Journal Article

Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Loring, Ralph H., Rameshbabu, Akshitha, Tilak, Bhargav, Swaminathan, Sweta, Adhikari, Ketaki, Ramesh, Vijay, Vinayakamoorthy, Remitha M., Iyer, Swetha, Tran, Phu V., Thulasi Raman, Sarulatha, Deshpande, Anish, Oza, Gauri, Rezvaya, Alexandra, Garg, Brijesh K., Wang, Zhiyuan, Dalvi, Parnika, Sukumaran, Sneha, Thummapudi, Jaya Prakash, Agarwal, Aayush

2020

Overview

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

• Publisher: MDPI AG,MDPI
• Subject: Acetylcholine receptors (nicotinic) / alpha7 Nicotinic Acetylcholine Receptor - biosynthesis / alpha7 Nicotinic Acetylcholine Receptor - genetics / alternate splice variants / Alternative splicing / Amino acids / Animals / Antibodies / antibody specificity / Brain - metabolism / Brain - pathology / Bungarotoxin / Bungarotoxins - pharmacology / Chaperones / Cloning / Deoxyribonucleic acid / DNA / Gene Expression Regulation / Gene Knockout Techniques / Genetic testing / HEK293 Cells / Humans / Immunoglobulins / in vitro vs. in vivo effects / Macrophages / Membrane Proteins - genetics / Membrane Proteins - metabolism / Mice / Mice, Knockout / multi-subunit membrane protein assembly / Neuroblastoma / Plasmids / protein folding / Proteins / Reading / receptor chaperone / Single-nucleotide polymorphism / Species / Western blotting