Members Of The SLC Family Regulated By AC3 And Their Roles In Mouse MOE

The main olfactory epithelium?MOE?is the main olfactory organ in mammals.Ade-nylyl cyclase 3?AC3?is one of the important members of cyclic adenosine monophosphate?cAMP?olfactory signaling pathway.AC3 deficiency affects the expression of olfactory sig-naling pathway.MOE is susceptible to pathogen infection when exposed to the external en-vironment;the function of MOE is also affected by solute changes in vivo.Most solutes in vivo are transported by transporters,the solute carriers?SLC?superfamily are membrane proteins,play an important role in solute transport in MOE.Whether AC3 deficiency affects the expression of SLC family members in MOE?And whether the changes on the expression of the SLC family members affect the function of MOE?These questions are yet to be re-solved.Here,qRT-PCR,Western blot,IF,Co-IP,LC-MS and other experimental methods were performed in wild-type controls(AC3^+/+),AC3 knock-out(AC3^-/-)and AC3 knock-in?hAC3?three genotypes mice MOE tissues and NIH3T3 cells to obtain the following results:1.qRT-PCR was performed for quantitative analysis of the expression of some SLC family members at the mRNA level,we observed that:?1?The expression of Slc8a1 and Slc6a3 were lower in AC3^-/-MOE than in AC3^+/+MOE,and higher in hAC3 MOE than in AC3^+/+MOE.?2?The expression of Slc9a2,Slc15a3 and Slc22a20 were higher in AC3^-/-MOE than in AC3^+/+MOE,and lower in hAC3 MOE than in AC3^+/+MOE.These demonstrated that the expression of some SLC family members were affected by AC3 at the mRNA level.2.Western blot and IF were performed for protein quantification and localization analy-sis of SLC8A1 and SLC6A3,we observed that:?1?The results at the protein level were consistent with the results at the tissue level.The results of protein and tissue levels indicated that the expression of SLC8A1 and SLC6A3were lower in AC3^-/-MOE than in AC3^+/+MOE,and higher in hAC3 MOE than in AC3^+/+MOE.?2?Localization analysis showed that the expression of AC3 did not affect the locali-zation of SLC8A1 and SLC6A3.SLC8A1 was mainly expressed in the sensory cilia,and colocalized with the ciliary marker protein AC3,and also showed the expression in dendrite,dendritic knobs and cell bodies.SLC6A3 was mainly expressed in the sensory cilia and sus-tentacular cells,colocalized with the sustentacular cells marker protein Ck18.These demonstrated that the expression of SLC8A1 and SLC6A3 were affected by AC3.3.Co-IP and LC-MS were performed for interacting proteins analysis of SLC8A1 and SLC6A3,we observed that,SLC8A1 and SLC6A3 interacting proteins were involved in the expression of many signaling pathways.Including cAMP olfactory signaling pathway and apoptosis signaling pathway which were related to our research.The interaction between SLC8A1 and anti-apoptotic protein HSP90AA1 was verified by experiments.These demon-strated that the apoptosis of MOE maybe affected by the expression of SLC8A1.4.Co-IP and Western blot were performed to prove relationship between SLC8A1 and HSP90AA1 in NIH3T3 cells,we observed that SLC8A1 interacted with HSP90AA1.And the expression of Caspase3 was significantly upregulated after knocking down the expression of SLC8A1 by RNAi in NIH3T3 cells,this indicated that decreased SLC8A1 expression led to increased apoptosis.These demonstrated that SLC8A1 interacted with HSP90AA1 to regulate apoptosis.