Nicotinic Acetylcholine Receptor Β2 Subunits Research Articles

A potential cost of evolving epibatidine resistance in poison frogs

BackgroundSome dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor β2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4β2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration–response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the β2 S108C into the α4β2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs.ResultsThe introduction of S108C into the α4β2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4β2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity.ConclusionsThe most parsimonious explanation of our current and previous work is that the S108C substitution renders the β2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while β2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4β2 receptor function.

Intravitreal Interleukin-2 modifies retinal excitatory circuits and retinocollicular innervation

Interleukin-2 is a classical immune cytokine whose neural functions have received little attention. Its levels have been found to be increased in some neuropathologies, such as Alzheimer's disease, multiple sclerosis and uveitis. Mechanistically, it has been demonstrated the role of IL-2 in regulating glutamate and acetylcholine transmission, thus being relevant for CNS physiology. In fact, our previous work showed that an acute intravitreal IL-2 injection during retinotectal development promoted contralateral eye axonal plasticity in the superior colliculus, but the involved mechanisms were not explored. So, our present study aimed to investigate the effect of increased intravitreal IL-2 levels on the retinal glutamatergic and cholinergic signalling required for retinotectal normal development. We showed through HRP neuronal tracing that intravitreal IL-2 also induces ipsilateral eye axonal sprouting. Protein level and/or immunolocalization analysis in the retina confirmed IL-2 pathway activation by increased expression of phospho-STAT-3, coupled to transient (24h) reduced levels of Egr1, PSD-95 and nicotinic acetylcholine receptor β2 subunit, suggesting reduced neural activity and synaptic sites. Also, AChE activity and GluN2B and GluA2 contents were reduced within 96h after IL-2 treatment. Therefore, IL-2-induced retinotectal plasticity might be driven by changes in cholinergic and glutamatergic pathways of the retina.

Spontaneous epileptic seizures in transgenic rats harboring a human ADNFLE missense mutation in the β2-subunit of the nicotinic acetylcholine receptor

We generated a transgenic rat strain with a missense mutation in V286L (V286L-TG), in the gene encoding the neuronal nicotinic acetylcholine receptor β2 subunit (CHRNB2) found in patients with autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). To confirm that V286L-TG rats exhibit seizures similar to those observed in humans, gene expression patterns and behavioral phenotypes were analyzed. In situ hybridization using a V286L Chrnb2-selective probe indicated that the transgene was expressed at higher levels in the cortex, hippocampus, and cerebellum of V286L-TG than wild-type littermates (non-TG). Spontaneous epileptic seizures with ictal discharges in electroencephalograms were detected in 45% of V286L-TG rats and the frequency of seizures was 0.73 times a week. This seizure type is similar to “paroxysmal arousals” that are observed in human ADNFLE. V286L-TG rats displayed nicotine-induced abnormal motor activity including seizures in comparison to non-TGs. Response time following nicotine administration occurred faster in V286L-TG than in non-TG rats.V286L-TG rats demonstrated spontaneous epileptic seizures, which are similar to human ADNFLE, and also showed a higher sensitivity to nicotine administration. Thus, the V286L-TG rat model could be a valuable tool for developing novel mechanism-driven treatment strategies for epilepsy and provide a better understanding of ADNFLE.

Nicotinic Acetylcholine Receptor β2 Subunit (CHRNB2) Gene and Short-Term Ability to Quit Smoking in Response to Nicotine Patch

Nicotinic Acetylcholine Receptor β2 Subunit (CHRNB2) Gene and Short-Term Ability to Quit Smoking in Response to Nicotine Patch

Nicotinic Acetylcholine Receptor β2 Subunit (CHRNB2) Gene and Short-Term Ability to Quit Smoking in Response to Nicotine Patch

Genes coding for nicotinic acetylcholine receptors may influence response to nicotine replacement therapy for smoking cessation. We examined the association of a 3' untranslated region polymorphism (rs2072661) in the nicotinic acetylcholine receptor beta2 subunit (CHRNB2) gene with quitting success in response to nicotine versus placebo patch during a short-term test of patch effects. In a within-subjects cross-over design, smokers of European descent (n = 156) received 21 mg nicotine and placebo patch in counter-balanced order, during two separate 5-day simulated quit attempts, each preceded by a week of ad libitum smoking. Abstinence was assessed daily by CO < 5 ppm. Smokers with the CHRNB2 GG genotype had more days of abstinence during the nicotine versus placebo patch week compared with those with the AG or AA genotypes (P < 0.01). Moreover, nicotine patch increased the probability of quitting on the target quit day, quitting anytime during the patch week, and avoiding relapse among those with the GG genotype but not the AA/AG genotypes, although the nicotine x genotype interaction was significant only for quitting on the target quit day (P < 0.05). Regardless of patch condition, quitting on the target quit day was more likely in those with the GG genotype versus AA/AG genotypes (P < 0.05). Genetic associations were not observed for craving or withdrawal responses to nicotine versus placebo patch. These findings are consistent with previous evidence of association of this variant with smoking cessation and suggest that polymorphisms in the nicotinic acetylcholine receptor beta2 subunit gene may influence therapeutic responsiveness to cessation medications.

Hidden function of neuronal nicotinic acetylcholine receptor β2 subunits in ganglionic transmission: comparison to α5 and β4 subunits

Neuronal nicotinic acetylcholine receptors (nAChR), which modulate fast excitatory postsynaptic potentials (f-EPSP), are located on both pre- and postganglionic sites in the autonomic nervous system (ANS). The receptor subunits α3, α5, α7, β2 and β4 are present in autonomic ganglia in various combinations and modulate acetylcholine (ACh) transmission. In the present study, autonomic functions were systemically examined in mice lacking β2 subunits (β2−/−) to further understand the functional role of β2 subunits in modulating ganglionic transmission. The results show normal autonomic functions, both under physiological conditions and in perturbed conditions, on thermoregulation, pupillary size, heart rate responses and ileal contractile reactions. This suggests that the function of β2-containing receptors in ganglionic transmission is hidden by the predominant β4 containing receptors and confirms previous studies which suggest that α3α5β4 nAChRs are sufficient for autonomic transmission. On the other hand, β2-containing receptors have only a minor function on postsynaptic responses to ACh, but may modulate ACh release presynaptically, although there is no evidence for this.

Mutational Analysis of Nicotinic Acetylcholine Receptor β2 Subunit Gene (CHRNB2) in a Representative Cohort of Italian Probands Affected by Autosomal Dominant Nocturnal Frontal Lobe Epilepsy

Twenty-four autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) probands were analyzed for the presence of V287L and V287M mutations in the CHRNB2 gene, which have been recently associated with the disease. In all patients, the involvement of the two additional loci reported as being associated with ADNFLE (CHRNA4 gene and chromosome 15q24 region) had been previously excluded. Mutational screening was performed by sequencing a polymerase chain reaction-amplified CHRNB2 DNA fragment, spanning the whole exon 5, which contains the V287L and V287M mutations and codes for approximately 65% of the mature protein. In none of the patients were mutations in the analyzed region of CHRNB2 found. These data, obtained in the largest ADNFLE cohort so far analyzed, demonstrate the rarity of the identified CHRNB2 mutations in ADNFLE patients.

Haplotypes of four novel single nucleotide polymorphisms in the nicotinic acetylcholine receptor β2‐subunit (CHRNB2) gene show no association with smoking initiation or nicotine dependence

Several types of evidence, including experiments with mice that lack the nicotinic acetylcholine receptor β2-subunit gene (CHRNB2), have suggested that a β2-containing nicotinic receptor is necessary for at least some of the reinforcing properties of nicotine. However, sequence variations in CHRNB2 have not been reported, and its role in influencing human smoking behavior and nicotine dependence is not known. We screened most of the introns and exons and found five novel single nucleotide polymorphisms (SNPs). We tested four of these SNPs in three large, carefully selected samples: nonsmokers (n = 317) and regular smokers low levels of nicotine dependence (ND, n = 238), or smokers with high-ND (n = 317). None of the four polymorphisms we tested, nor their estimated haplotypes, were associated with smoking initiation or progression to nicotine dependence. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:646–653, 2000. © 2000 Wiley-Liss, Inc.

The neuron-restrictive silencer element: a dual enhancer/silencer crucial for patterned expression of a nicotinic receptor gene in the brain.

The neuron-restrictive silencer element (NRSE) has been identified in several neuronal genes and confers neuron specificity by silencing transcription in nonneuronal cells. NRSE is present in the promoter of the neuronal nicotinic acetylcholine receptor beta2-subunit gene that determines its neuron-specific expression in the nervous system. Using transgenic mice, we show that NRSE may either silence or enhance transcription depending on the cellular context within the nervous system. In vitro in neuronal cells, NRSE activates transcription of synthetic promoters when located downstream in the 5' untranslated region, or at less than 50 bp upstream from the TATA box, but switches to a silencer when located further upstream. In contrast, in nonneuronal cells NRSE always functions as a silencer. Antisense RNA inhibition shows that the NRSE-binding protein REST contributes to the activation of transcription in neuronal cells.