Ca2+ signaling and HCO3- secretion by exocrine glands

National Institute Of Dental & Craniofacial Research

Investigators

Linked publications & trials

Abstract

Project 1: TRPC3 channel gating by lipids requires localization at the membrane contact site ER-PM junctions defined by STIM1 TRPC3, a member of the Transient Receptor Potential (TRP) superfamily of ion channels, is a lipid-regulated, non-selective Ca2+ permeable channel that mediates essential component of the receptor evoked Ca2+ signal. The modes and mechanisms by which lipids regulate TRPC3 and other members of the TRPC channel family are not well understood. Here, we used a structure-guided approach to report that PI(4,5)P2 regulates TRPC3 in three independent modes. PLC-dependent hydrolysis generates diacylglycerol (DAG) that interacts with lipid binding site 2 in the channel pore. PI(4,5)P2 interacts with lipid site 1 previously identified in the Cryo-EM structure to inhibit TRPC3 opening and regulate access of DAG to the pore lipid site 2. The activation and regulation of TRPC3 by PI(4,5)P2 requires recruitment of TRPC3 to the membrane contact site ER/PM junctions at a PI(4,5)P2-rich domain. Accordingly, we identified an FFAT site at the TRPC3 N terminus loop within the ankyrin repeats, a region that envelopes the pole helix. The FFAT site interacts with the ER resident VAPB to recruit TRPC3 to the ER/PM junctions and control its receptor-mediated activation. The TRPC3 lipid interacting and regulatory sites are completely conserved in TRPC6 and TRPC7 and in part in other TRPC channels. These findings provide fundamental information on regulation of ion channels by lipids that is needed for drug targeting in treating epithelial, muscular and neurodegenerative diseases known to be affected by aberrant TRPC channels function. These studies are under review. Future plans for STIM1, Orai1 and TRPC channels: a) We started examining regulation of STIM1-Orai1 and the ER/PM junctions by lipids. Currently, we are studying the role of the ER/PM junctions in regulation of STIM1 clustering by PtdSer at restricted and specific micro/nanodomains. b) We continue to examine how STIM1 gates the TRPC and Orai1 channels. Project 2: Regulation of Cl- signaling and ion transport by IRBIT-mediated recruitment of multiple kinases and phosphatases. IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B, CFTR and the Ca2+-activated Cl- channels Bestrophin 2 (Best2) and Anoctamin 1 (TMEM16A, ANO1). Interaction with IRBIT increases their activity and regulation by second messengers. Remarkably, IRBIT targets all transport proteins to the ER/PM junctions to increase signaling repertoire by making the Ca2+-activated Cl- channels regulated by both Ca2+ and cAMP. This is achieved by exposing multiple PKA phosphorylable sites in the proteins that can either activate or inhibit their activity. Future plans for fluid and HCO3- secretion: a) We continue to study the impact of using FDA approved CFTR potentiators and correctors to correct the aberrant fluid secretion in mouse models of Sjogrens syndrome and pancreatitis. b) 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 protein IRBIT makes 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. c) 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.