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

Alpha7 nicotinic acetylcholine receptors (alpha 7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [alpha BGT] 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 alpha 7nAChR expression are reported. Finally, antibodies against alpha 7nAChRs have shown various deficiencies. We find mouse macrophages bind alpha BGT but lack NACHO. We also report on a new alpha 7nAChR 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 alpha BGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of alpha BGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes alpha BGT binding to alpha 7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of alpha 7nAChRs.