Immunolabeling demonstrates the interdependence of mouse brain α4 and β2 nicotinic acetylcholine receptor subunit expression

Abstract

Immunolabeling of beta2 and alpha4 subunits was quantitated in brain sections (14 mum) using [(125)I]mAb 270 and [(125)I]mAb 299, respectively. Specificity was demonstrated by signal loss in beta2(-/-) and alpha4(-/-) brain sections, respectively. Even mild paraformaldehyde fixation severely affected immunolabeling, so this study used unfixed sections. Immunolabeling autoradiography was used to map and quantitate the effects of beta2 and alpha4 subunit-null mutations on their putative partner subunits' protein expression. [(125)I]mAb 299 labeling was nearly eliminated in beta2(-/-) sections, although dorsal interpeduncular nucleus (IPN) retained a faint signal. Therefore, alpha4 subunit expression is almost universally beta2-dependent. In contrast, alpha4-null mutation effects on [(125)I]mAb 270 immunolabeling varied widely among brain regions. In corticothalamic regions, [(125)I]mAb 270 labeling was eliminated. However, in habenulopeduncular regions, alpha4 genotype had no effect. Other (predominantly dopaminergic and optic tract) nuclei also retained reduced [(125)I]mAb 270 labeling in alpha4(-/-) sections. Thus, although most beta2 subunit protein expression is alpha4-dependent, this dependence is not universal. Presumably, residual beta2 subunits are found in non-alpha4* subtypes. Together, these results show that immunolabeling is applicable to reliable, quantitative investigations of neuronal nAChRs, and that subunit-null mutants can be appropriate controls for such experiments. In situ mRNA hybridization was also performed to determine if altered mRNA transcription mediated the interdependence of alpha4 and beta2 subunit expression. alpha4-Null mutation did not affect beta2 mRNA expression, nor did beta2 genotype affect alpha4 mRNA expression. Consequently, it seems that the two subunits' effects on each other's expression are mediated at the protein, rather than gene expression level.

Chronic Nicotine Cell Specifically Upregulates Functional α4* Nicotinic Receptors: Basis for Both Tolerance in Midbrain and Enhanced Long-Term Potentiation in Perforant Path

“…Moderate levels of ␣4 expression were found in the superior colliculus, cerebral cortex, retrosplenial granular cortex, and subiculum. This pattern agrees well with previous data (Swanson et al, 1987;Marks et al, 1992;Whiteaker et al, 2006). When ␣4YFP fluorescence is totaled for these 16 brain regions, the Het mouse brain fluorescence is approximately one-half (45%) that of Hom, consistent with straightforward gene dosage.…”

“…2B), indicating that the expression of nicotinic binding sites was unaffected by YFP insertion. Consistent with these observations, binding of 125 I-mAb299, which specifically labels the ␣4 subunit (Whiteaker et al, 2006) was unaffected by YFP insertion (Fig. 2C).…”

“…Specific 125 I-mAb 299 labeling in each region was defined by subtracting labeling in the corresponding region of ␣4 KO brain sections. Studies on ␣4 knock-out mice with this procedure show the specificity of our method (Whiteaker et al, 2006).…”

“…125 I-mAb autoradiography was performed as described (Whiteaker et al, 2006). Sections were allowed to equilibrate to room temperature, and then rehydrated for 15 min in 100 mM NaCl, 10 mM sodium phosphate buffer, pH 7.5 (PBS).…”

Intracellular complexes of the β2 subunit of the nicotinic acetylcholine receptor in brain identified by proteomics

“…We compared the constituents of the immunoprecipitated ␤2 subunit complex between WT and ␤2 Ϫ/Ϫ mice to eliminate the nonspecific binding of mAb 270 in brain tissue (32). A representative Coomassie blue-stained gel shows the results of an immunoprecipitation experiment conducted in WT and ␤2 Ϫ/Ϫ mice (Fig.…”