokers. This concentration would desensitize 60% of these AChRs and permit smoldering activation of the remainder. The low sensitivity to activation and desensitization of (α3β4)₂α5 AChRs by nicotine results in a broad region of overlap centered near 10 μM. Thus, at the nicotine concentrations in smokers, negligible activation or desensitization of this subtype would occur.

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Figures

Fig. 1.

α5 Asn 398 exhibited lower Ca2+ permeability than α5 Asp 398 in (α4β2)2α5 AChRs, but this variation had no effect on Ca2+ permeabilities of α3* AChRs. Human AChRs were expressed in X. laevis oocytes. (α4β2)2α5 AChRs were expressed using a tetrameric concatamer β2-α4-β2-α4 plus free α5 subunits. α3* AChR subtypes used all free subunits. The relative response is the current obtained using 1.8 mM Ca2+ as the sole cation compared with the current obtained using a buffer containing 90 mM Na+, 2.5 mM K+, and 1.8 mM Ca2+. Thus, currents reflect only Ca2+ flow through AChRs. Maximum responses were obtained using 10 μM ACh for the sensitive (α3β2)2α5 and (α4β2)2α5 subtypes and 1000 μM ACh for the less sensitive (α3β4)2α5 subtype.

Fig. 2.

Assay of effects of the α5 D398N variation on sensitivity to activation by ACh and short-term desensitization of the (α4β2)2α5, (α3β2)2α5, and (α3β4)2α5 AChR subtypes. Human AChRs were expressed in X. laevis oocytes as in Fig. 1. The (α4β2)2α5 subtype is shown in A to C. A, there is no significant difference in sensitivity to activation by ACh in the two α5 variants. Each response is the average of four to five oocytes. B and C, kinetics of short-term desensitization in a single oocyte expressing each variant exposed to some of the ACh concentrations used in the dose/response curve. The averaged desensitization of four to five oocytes at 3 s after application of ACh are shown in Table 1, revealing that the N variant desensitizes significantly more. The responses in the dose-response curve were the peak responses. The (α3β2)2α5 subtype is shown in D to F. D, there is no significant difference in sensitivity to activation in the two α5 variants. E and F, kinetics of desensitization. It is much faster and more extensive than in (α4β2)2α5 AChRs. The extent of desensitization by the two α5 variants in the (α3β2)2α5 subtype is not significant, as shown in Table 1. The (α3β4)2α5 subtype is shown in G to I. G, there is no significant difference in sensitivity to activation in the two α5 variants. H and I, kinetics of desensitization. It is slower and less extensive than in (α3β2)2α5 AChRs. The extent of desensitization by the two α5 variants is not significant, as shown in Table 1.

Fig. 3.

Nicotine sensitivities differ dramatically among the (α4β2)2α5, (α3β2)2α5, and (α3β4)2α5 subtypes. Human AChRs were expressed in permanently transfected human embryonic kidney cell lines (Wang et al., 1998; Kuryatov et al., 2008). Responses were assayed in microwell cultures using a Flexstation microplate fluorometer and a fluorescent probe sensitive to membrane potential. Desensitization was measured after 1.5 h. This allows enough time for equilibrium desensitization but not enough time for up-regulated AChRs to be expressed on the cell surface. The fraction of AChRs remaining activatable after desensitization was assayed using 1 mM ACh. The overlapping region between concentration/response curves for desensitization and activation is highlighted in gray. AChRs remaining undesensitized could undergo smoldering activation in this concentration range. The potential smoldering response at each concentration of nicotine was calculated by multiplying the short-term response to nicotine at that concentration by the fractional response remaining after desensitization. The area under the smoldering response curve below 0.2 μM was blackened to indicate the range of responses that might be sustained in smokers.

References

1. Amos CI, Wu X, Broderick P, Gorlov IP, Gu J, Eisen T, Dong Q, Zhang Q, Gu X, Vijayakrishnan J, et al. (2008) Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1. Nat Genet 40:616–622 - PMC - PubMed
1. Bailey CD, De Biasi M, Fletcher PJ, Lambe EK. (2010) The nicotinic acetylcholine receptor α5 subunit plays a key role in attention circuitry and accuracy. J Neurosci 30:9241–9252 - PMC - PubMed
1. Benowitz NL. (1996) Pharmacology of nicotine: addiction and therapeutics. Annu Rev Pharmacol Toxicol 36:597–613 - PubMed
1. Berrettini W, Yuan X, Tozzi F, Song K, Francks C, Chilcoat H, Waterworth D, Muglia P, Mooser V. (2008) α5/α3 nicotinic receptor subunit alleles increase risk for heavy smoking. Mol Psychiatry 13:368–373 - PMC - PubMed
1. Bertrand D, Gopalakrishnan M. (2007) Allosteric modulation of nicotinic acetylcholine receptors. Biochem Pharmacol 74:1155–1163 - PubMed

Acetylcholine receptor (AChR) α5 subunit variant associated with risk for nicotine dependence and lung cancer reduces (α4β2)₂α5 AChR function - PubMed (original) (raw)