Regulation of Ca2+ signaling by STIM1. Ca2+ influx by store-operated Ca2+ channels is a key component of the receptor-evoked Ca2+ signal. In all cells examined, transient receptor potential canonical (TRPC) channels mediate a significant portion of the receptor-stimulated Ca2+ influx. Recent studies have revealed how STIM1 activates TRPC1, but the role of STIM1 in TRPC channel activation by receptor stimulation is not fully understood. We established mutants of TRPC channels that could not be activated by STIM1 but were activated by the """"""""charge-swap"""""""" mutant STIM1(K684E,K685E). WT but not mutant TRPC channels were inhibited by scavenging STIM1 while mutant TRPC channels were robustly activated by receptor stimulation. Moreover, STIM1 and STIM1(K684E,K685E) reciprocally affected receptor-activated WT and mutant TRPC channels. Together, these findings indicate that TRPC channels can function as STIM1-dependent and STIM1-independent channels, which increases the versatility of TRPC channel function and their role in receptor-stimulated Ca2+ influx. To understand the role of the native STIM1 and Orai1 in polarized Ca2+ signals in secretory epithelial cells we found that inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca2+ influx and frequency of Ca2+ oscillations. The native Orai1 localization is restricted to the apical pole of the lateral membrane and does not cluster in response to store depletion. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. TRPC1 is expressed in both apical and basolateral regions of the plasma membrane. Co-IP of STIM1/Orai1/IP3Rs/TRPCs is enhanced by cell stimulation and disrupted by 2APB. The polarized localization and recruitment of these proteins results in preferred Ca2+ entry that is initiated at the apical pole. The role of Ca2+ influx channels in disease was shown by demonstrating that genetic and pharmacological of the Ca2+ influx channel TRPC3 protect pancreas and salivary glands from the toxic effect of excessive Ca2+ influx. Another type of Ca2+ channels are the TRPMLs. Mutations in TRPML1, a lysosomal Ca2+-permeable TRP channel, lead to mucolipidosis type IV with achlorhydria. We produced Trpml1 null mice and demonstrated critical requirement for this channel in gastric acid secretion. Histologic and ultrastructural analyses revealed that Trpml1-/- parietal cells are damaged, although the intralysosomal Ca2+ content were unaffected in these cells. Cells express two organellar Ca2+ channels, TRPMLs and the NAADP-activated TPCs. A critical question is whether the two channels are related. We used molecular, biochemical and functional assays to show that the TRPMLs and TPCs are indepent channels. Mechanism and Regulation of Epithelial HCO3- secretion: This lab also study epithelial HCO3- transport in health and disease. Members of the SLC26 family of anion transporters play a critical role in epithelial HCO3- secretion. We characterize several features of the electrogenic members of the family to identify structural motifs that determine multiple functional modes of these transporters and explain how the transporters can function either as coupled or as uncoupled transporters. Fluid and HCO3- secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition. Secretion of ductal fluid and HCO3- in secretory glands is fueled by Na+/HCO3- cotransport mediated by basolateral NBCe1-B and by Cl-/HCO3- exchange mediated by luminal Slc26a6 and CFTR. To understand the mechanisms governing ductal secretion we showed that ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator IRBIT and by inhibition of PP1. In contrast, silencing the WNK and SPAK kinases increased secretion. Molecular analysis revealed that the WNK kinases acted as scaffolds to recruit SPAK, which phosphorylated CFTR and NBCe1-B, reducing their cell surface expression. IRBIT opposed the effects of WNKs and SPAK by recruiting PP1 to the complex to dephosphorylate CFTR and NBCe1-B, restoring their cell surface expression, in addition to stimulating their activities. These findings stress the pivotal role of IRBIT in epithelial fluid and HCO3- secretion and provide a molecular mechanism by which IRBIT coordinates these processes. They also have implications for WNK/SPAK kinase-regulated processes involved in systemic fluid homeostasis, hypertension, and cystic fibrosis.

Project Start
Project End
Budget Start
Budget End
Support Year
4
Fiscal Year
2013
Total Cost
$2,588,531
Indirect Cost
Name
National Institute of Dental & Craniofacial Research
Department
Type
DUNS #
City
State
Country
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Park, Soonhong; Ahuja, Malini; Kim, Min Seuk et al. (2016) Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV. EMBO Rep 17:266-78
Son, Aran; Park, Seonghee; Shin, Dong Min et al. (2016) Orai1 and STIM1 in ER/PM junctions: roles in pancreatic cell function and dysfunction. Am J Physiol Cell Physiol 310:C414-22
Hegyi, Péter; Wilschanski, Michael; Muallem, Shmuel et al. (2016) CFTR: A New Horizon in the Pathomechanism and Treatment of Pancreatitis. Rev Physiol Biochem Pharmacol 170:37-66
Hong, Jeong Hee; Muhammad, Emad; Zheng, Changyu et al. (2015) Essential role of carbonic anhydrase XII in secretory gland fluid and HCO3 (-) secretion revealed by disease causing human mutation. J Physiol 593:5299-312
Shcheynikov, Nikolay; Son, Aran; Hong, Jeong Hee et al. (2015) Intracellular Cl- as a signaling ion that potently regulates Na+/HCO3- transporters. Proc Natl Acad Sci U S A 112:E329-37
Cao, Xu; Choi, Seok; Maléth, Jozsef J et al. (2015) The ER/PM microdomain, PI(4,5)P₂ and the regulation of STIM1-Orai1 channel function. Cell Calcium 58:342-8
Jha, Archana; Brailoiu, Eugen; Muallem, Shmuel (2014) How does NAADP release lysosomal Ca (2+)? Channels (Austin) 8:
Lee, Kyu Pil; Choi, Seok; Hong, Jeong Hee et al. (2014) Molecular determinants mediating gating of Transient Receptor Potential Canonical (TRPC) channels by stromal interaction molecule 1 (STIM1). J Biol Chem 289:6372-82
Muallem, Shmuel (2014) CFTR does it again: control of insulin secretion. Sci China Life Sci 57:1046
Hong, Jeong Hee; Park, Seonghee; Shcheynikov, Nikolay et al. (2014) Mechanism and synergism in epithelial fluid and electrolyte secretion. Pflugers Arch 466:1487-99

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