Orai1 and STIM1 in ER/PM junctions: roles in pancreatic cell function and dysfunction

Son, Aran,Park, Seonghee,Shin, Dong Min,Muallem, Shmuel American Physiological Society 2016 American journal of physiology. Cell physiology Vol.310 No.6

<P>Membrane contact sites (MCS) are critical junctions that form between the endoplasmic reticulum (ER) and membranes of various organelles, including the plasma membrane (PM). Signaling complexes, including mediators of Ca2+ signaling, are assembled within MCS, such as the ER/PM junction. This is most evident in polarized epithelial cells, such as pancreatic cells. Core Ca2+ signaling proteins cluster at the apical pole, the site of inositol 1,4,5-trisphosphate-mediated Ca2+ release and Orai1/transient receptor potential canonical-mediated store-dependent Ca2+ entry. Recent advances have characterized the proteins that tether the membranes at MCS and the role of these proteins in modulating physiological and pathological intracellular signaling. This review discusses recent advances in the characterization of Ca2+ signaling at ER/PM junctions and the relation of these junctions to physiological and pathological Ca2+ signaling in pancreatic acini.</P>

TRPML1 as lysosomal fusion guard

Ahuja, Malini,Park, Soonhong,Shin, Dong Min,Muallem, Shmuel Informa UK (TaylorFrancis) 2016 Channels Vol.10 No.4

The Ca²⁺ Channel TRPML3 Regulates Membrane Trafficking and Autophagy

Kim, Hyun Jin,Soyombo, Abigail A.,Tjon-Kon-Sang, Sandra,So, Insuk,Muallem, Shmuel Blackwell Publishing Ltd 2009 Traffic Vol.10 No.8

<P><B>TRPML3 is an inward rectifying Ca<SUP>2+</SUP> channel that is regulated by extracytosolic H<SUP>+</SUP>. Although gain-of-function mutation in TRPML3 causes the varitint-waddler phenotype, the role of TRPML3 in cellular physiology is not known. In this study, we report that TRPML3 is a prominent regulator of endocytosis, membrane trafficking and autophagy. Gradient fractionation and confocal localization reveal that TRPML3 is expressed in the plasma membrane and multiple intracellular compartments. However, expression of TRPML3 is dynamic, with accumulation of TRPML3 in the plasma membrane upon inhibition of endocytosis, and recruitment of TRPML3 to autophagosomes upon induction of autophagy. Accordingly, overexpression of TRPML3 leads to reduced constitutive and regulated endocytosis, increased autophagy and marked exacerbation of autophagy evoked by various cell stressors with nearly complete recruitment of TRPML3 into the autophagosomes. Importantly, both knockdown of TRPML3 by siRNA and expression of the channel-dead dominant negative TRPML3(D458K) have a reciprocal effect, reducing endocytosis and autophagy. These findings reveal a prominent role for TRPML3 in regulating endocytosis, membrane trafficking and autophagy, perhaps by controlling the Ca<SUP>2+</SUP> in the vicinity of cellular organelles that is necessary to regulate these cellular events.</B></P>

Homer2 Protein Regulates Plasma Membrane Ca²⁺-ATPase-mediated Ca²⁺ Signaling in Mouse Parotid Gland Acinar Cells

Yang, Yu-Mi,Lee, Jiae,Jo, Hae,Park, Soonhong,Chang, Inik,Muallem, Shmuel,Shin, Dong Min American Society for Biochemistry and Molecular Bi 2014 The Journal of biological chemistry Vol.289 No.36

<P>Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca<SUP>2+</SUP>-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca<SUP>2+</SUP> signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca<SUP>2+</SUP> signaling in parotid gland acinar cells using <I>Homer2</I>-deficient (<I>Homer2</I><SUP>−/−</SUP>) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of <I>Homer2</I> did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca<SUP>2+</SUP>-ATPase pumps. In contrast, <I>Homer2</I> deletion markedly increased expression of plasma membrane Ca<SUP>2+</SUP>-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, <I>Homer2</I> deficiency increased Ca<SUP>2+</SUP> extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PP<I>XX</I>F-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PP<I>XX</I>F-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca<SUP>2+</SUP> clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca<SUP>2+</SUP> signaling in parotid acinar cells.</P>

Convergence of IRBIT, phosphatidylinositol (4,5) bisphosphate, and WNK/SPAK kinases in regulation of the Na⁺-HCO₃⁻ cotransporters family

Hong, Jeong Hee,Yang, Dongki,Shcheynikov, Nikolay,Ohana, Ehud,Shin, Dong Min,Muallem, Shmuel National Academy of Sciences 2013 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.110 No.10

<P>Fluid and HCO<SUB>3</SUB><SUP>−</SUP> secretion is a vital function of secretory epithelia, involving basolateral HCO<SUB>3</SUB><SUP>−</SUP> entry through the Na<SUP>+</SUP>-HCO<SUB>3</SUB><SUP>−</SUP> cotransporter (NBC) NBCe1-B, and luminal HCO<SUB>3</SUB><SUP>−</SUP> exit mediated by cystic fibrosis transmembrane conductance regulator (CFTR) and solute carrier family 26 (SLC26) Cl<SUP>−</SUP>/HCO<SUB>3</SUB><SUP>−</SUP> exchangers. HCO<SUB>3</SUB><SUP>−</SUP> secretion is highly regulated, with the WNK/SPAK kinase pathway setting the resting state and the IRBIT/PP1 pathway setting the stimulated state. However, we know little about the relationships between the WNK/SPAK and IRBIT/PP1 sites in the regulation of the transporters. The first 85 N-terminal amino acids of NBCe1-B function as an autoinhibitory domain. Here we have identified a positively charged module within NBCe1-B(37-65) that is conserved in NBCn1-A and all 20 members of the NBC superfamily except NBCe1-A. This module is required for the interaction and activation of NBCe1-B and NBCn1-A by IRBIT and their regulation by phosphatidylinositol 4,5-bisphosphate (PIP<SUB>2</SUB>). Activation of the transporters by IRBIT and PIP<SUB>2</SUB> is nonadditive but complementary. Phosphorylation of Ser65 mediates regulation of NBCe1-B by SPAK, and phosphorylation of Thr49 is required for regulation by IRBIT and SPAK. Sequence searches using the NBCe1-B regulatory module as a template identified a homologous sequence in the CFTR R domain and Slc26a6 sulfat transporter and antisigma factor antagonist (STAS) domain. Accordingly, the R and STAS domains bind IRBIT, and the R domain is required for activation of CFTR by IRBIT. These findings reveal convergence of regulatory modalities in a conserved domain of the NBC that may be present in other HCO<SUB>3</SUB><SUP>−</SUP> transporters and thus in the regulation of epithelial fluid and HCO<SUB>3</SUB><SUP>−</SUP> secretion.</P>

Molecular Mechanism of Pancreatic Bicarbonate Secretion

Lee, Min-Goo,Kim, Je-Woo,Kim, Kyung-Hwan,Muallem, Shmuel The Korean Society of Pharmacology 2002 The Korean Journal of Physiology & Pharmacology Vol.6 No.3

Thanks to recent progress in availability of molecular and functional techniques it became possible to search for the basic molecular and cellular processes that mediate and control $HCO_3{^-}$ and fluid secretion by the pancreatic duct. The coordinated action of various transporters on the luminal and basolateral membranes of polarized epithelial cells mediates the transepithelial $HCO_3{^-}$ transport, which involves $HCO_3{^-}$ absorption in the resting state and $HCO_3{^-}$ secretion in the stimulated state. The overall process of HCO3 secretion can be divided into two steps. First, $HCO_3{^-}$ in the blood enters the ductal epithelial cells across the basolateral membrane either by simple diffusion in the forms of $CO_2$ and $H_2O$ or by the action of an $Na^+-coupled$ transporter, a $Na^+-HCO_3$ cotranporter (NBC) identified as pNBC1. Subsequently, the cells secrete $HCO_3{^-}$ to the luminal space using at least two $HCO_3{^-}$ exit mechanisms at the luminal membrane. One of the critical transporters needed for all forms of $HCO_3{^-}$ secretion across the luminal membrane is the cystic fibrosis transmembrane conductance regulator (CFTR). In the resting state the pancreatic duct, and probably other $HCO_3{^-}$ secretory epithelia, absorb $HCO_3{^-}.$ Interestingly, CFTR also control this mechanism. In this review, we discuss recent progress in understanding epithelial $HCO_3{^-}$ transport, in particular the nature of the luminal transporters and their regulation by CFTR.

Palmitoylation controls trafficking of the intracellular Ca²⁺ channel MCOLN3/TRPML3 to regulate autophagy

Kim, So Woon,Kim, Dong Hyun,Park, Kyoung Sun,Kim, Mi Kyung,Park, Yun Min,Muallem, Shmuel,So, Insuk,Kim, Hyun Jin Informa UK (TaylorFrancis) 2019 AUTOPHAGY Vol.15 No.2

Molecular determinants of fast Ca2+-dependent inactivation and gating of the Orai channels.

Lee, Kyu Pil,Yuan, Joseph P,Zeng, Weizhong,So, Insuk,Worley, Paul F,Muallem, Shmuel National Academy of Sciences 2009 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF Vol.106 No.34

<P>Ca(2+) influx by store-operated Ca(2+) influx channels (SOCs) mediates many cellular functions regulated by Ca(2+), and excessive SOC-mediated Ca(2+) influx is cytotoxic and associated with disease. One form of SOC is the CRAC current that is mediated by Orai channels activated by STIM1. A fundamental property of the native CRAC and of the Orais is fast Ca(2+)-dependent inactivation, which limits Ca(2+) influx to guard against cellular damage. The molecular mechanism of this essential regulatory mechanism is unknown. We report here the fast Ca(2+)-dependent inactivation is mediated by three conserved glutamates in the C termini (CT) of Orai2 and Orai3, which show prominent fast Ca(2+)-dependent inactivation compared with Orai1. Transfer of the CT between the Orais transfers both the extent of channel opening and the mode of fast Ca(2+)-dependent inactivation. Fast Ca(2+)-dependent inactivation of the Orais also requires a domain of STIM1; fragments of STIM1 that efficiently open Orai channels do not evoke fast inactivation unless they include an anionic sequence that is C-terminal to the STIM1-Orai activating region (SOAR). Our studies suggest that Orai CT are necessary and sufficient to control pore opening and uncover the molecular mechanism of fast Ca(2+)-dependent inactivation that has implications for Ca(2+) influx by SOC in physiological and pathological states.</P>

RANKL-mediated Reactive Oxygen Species Pathway That Induces Long Lasting Ca²⁺Oscillations Essential for Osteoclastogenesis

Kim, Min Seuk,Yang, Yu-Mi,Son, Aran,Tian, Yu Shun,Lee, Syng-Ill,Kang, Sang Won,Muallem, Shmuel,Shin, Dong Min American Society for Biochemistry and Molecular Bi 2010 The Journal of biological chemistry Vol.285 No.10

Native Store-operated Ca2+ Influx Requires the Channel Function of Orai1 and TRPC1.

Kim, Min Seuk,Zeng, Weizhong,Yuan, Joseph P,Shin, Dong Min,Worley, Paul F,Muallem, Shmuel American Society for Biochemistry and Molecular Bi 2009 The Journal of biological chemistry Vol.284 No.15

<P>With the discovery of STIM1 and Orai1 and gating of both TRPC and Orai1 channels by STIM1, a central question is the role of each of the channels in the native store-operated Ca(2+) influx (SOCs). Here, we used a strategy of knockdown of Orai1 and of TRPC1 alone and in combination and rescue by small interfering RNA-protected mutants (sm) of smOrai1 and smTRPC1 to demonstrate that in human embryonic kidney (HEK) cells, rescue of SOCs required co-transfection of low levels of both smOrai1 and smTRPC1. The pore mutant Orai1(E106Q) failed to rescue the SOCs in the presence or absence of TRPC1 and, surprisingly, the pore mutant TRPC1(F562A) failed to rescue the SOCs in the presence or absence of Orai1. TRPC1 is gated by electrostatic interaction between TRPC1(D639D,D640D) with STIM1(K684K, K685K). Strikingly, the channel-dead TRPC1(D639K,D640K) that can be rescued only by the STIM1(K684E,K685E) mutant could restore SOCs only when expressed with Orai1 and STIM1(K684E,K685E). Accordingly, we found a mutual requirement of Orai1 and TRPC1 for their interaction with the native STIM1 in HEK cells. By contrast, SOC and the CRAC current in Jurkat cells were inhibited by knockdown of Orai1 but were not influenced by knockdown on TRPC1 or TRPC3. These findings define the molecular makeup of the native SOCs in HEK cells and the role of a STIM1-Orai1-TRPC1 complex in SOC activity.</P>