TY - JOUR
T1 - Why does knocking out NACHO, but not RIC3, completely block expression of α7 nicotinic receptors in Mouse brain?
AU - Deshpande, Anish
AU - Vinayakamoorthy, Remitha M.
AU - Garg, Brijesh K.
AU - Thummapudi, Jaya Prakash
AU - Oza, Gauri
AU - Adhikari, Ketaki
AU - Agarwal, Aayush
AU - Dalvi, Parnika
AU - Iyer, Swetha
AU - Raman, Sarulatha Thulasi
AU - Ramesh, Vijay
AU - Rameshbabu, Akshitha
AU - Rezvaya, Alexandra
AU - Sukumaran, Sneha
AU - Swaminathan, Sweta
AU - Tilak, Bhargav
AU - Wang, Zhiyuan
AU - Tran, Phu V.
AU - Loring, Ralph H.
N1 - 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.
PY - 2020/3
Y1 - 2020/3
N2 - 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.
AB - 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.
KW - Alternate splice variants
KW - Antibody specificity
KW - In vitro vs
KW - In vivo effects
KW - Multi-subunit membrane protein assembly
KW - Protein folding
KW - Receptor chaperone
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U2 - 10.3390/biom10030470
DO - 10.3390/biom10030470
M3 - Article
C2 - 32204458
AN - SCOPUS:85082285316
SN - 2218-273X
VL - 10
JO - Biomolecules
JF - Biomolecules
IS - 3
M1 - 470
ER -