Ca2+ signaling and HCO3- secretion by exocrine glands

National Institute Of Dental & Craniofacial Research

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Abstract

Project 1: ANO1 plays a crucial role in determining numerous physiological functions, including epithelial secretion, yet its regulatory mechanisms remain incompletely understood. Here, we describe a fundamental dynamic regulation of ANO1 surface expression and Ca2+-dependent gating via the cAMP/PKA pathway at the STIM1 ER/PM junctions. At these junctions, STIM1 assembles AC-AKAP-PKA complexes, while E-Syt1 mediates formation of ANO1-VAPA-IRBIT-E-Syt1-AC8-AKAP5-PKA complex, that phosphorylates ANO1 S673, increasing ANO1 Ca2+ affinity. Within these complexes, the Ca2+ and cAMP pathways act synergistically to enhance ANO1 function. By contrast, E-Syt2 dissociates the ANO1-VAPA interaction, forming ANO1-IRBIT-E-Syt2-AC6-AKAP11-PKA complex that phosphorylates ANO1 S221, which markedly reduces ANO1 Ca2+ affinity. The effects of the E-Syts are primarily mediated by their reciprocal regulation of junctional PI(4)P, PI(4,5)P2 and PtdSer. Accordingly, IRBIT deletion in mice impairs receptor-stimulated activation of ANO1 and fluid secretion. These findings should have broad implications for ANO1 roles and functions across various tissues. Project 2: Endoplasmic reticulum/plasma membrane (ER/PM) junctions are a major site of cellular signal transduction including in epithelia; however, whether their lipid membrane environment affects junctional ion transporters function remains unclear. Here, we show that epithelial secretion is governed by phosphatidylserine (PtdSer) levels in ER/PM nanodomains, specified by the antagonistic action of the lipid transfer proteins E-Syt3 and ORP5, which transduce cAMP signals to the chloride channel CFTR and activate the sodium-bicarbonate cotransporter NBCe1-B by IRBIT. Lipid transfer by E-Syt3, along with restricted plasma membrane localization by the E-Syt3 C2C domain, are essential for E-Syt3 function, as removal of PtdSer from junctions by E-Syt3 dissociated the cAMP signaling pathway complex, preventing CFTR activation, and prevented NBCe1-B activation by IRBIT. CFTR and NBCe1-B PtdSer sensor domains responded to PtdSer reduction by E-Syt3; which was reversed by exogenous PtdSer or by PtdSer supplied by ORP5. In mice, E-Syt3 depletion improved chloride flux and fluid secretion in salivary glands and isolated pancreatic ducts. These findings provide a framework for understanding the role of junctional lipids in the assembly of functional ion protein complexes and cellular communication at epithelial signaling hubs. Future studies: Explore the role of the ER/PM junction tethers Anoctamin 8 in junctional lipid metabolism and Ca2+ signaling. 3. The core functions of the anoctamins are Cl- channel activity and phosphatidylserine (and perhaps other lipids) scrambling. These functions have been extensively studied in various tissues and cells. However, another function of the anoctamins that is less recognized and minimally explored is as tethers at membrane contact sites. This short review aims to examine evidence supporting the localization of the anoctamins at membrane contact sites, their tether properties, and their functions as tethers. Future plans for fluid and HCO3- secretion: a) Synergism between signaling pathways, the Ca2+ and cAMP signaling, is the physiological mode of cell signaling that is aberrant in pathology. We are exploring the molecular mechanism of Ca2+ and cAMP signaling synergism and expansion of signaling repertoire by determining how the scaffolding proteins VAPA and IRBIT make the Ca2+-activated Cl- and HCO3- permeable channel Best2 and the Ca2+-activated Cl- ANO1 to became Ca2+ and cAMP regulated and the physiological significance of this regulation. b) We are studying how targeting the Cl- channel CFTR and the Na+-HCO3- co-transporter NBCe1-B to the ER/PM junctions affect their function and regulation by PtdSer.