Control of intracellular chloride signaling by IRBIT-mediated recruitment of multiple kinase and phosphatase pathways

Cl − is the major anion and important osmolyte in most living cells and has a prominent role in cellular homeostasis and intracellular chloride (Cl − in ) affect the activity of several transporters. IRBIT is a multifunctional protein that controls the activity of IP3 receptors and multiple Cl − and HCO 3 − transporters by unknown mechanism. In the case of the ubiquitous NBCe1‐B, IRBIT interacts with the NBCe1‐B N terminus autoinhibitory domain (AID), exposing two cryptic Cl‐interacting sites to confer regulation of NBCe1‐B by Cl − in . Here, we report that IRBIT do so by recruiting a panel of phosphatases and kinases that both regulate signaling by Cl − in and modulate NBCe1‐B conformation to open the transport pathway. Thus, IRBIT recruits the phosphatase PP1 and the kinase SPAK to control phosphorylation of S65 to control regulation of NBCe1‐B by Cl − in at a 32 GXXXP 36 site. IRBIT recruits the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of S12 to control regulation of NBCe1‐B by Cl − in at a 195 GXXXP 199 site. IRBIT then acts on S232, S233, S235 to generate multiple conformations with either dephosphorylated S232/S233, S232/S235 or S233/S235. Each conformation has different activation by IRBIT and regulation by Cl − in . S232/S233/S235 are conserved in several members of the NBC transporters. Mutation of serine to alanine resulted in a fully activated conformation, while mutation of serine to phosphomimetic aspartate inhibited transport and eliminated IRBIT mediated activation, suggesting that S232/S233/S235 determine whether NBC transporter is in an opened or closed conformation. These findings together with structural modeling of NBCe1‐B uncover an intricate mechanism by Cl − in signals through IRBIT‐mediated recruitment of kinases and phosphatases to regulate key transporters involved in epithelial fluid and electrolyte secretion. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .