Department of Pharmaceutical Sciences, Northeastern University, Boston, MA 02115, USA.
Center for Neurobehavioral Development, Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA.
Biomolecules. 2020 Mar 19;10(3):470. doi: 10.3390/biom10030470.
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 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 ( 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 KOs show subtle αBGT binding changes across different mouse brain regions, while 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.
α7 烟碱型乙酰胆碱受体 (α7nAChRs) 不仅因其生理作用而引人注目，还因为这种受体需要伴侣蛋白来运输到细胞膜表面（通过 α-银环蛇毒素 [αBGT] 结合来衡量）。虽然针对编码蛋白伴侣 NACHO 的基因敲除 (KO) 动物和跨物种反应的抗体存在，但跨物种进行 Western blot 分析的商业可得的 RIC3 伴侣蛋白抗体尚未广泛应用。此外，尚未报道 RIC3 功能缺失 (KO) 对 α7nAChR 表达的影响。最后，针对 α7nAChRs 的抗体存在各种缺陷。我们发现小鼠巨噬细胞结合 αBGT，但缺乏 NACHO。我们还报告了一种新的 α7nAChR 抗体，并测试了跨物种反应的商业可得的抗 RIC3 抗体，这些抗体允许对体外培养物进行 Western blot 分析。这些抗体还与特定的 RIC3 剪接变体和单核苷酸多态性反应。初步放射自显影分析显示，KO 在不同的小鼠脑区显示出微妙的 αBGT 结合变化，而 KO 则完全丧失了 αBGT 结合。这些发现与体外观察到的效果不一致，因为 RIC3 促进了在 HEK 细胞中表达的 αBGT 与 α7nAChRs 的结合，即使在没有 NACHO 的情况下也是如此。综上所述，可能有其他调节因子参与了 α7nAChRs 的体内表达。
