Why does knocking out NACHO, but not RIC3, completely block expression of α7 nicotinic receptors in Mouse brain?

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

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Article number470
JournalBiomolecules
Volume10
Issue number3
DOIs
StatePublished - Mar 2020

Bibliographical note

Funding Information:
Funding: This research was funded by the Minnesota Medical Foundation and the University of Minnesota OVPR Grant-In-Aid to PVT.

Publisher Copyright:
© 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Keywords

  • Alternate splice variants
  • Antibody specificity
  • In vitro vs
  • In vivo effects
  • Multi-subunit membrane protein assembly
  • Protein folding
  • Receptor chaperone

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't

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