Ca2+ signaling and HCO3- secretion by exocrine glands

National Institute Of Dental & Craniofacial Research

Investigators

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Abstract

Project 1: Recent structural studies have shown that the carboxyl-terminus of many TRP channels, including TRPC3, are folded into a horizontal rib helix that is connected to the vertical pole helix, which play roles in inter-structural interactions and multimerization. In a previous work we identified I807 located in the pole helix with a role in regulation of TRPC3 by STIM1 (Lee et al., 2014, Liu et al., 2022). To further determine the role of the pole helix in TRPC3 function, here we identified key hydrophobic residues in the pole helix that form tight tunnel-like structure and used mutations to probe their role in TRPC3 regulation by Ca2+ and Calmodulin. Our findings suggest that the hydrophobic starch formed by the I807-L818 residues has several roles, it modulates gating of TRPC3 by Ca2+, affects channel selectivity and the channel Ca2+ permeability. Mutations of I807, I811, L814 and L818 all attenuated the Ca2+-dependent inactivation (CDI) of TRPC3, with I807 having the most prominent effect. The extent of modulation of the CDI depended on the degree of hydrophobicity of I807. Moreover, the TRPC3(I807S) mutant showed altered channel monovalent ion selectivity and increased Ca2+ permeability, without affecting the channel permeability to Mg2+ and Ba2+ and without changing the pore diameter. The CDI of TRPC3 was reduced by an inactive calmodulin mutant and by a pharmacological inhibitor of calmodulin, which was eliminated by the I807S mutation. Notably, deletion of STIM1 caused similar alteration of TRPC3 properties. Taken together, these findings reveal a role of the pole helix in CDI, in addition to its potential role in channel multimerization that required gating of TRPC3 by STIM1. Since all TRPC and most TRP channels have pole helix structures, our findings raise the possibility that the pole helix may have similar roles in all the TRP family. 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.