Regulatory effects of inositol 1,4,5-trisphosphate receptor genes knockdown on intracellular Ca²⁺ concentration and ion channel-related gene expression in duck uterine epithelial cells.

This study employed primary uterine epithelial cells from Sansui ducks (Anas platyrhynchos) at the peak of lay as an in vitro model. Using shRNA-mediated interference, we generated single knockdowns of inositol 1,4,5-trisphosphate receptor type 1 (IP3R1), inositol 1,4,5-trisphosphate receptor type 2 (IP3R2), and inositol 1,4,5-trisphosphate receptor type 3 (IP3R3), as well as dual knockdowns of IP3R1+IP3R2, IP3R1+IP3R3, and IP3R2+IP3R3, and a triple knockdown targeting all three IP3R isoforms. These treatments were designed to elucidate the regulatory effects of IP3R expression on intracellular calcium ion (Ca²⁺) concentrations and the expression of genes related to ion transport. After 48 h of transfection, fluorescence labeling with the Fluo-3/AM probe showed that intracellular Ca²⁺ concentrations were significantly higher in all treatment groups compared with the control group (P < 0.01). The real-time qPCR (RT-qPCR) analysis of ten ion transport-related genes revealed that the expression of transient receptor potential cation channel subfamily V member 6 (TRPV6), ORAI calcium release-activated calcium modulator 1 (ORAI1), epithelial sodium channel gamma subunit (SCNN1G), solute carrier family 8 member A1 (SLC8A1), ATPase plasma membrane Ca²⁺-transporting 1 (ATP2B1), and ATPase sarcoplasmic/endoplasmic reticulum Ca²⁺-transporting 2 (ATP2A2) was significantly downregulated in multiple IP3R knockdown groups compared with the NC group (P < 0.05). The expression levels of these genes also showed significant correlations with intracellular Ca²⁺ levels (P < 0.05), including both positive and negative correlations. In the IP3R1 knockdown group, TRPV6, ORAI1, and SCNN1G were negatively correlated with Ca²⁺ concentration (P < 0.05). In contrast, ATP2B1 exhibited a positive correlation with Ca²⁺ in the IP3R2 knockdown group (P < 0.01), whereas SLC8A1 expression was positively correlated with Ca²⁺ in the IP3R1 + IP3R3 dual knockdowns group (P < 0.05). Collectively, the knockdown of IP3Rs not only reduced intracellular calcium ion (Ca²⁺) release but also altered the expression of ion channel-related genes, thereby affecting Ca²⁺ homeostasis. These findings provide new molecular evidence for the regulatory role of IP₃Rs in ion transport networks and Ca²⁺ balance in uterine epithelial cells of laying ducks.