The A-kinase anchoring protein Yotiao decrease the ER calcium content by inhibiting the store operated calcium entry

•The scaffolding protein Yotiao serves as a novel inhibitor of store-operated calcium entry (SOCE) and reduces ER Ca2+ levels.•The interaction between Yotiao and adenylyl cyclase type 9 (AC9) is crucial for Yotiao's ability to decrease ER Ca2+ levels.•Yotiao decreases ER Ca2+ levels by inhibiting SOCE through its actions on Orai1 C terminus. The meticulous regulation of ER calcium (Ca2+) homeostasis is indispensable for the proper functioning of numerous cellular processes. Disrupted ER Ca2+ balance is implicated in diverse diseases, underscoring the need for a systematic exploration of its regulatory factors in cells. Our recent genomic-scale screen identified a scaffolding protein A-kinase anchoring protein 9 (AKAP9) as a regulator of ER Ca2+ levels, but the underlying molecular mechanisms remain elusive. Here, we reveal that Yotiao, the smallest splicing variant of AKAP9 decreased ER Ca2+ content in animal cells. Additional testing using a combination of Yotiao truncations, knock-out cells and pharmacological tools revealed that, Yotiao does not require most of its interactors, including type 1 inositol 1,4,5-trisphosphate receptors (IP3R1), protein kinase A (PKA), protein phosphatase 1 (PP1), adenylyl cyclase type 2 (AC2) and so on, to reduce ER Ca2+ levels. However, adenylyl cyclase type 9 (AC9), which is known to increases its cAMP generation upon interaction with Yotiao for the modulation of potassium channels, plays an essential role for Yotiao's ER-Ca2+-lowering effect. Mechanistically, Yotiao may work through AC9 to act on Orai1-C terminus and suppress store operated Ca2+ entry, resulting in reduced ER Ca2+ levels. These findings not only enhance our comprehension of the interplay between Yotiao and AC9 but also contribute to a more intricate understanding of the finely tuned mechanisms governing ER Ca2+ homeostasis. [Display omitted]