shmuel Muallem - US grants

The principal object of this project is to extend our studies on the effect of Ca2+ mobilizing hormones on the Ca2+ permeability and Ca2+ pump of the intracellular stores of pancreatic acinar cells. This constitutes as essential stage towards understanding the mechanisms of release and reloading of the intracellular stores with Ca2+upton cell stimulation and removal of stimuli. To date, we have developed techniques; to measure free cytosolic Ca2+ in intact pancreatic acinar cells using the Ca2+ sensitive dye FURA 2; to specifically label the hormone mobilizable intracellular pool with 45Ca in intact cells; to measure Ca2+ channel mediated - 45Ca fluxes in intact and permeabilized cells; and to measure Ca2+ pump mediated -45Ca fluxes in permeabilized cells. Using these techniques, the immediate aims are (a) demonstration that Ca2+ release from the intracellular pool is sufficient to activate the plasma membrane Ca2+ channel. Activation of this channel is not directly mediated and does not require the presence of the hormone. (b) Show that the hormone, like IP3, activates a Ca2+ channel in the endoplasmic reticulum membrane. (c) Compare the effects of IP3, GTP and arachidonic acid with the hormone activated Ca2+ release from the intracellular pool. (d) Study the contribution of IP3, GTP and arachidonic acid to the hormone dependent Ca2+ release (e) define the requirements for the newly discovered, activation of the endoplasmic reticulum Ca2+ pump by the Ca2+ mobilizing hormone (f) attempt to characterize the mechanism of activation of the endoplasmic reticulum Ca2+ pump by the Ca2+ pump by the Ca2+ mobilizing hormones. Our basic studies are providing techniques to test hypotheses on hormonal regulation of free cytosolic Ca2+ and Ca2+ content in the intracellular stores of pancreatic acinar cells both during and at the termination of cell stimulation.

One of the critical functions of the osteoblast is matrix mineralization. To accomplish this task, the osteoblast must be able to influence the pH and ion current of the fluid in the demarcation zone. It is likely that the osteoblasts are capable of generating and maintaining gradients of HCO3-, Cl-, Na+, K+ and Ca2+ between the plasma and bone extracellular fluid of the demarcation zone. These cells also undergo marked shape changes during hormonal stimulation. Maintenance of the ionic gradients, in particular Ca2+ and HCO3-, are essential for bone remodeling. Establishing vectorial transport and transcellular ionic gradients require polarized localization of ion transport pathways. The nature of the ionic pathways and their localization in the osteoblasts are not known. It is also not known whether the shape changes observed in response to calcitropic hormones are also accompanied by volume changes. We will use the osteosarcoma cell line UMR 106, and primary cultures of osteoblast-like cells from long bone to: a) identify and then kinetically characterize the ionic pathways which participate in cytosolic pH regulation by the osteoblast. These ionic pathways include Cl- dependnt HCO3- secretion (Cl-/HCO3-), Na+/H+ exchanger K+ and Cl- conductances nd KCl or 3NaKCl2 cotransporters. This will be achieved by measurements of pHi and (Ca2+)i with the pH and Ca2+ sensitive dyes BCECF and Fura 2, respectively, and net, undirectional and exchange transport of 36 Cl-, 22Na, and 86Rb.; b) localize these ionic pathways to the membrane facing the BECF or the membrane facing plasma. UMR-106 and normal rat osteoblast-like cells will be grown on collagen coated filters and placed in chamber. The ionic transporters facing the collagen matrix or the medium will be identified. In parallel, the ionic transport characteristics of vesicles prepared from different portions of the osteoblast plasma membrane will be studied. These ionic pathways will be examined within the framework of cell volume regulation by these cells. Finally, the effect of hormones such as PTH2+ Vitamin D, prostoglandins, epidermal growth factor, and second messengers such as Ca2+, cAMP, and protein Kinase C activation, on these ionic pathways will be studied as outlined above.

The principal objective of this proposal is to extend our studies of Ca2+ homeostasis in resting and stimulated pancreatic acinar cells. This constitutes an essential stage towards understanding the mechanisms of release and reloading of intracellular Ca2+ stores during [Ca2+]i oscillation, and upon termination of cell stimulation. Over the last few years we have developed techniques: to specifically label the agonist-- mobilizable intracellular pool with (45)Ca in intact cells; to measure (45)Ca fluxes mediated by conductive pathways in intact and permeabilized cells and Ca2+ pump-mediated-(45)Ca fluxes in permeabilized cells; to measure free cytosolic Ca 2+ concentration with Fura 2 in cell suspension and in single pancreatic acinus. We have also identified the presence of a cytosolic Ca2+ binding protein which inhibits IP3-mediated Ca 2+ release. Using these techniques the immediate aims of the proposal are to 1) determine the route of Ca 2+ entry across the plasma membrane during stimulation and reloading. This should reveal the role of the cytosolic Ca 2+ pool in controlling reloading of intracellular Ca 2+ stores, 2) characterize [Ca2+]i oscillation of pancreatic acinus and single cells within the acinus in an attempt to determine the role of pool Ca 2+ content and [Ca2+]i in regulating Ca2+ oscillation, 3) define the activation of the intracellular Ca2+ pump by agonists and protein kinases, 4) study the overall regulation of IP3 binding and IP3-mediated Ca 2+ release by the cytosolic inhibitory Ca 2+ binding protein, 5) purify the inhibitory protein and characterize its interaction with the IP3-mobilizable Ca 2+ pool and its distribution in various cells. Regulation of the interaction of this protein with the IP3-activated Ca 2+ channel has the potential of providing a temporal aspect to regulation of IP3-mediated Ca 2+ release and thus resulting in Ca2+ oscillation. We hope that our basic studies are providing techniques to test hypotheses on hormonal regulation of cellular Ca2+ pathways controlling free cytosolic Ca 2+ and will bring us closer toward understanding Ca 2+ homeostasis and Ca 2+ oscillation by the stimulated pancreatic acinar cell.

The principal objective of this proposal is to understand the mechanisms of fluid and electrolyte secretion, by the exocrine pancreas. Pancreatic fluid and electrolyte secretion has an acinar and ductal components which most probably occur by different mechanisms. In addition, the secretory mechanism is likely to differ along the ductal tree. There are significant species variability; the rat pancreas represents one extreme secreting fluid relatively rich in Cl- whereas the guinea pig pancreas secretes fluid poor in Cl- and rich in HCO3-. We therefore propose to make use of these naturally occurring variabilities to identify and characterize the key mechanisms responsible for pancreatic fluid and electrolyte secretion. Towards achieving this goal, we have developed the techniques required to: prepare isolated interlobular ducts with or without acini attached to them, microdissect and perfuse intralobular ducts, and perfuse main ducts; measure the intracellular concentrations of and Cl-, H+/HCO3- and Ca 2+ using photon counting from single cells or image acquisition and analysis of cells loaded with fluorescent dyes; measure ion fluxes mediated by specific transporters. We have also developed a new and simple technique for the simultaneous recording of intracellular ionic activities and cell volume of single cells with high time resolution. These techniques will be used to 1) Study and compare H+/HCO3- transport mechanisms in rat and guinea pig pancreatic ducts, their regulation by specific agonists and relation to cell volume regulation. These studies are aimed at discovering the active mechanisms responsible for HCO3- secretion to the duct lumen. 2) Determine Cl- transport mechanisms by the various ductal (and acinar) cells. It is hoped that we will be able to determine the mechanism of active transepithelial Cl- absorption by duct cells and the basis for cellular and species variations. 3) Localize acid base and Cl- transporters to the serosal and/or luminal ductal cell membranes using perfused ducts. The purpose of these studies is to develop and test physiologically and thermodynamically sound models for acinar and ductal fluid and electrolyte secretion. 4) Study mechanisms of cell volume regulation in single pancreatic duct (and acinar) cells to understand the cellular volume compensatory mechanism and interrelationships between the transporters during the overall process of fluid and electrolyte secretion. I hope that the proposed studies will extend the knowledge and determine the underlying mechanisms of fluid and electrolyte secretion by the exocrine pancreas.

Abnormal function of salivary gland occurs in radiation, drug therapies, Sjogren's syndrome and Cystic Fibrosis. Saliva is formed by secretion of proteins and fluid by acinar cells. The ductal system absorbs the Na+ and Cl- and secretes K+ and HCO3- to form the final saliva. Over the last few years, we came to appreciate the central role of CFTR-regulated HCO3- transport in epithelial fluid and electrolyte secretion, including that by salivary glands. CFTR supports Cl--dependent HCO3- transport, and CF-causing mutations with normal C1- channel activity have aberrant HCO3- transport. In the preliminary data we show that SMG express several members of the new SLC26 family of Cl-/HCO3- exchangers-DRA, PDS, SMCBT-and BTR1, that function in HCO3- secretion. SMG also express splice variants on the NBCn1 family that function in HCO3- salvage. The proteins of both families are regulated by CFTR. Based on these findings, we developed a new hypothesis to propose that transcellular HCO3- transport is central to salivary gland function and that CFTR coordinates HCO3- transport at rest and during stimulation. The hypothesis will be tested by following four aims. In aim 1 we will study the reciprocal regulatory interaction between CFTR and mDRA. Preliminary data shows that CFTR markedly stimulates mDRA activity and that mDRA may affect anion selectivity of CFTR. We will use several CFTR and DRA mutants to a) characterize the mechanism by which CFTR activates mDRA b) study how mDRA affects CFTR channel properties and c) study the reciprocal regulation in vivo in WT and deltaF mice. In aim 2 we will probe interactions between CFTR and the SLC6 family members Pendrin and SLC26A6 that are expressed at high levels in SMG ducts. After basic characterization of SLC26A6 C1-/HCO3- exchange activity, we will probe whether CFTR regulates PDS and SMCBT in vitro and in vivo as it controls mDRA. In aim 3 we will study the role of BTR1 in SMG function. BTR1 is the first member of a new family of HCO3- transporters that is expressed in SMG duct and acinar cells. We propose to characterize Cl- and HCO3- transport by BTR1 as the potential HCO3- transporter or channel in the LM of the SMG that generates the final 140 mM HCO3- in saliva. In aim 4 we will characterize regulation of the NBCn1 splice variants by CFTR and their role in HCO3- salvage by the SMG. We will characterize the activity of individual and combinations of NBCn1 isoforms and the mechanism of their inhibition by CFTR in vitro and in vivo using the deltaF mouse. Successful completion of the experiments should considerably clarify the role of CFTR in regulating HCO3- homeostasis in the resting and stimulated states. The studies may also shift the emphasis from efforts to correct Cl- transport to efforts to correct Cl- and HCO3- transport in diseases of secretory epithelia such as CF and Sjogren's syndrome.

The polarized nature of acinar cell function requires polarized organization and function of signaling complexes. Understanding assembly of signaling proteins into complexes within cellular microdomains is the central theme of this proposal. Based on our preliminary findings of: a) the role of RGS proteins in Ca2+ signaling; and b) the regulation of ICRAC by IP3R we will test the hypothesis of organization of Ca2+ signaling proteins into complexes by 1. Study the role of RGS proteins in conferring signaling specificity and assembly of Ca2+ signaling complexes. This will be achieved by finding the N-terminal sequence responsible for receptor recognition. Mapping and isolation of signaling complexes with RGS box and the N-terminus. Study the role of native pancreatic RGS proteins in Ca2+ oscillations. These studies will reveal the role of RGS proteins in Ca2+ signaling and determine complexes composition by identifying known and novel proteins in the isolated complexes. 2. Explore Ca2+ signaling complexes at the PM/ER junction - Coupling through ICRAC. For that we will determine if the newly discovered miniature Ca2+ channel Imin is ICRAC and use deletion mutations to identify the domain in IP3R that regulate Imin and the ICRAC analogue Htrp3. This domain will be used as a bait to identify its binding partner in Htrp3 and maybe to Imin. These studies will assess the validity of the coupling hypothesis to capacitative Ca2+ entry and the role of PM/ER junction in assembly of Ca2+ signaling complexes. 3. SERCA3 minus/minus mice and Ca2+ signaling complexes. An alternative approach to organization of Ca2+ signaling complexes is to probe the role of SERCA3 in Ca2+ signaling. To determine the mechanism of receptor-selective impaired Ca2+ signaling in SERCA3 minus/minus cells we will compare a) the biochemical, transport and spatiotemporal properties of Ca2+ signaling b) the coupling of m3 receptors in signaling complexes and c) storage and behavior of ER Ca2+ in WT and SERCA3 minus/minus cells. These studies will provide the ultimate proof for the autonomous functioning of Ca2+ signaling complexes. 4. Prob cellular microdomains with targeted cameleons. ER-cameleon and cameleons fused to the N and C termini of VAMP2 and Syncollin will be packaged into adenoviruses and infected into acinar cells. These constructs should probe dynamics of ER Ca2+ and the [Ca2+] next to signaling complexes to determine their role in local control of [Ca2+]. I believe that the tools developed in the previous grant period, the expertise of the personnel in my lab and the collaborations we established will allow us to achieve our goals and provide a new level of understanding of Ca2+ signaling of general relevance to cell signaling and to the function of exocrine cells.

DESCRIPTION (provided by applicant) Funds are sought to cover partial support of the Gordon Research Conference on Calcium Signaling, to be held at Oxford, UK, September 27, 2001. The meeting provides a unique multi-disciplinary forum for interchange of ideas and information in the field of Ca2,+ signaling. Since Ca2+ Signaling is a fundamental means of cellular control common to all eukaryotic organisms from yeast to mammalian cells, the meeting derives particular strength by stimulating the interchange of basic information on cell regulation from workers using a rich variety of different organisms and cellular systems. The primary objective is to facilitate successful interactions between new and established investigators. Highest priority will be given to the selection and provision of funds to support junior scientists (student and postdoctoral trainees), women scientists, under-represented minority scientists and international participation. The conference will include nine discussion sessions, each lead by a prominent member of the field with experience in maximizing interactive exchange. There will also be two poster sessions. The main sessions will emphasize the role of Ca2+ in controlling fundamental cell functions. These include the regulation of Ca2+ fluxes at the plasma membrane, the spatial and temporal control of Ca2+, signaling events, and the role of Ca2+ signaling in exocytosis, fertilization, cellular energetic and cell death. Participants include a combination of established and successful younger investigators using an array of cellular systems and different techniques to observe the generation and propagation of Ca2+ signals, ranging from molecular biological analysis, manipulation of the Ca2+ signaling machinery in model systems, in vivo, to sophisticated and pioneering micro- imaging technology in subcellular compartments. Basic disciplines of participants include biochemistry, physiology, cell biology, molecular biology, biophysics and pharmacology; however, all have a primary interest in understanding the mechanisms and significance of Ca2+ signaling events in cells. The topics to be addressed at this meeting are important to our understanding of a number of health problems, including issues related to environmental toxins that affect health, cancer biology, cardiac and neuronal functions, diabetes and aging to name a few.

DESCRIPTION (provided by applicant): The polarized nature of salivary gland functions requires polarized organization and function of signaling complexes. Understanding assembly of signaling proteins into complexes within cellular microdomains is the central theme of this proposal. Based on our preliminary findings of polarized expression of Ca2+ signaling proteins in secretory cells, expression of multiple P2Rs in a membrane limited manner in salivary gland cells, regulation of GPCR signaling by RGS proteins and the regulation of Icrac channels by IP3R we will test the central hypothesis by 1. Studying the spatial features of Ca2+ signaling by P2Rs and GPCR. Imaging of Fura2, BTC and Mg-Fura2 will be used to capture Ca2+ waves and gradients in SMG cells and correlate them with expression patterns of P2Rs and Ca2 about signaling complexes. Function of individual P2Rs will be probed further by characterizing ionic current activated by ATP and by partial P2R agonist and the sensitivity of these currents to P2Rs inhibitory Abs. 2. Determine interaction of RGS proteins with GPCR. This will be achieved by measuring the potency of several RGS proteins to inhibit Ca2+ signaling evoked by GPCR in SMG cells. RGS proteins specific antibodies and dominant negatives will be used to identify the active RGS proteins in SMG cells and their physiological role. Known constitutively active mutants of the alfa-1BAR will be used in an attempt to identify the site of interaction of RGS proteins with GPCR. 3. Explore Ca2+ signaling complexes at the PM/ER junction: icrac-IP3R complexes. Determine if Imin is Icrac and characterize single channel properties of Icrac in excised patches from native SMG cells. Determine whether Icrac in SMG cells is gated by the newly discovered conformational coupling mode. Study regulation of Icrac by the Homer family of scaffolding proteins in the context of their role in assembly of 1crac-IP3R and signaling complexes. I believe that the tools available in my lab for the proposed projects, the expertise of the personnel in my lab and the collaborations we established will allow us to achieve our goals and provide insights of general relevance to cell signaling and to the function of salivary gland cells.

DESCRIPTION (provided by applicant): Funds are sought to cover partial support of the Gordon Research Conference on Calcium Signaling to be held at Mount Holyoke College, South Hadley, Massachusetts, July 6-11. The meeting provides a unique multidisciplinary forum for exchange of ideas and information in the field of Ca>signaling, a fundamental form of cellular control common to all eukaryotic organisms, from yeast to mammals. A particular strength of the meeting is its ability to stimulate international interactions between workers using a variety of organisms and cellular systems and with interests that range from fundamental mechanisms to disease states. The primary objective is to facilitate successful interactions between new and established investigators. Highest priority will be given to the selection of, and provision of funds for, junior scientists (student and postdoctoral trainees), women scientists, under-represented minority scientists and international participants. The conference will include 8 discussion sessions, each lead by a prominent member of the field (established and young) with experience in maximizing interactive exchange. There will also be two poster sessions. The main sessions will emphasize the role of Ca 2+ in controlling fundamental cell functions. These include the regulation of Ca 2+ fluxes at the plasma membrane, the spatial and temporal control of Ca 2+ signaling events, and the roles of Ca 2+ signaling in sensory systems (olfaction, taste, smell, temperature), exocytosis, cellular energetics, cell death and cardiac and neuronal diseases. Chairs and speakers include female scientists (3/8 chairs and 6/24 speakers) and a combination of established and successful younger investigators. They represent an array of cellular systems and different techniques ranging from structural and molecular biological analyses and manipulation of the Ca2+ signaling machinery in model systems and in vivo, to pioneering micro-imaging technology in subcellular compartments to observe the generation and propagation of Ca2+ signals. Participants are drawn from backgrounds that include biochemistry, physiology, cell biology, molecular biology, biophysics and pharmacology; all share a primary interest in understanding the mechanisms and significance of Ca 2+ signaling in cells. The topics included at this meeting are both fundamentally important and directly relevant to an understanding of significant diseases, including cardiovascular, neurodegenerative and sensory diseases, ageing, diabetes, cancer, and the effects of environmental toxins on human health.

DESCRIPTION (provided by applicant): The polarized nature of pancreatic acinar cell functions require polarized organization and function of signaling complexes. Understanding assembly and regulation of signaling proteins within complexes in cellular microdomains is the central theme of this proposal. Based on the findings of polarized expression of Ca2+ signaling proteins in secretory cells, the discovery of direct regulation of IP3Rs channel activity by GBy, binding of RGS proteins and the third intracellular loop of GPCRs (31L) to the scaffolding protein spinophilin, the role of Homer proteins in mediated communication between TRPC channels and IP3Rs at the PM/ER junction and the mislocalization of Ca2+ transporting proteins and organelles in PERK1-/- and MISTI -/- mice, we will test the central hypothesis by I. Study gating of IP3Rs by G(( and its relevance to Ca 2+ signal in pancreatic acini. This new mode of Ca 2+ signaling by GPCRs will be examined by identifying the IP3Rs and G(( domains that mediate the regulation and the mechanism of their interaction. More important, the domains and peptides and mutations thereof will be used to determine the significance of this mode of Ca2+ signaling to Ca2+ signaling by pancreatic acini. 2. Study the role of spinophilin (SPL) in mediating communication between RGS proteins and GPCRs, This will be achieved by refining the SPL domains that interact with RGS proteins, the role of SPL in RGS proteins GAP activity and identify dominant negatives that interfere with the interaction of SPL with RGS proteins and GPCRs. These tools will be used to examine the role of SPL interactions in vivo. In a second approach, constitutively active and signaling impaired mutants in the 31L of the M3R will be used to further map the site of communication between RGS proteins and GPCR. 3. Study the role of Homer proteins in Ca 2+ signaling at the PM/ER junction. This will be achieved by examining the role of Homers in activation of TRPCI, TRPC3 and TRPC6 that are expressed in pancreatic acini by IP3Rs and by lipids. In another approach, Ca2+ signaling will be characterized in pancreatic acini of Homer I -/- mice. 4. Characterize Ca 2+ signaling in PERK1 -I-and MIST1-I- mice. Ca2+ transporting proteins and organelles are mislocalized in pancreatic acini of these mice, offering us the first opportunity to examine the role of assembly of Ca2+ signaling complexes in cellular microdomains in the initiation, propagation and generation of polarized Ca2+ waves. More important, the pancreas of the mutant mice develops with time morphological characteristics of pancreatitis. Hence, these mice may provide an animal model to study the role of aberrant Ca 2+ signaling in chronic pancreatitis. I believe that the tools available in my lab, the expertise of the personnel and the collaborations we established will allow us to achieve our goals and provide insights of general relevance to cell signaling and to the function and dysfunction of the pancreas.

[unreadable] DESCRIPTION (provided by applicant): Ca2+ signaling regulate fluid and electrolyte secretion by salivary gland (SG) cells. This polarized function of SG dictates polarized organization and functioning of Ca2+ signaling complexes in cellular microdomains. Scaffolding proteins plays critical roles in the assembly AND regulation of Ca2+ signaling proteins within the complexes. A central component of the Ca2+ signaling complexes is Ca2+ influx, which is mediated by TRPC channels. TRPC3 and TRPC6 are the dominant channels in SG. How the scaffolds Spinophilin (SPL), Neurabin (NRB) and Homerl regulates the action of GPCRs and TRPC3/6 channels is the theme of this proposal. Towards achieving our goals we found the regulation of Ca2+ signaling by the SPL/NRB pair, the role of Homerl in trafficking of TRPC channels, regulation of TRPC6 activity by SPL and by the newly discovered STIM1. These findings led to development of the following specific aims to probe the role of scaffolds in SG Ca2+ signaling. 1. Determine the role of SPL/NRB in regulating GPCRs Ca2+ signaling by RGS proteins in SG cells. This will be achieved by a) Identifying the NRB domain that binds RGS proteins and the relationship between SPL and NRB binding of RGS proteins, b) Determining the role of NRB in Ca2+ signaling in RGS2-/- and NRB-/- cells and c) Characterizing Ca2+ signaling in SG cells from SPL-/- and NRB-/- mice. 2. Explore the role of Homerl in TRPC channels translocation and Ca2+ influx by: a) studying translocation and retrieval of TRPC3/6 in SG cells and the role of store depletion and Homerl in both activities; b) Extend the findings in native cells by studying translocation of TRPC3/6-YFP expressed in HEK cells by biotinylation and TIRF assays. 3. Study regulation of TRPC3/6 by SPL/NRB by: a) Identifying the two SPL/NRB domains that interact with TRPC3/6; b) determine the effect of SPL/NRB in TRPC3/6 translocation and retrieval; c) characterize the regulation of TRPC3/6 channel activity by SPL/NRB in vivo using SPL-/- and NRB-/- cells. 4. Study regulation of TRPC3/6 by STIM1 by: a) determine the regulation of NATIVE and expressed TRP3/6 channels by STIM1, b) explore the mechanism by which STIM1 regulates TRPC3/6. The proposed work explores novel regulatory mechanisms in Ca2+ signaling and their relevance to regulation of SG fluid and electrolyte secretion. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The chief function of pancreatic acinar cells is the secretion of digestive enzymes and a small amount of isotonic fluid. Both functions are regulated by hormones and neurotransmitters that use Ca2+ as the second messenger. At the same time, aberrant Ca2+ signaling is the nodal point in all forms of pancreatitis. The hypothesis of this proposal is that specifically aberrant Ca2+ influx by store-operated Ca2+ channels (SOCs) is responsible for pancreatitis. Therefore, understanding regulation and function of Ca2+ influx channels is critical for understanding acinar cell function and dysfunction in the context of pancreatitis. Acinar cells have several Ca2+ influx channels, TRPC1, TRPC3, TRPC4, TRPC6 and Orai1, all of which are gated by the ER Ca2+ sensor STIM1. TRPCs are also gated by the scaffold Homer1 that inhibits their activity. Considering the involvement of SOCs in pancreatitis, the overall goal of this proposal is to understand gating mechanism of the pancreatic SOCs by STIM1 and Homer1 and their role in pancreatitis. This will be achieved in four aims: Aim 1: Decipher the molecular mechanism of TRPCs gating by STIM1 and Homer1. Homer1 interacts with a PPXF motif to keep the channels in a close state. STIM1 opens the TRPCs by interacting with two conserved residues that are only 4 aresidues upstream of the Homer1 binding motif. We propose to determine a) the gating mechanism of TRPCs by STIM1;b) how Homer1 and STIM1 function in tandem to gate the TRPCs. Aim 2: Determine molecular mechanism of Orai1 gating by STIM1. Agonist-stimulated pancreatic Ca2+ influx is mediated by a combination of TRPCs and Orai1 that are differentially gated by STIM1. We discovered that the STIM1(234-535) fragment is sufficient for full activation of Orai1. Hence we will: a) determine the minimal STIM1 domain that gates Orai1;b) how STIM1 opens Orai1;c) what is the relationship between the NATIVE TRPCs and NATIVE Orai1. Aim 3: The role of TRPCs, Orai1 and STIM1 in physiological and pathological acinar cells Ca2+ signaling and exocytosis will be determined by characterizing Ca2+ signaling and exocytosis in acinar cells from TRPC1-/-, TRPC3-/-, TRPC6-/-, Orai1-/- and STIM1-/- mice. Aim 4: The role of TRPC channels, Orai1, STIM1 will be studies in vitro-induced pancreatitis and measure cellular reporters of cell stress and cell damage. Then the KO mice will be used to evaluate the role of the Ca2+ influx channels in careluin and bile acid models of pancreatitis. PUBLIC HEALTH RELEVANCE: The pancreas secretes digestive enzymes and fluid in response to stimulation by hormones and neurotransmitters. The stimulants tell the pancreatic cells how to do so by changing the concentration of Ca2+ ions within the cells. Most of the Ca2+ enters the cells from the extracellular environment. When this process is aberrant, the digestive enzymes remain trapped within the cells and the cells dye and the patient develop the disease called pancreatitis. The aims of this proposal are to understand how Ca2+ ions enter the cells and how the aberrant Ca2+ entry causes the pancreatitis. This will be achieved by studying Ca2+ entry into the cells in model cell systems and in the cells of the pancreas of mice from which the genes that are responsible for the entry of Ca2+ into the cells have been deleted.

DESCRIPTION (provided by applicant): Fluid and electrolyte transport by the pancreatic duct is unique among secretory glands. The duct does not absorb Na and secrets nearly 140 mM HCO3-. Ductal function is critical for pancreatic health, as evident in CF and pancreatitis. Although the Cl function of CFTR is required for ductal secretion, recent findings show that CFTR is a global regulator of epithelial function by regulating several Cl- and HCO3- transporters that participate in duntal fluid and HCO3- secretion, in particular members of the SLC26 transporters family (SLC26Ts). Indeed, we have shown that a) the electrogenicity and isoform specific stoichiometry of the SLC26Ts, b) the function of the SLC26Ts in ductal HCO3- secretion, c) the mutual regulation of the SLCTs and CFTR, d) the interaction between the CFTR R domain and SLC26Ts STAS domain, e) the regulation of the SLC26Ts by the WNKs kinases. These findings led to a new hypothesis of ductal fluid and HCO3- secretion in which the bulk of HCO3- secretion is mediated by different SLC26Ts that are regulated by CFTR. In turn, the SLC26Ts supress CFTR activity in the resting state and enhance CFTR activity at the stimulated state. CFTR determines the final HCO3- concentration in the pancreatic juice by controlling the membrane potential of the duct. We will test this hypothesis in model systems and native pancreatic ducts of WT and slc26a6-/- mice and the role of HCO3- secretion in pancreatitis in four specific aims: 1. Determine the role of coserved Ser/Thr/Tyr phosphortlation sites suggested by the NMR structure od STAS-R domains in the STAS-R domain interaction and mutual regulation of CFTR and slc26a3/6. 2. Study regulation of slc26a3/6 by the WNKs kinases and in the reciprocal regulation of CFTR and slc26a3/6 in vitro and in vivo. 3. study the role of kinases and signaling pathways upstream of the WNKs and kinases downstrean of the WNKs in the regulation of slc26a3/6 in vivtro and in vivo. 4. Examine the role of slc6a6 and HCO3- secretion in pancreatitis. The slc26a6-/- mice in which HCO3- secretion is compromized, will be used to determine the role of HCO3- secretion in induction and progression of acute pancreatitis. The proposed work should advance our understanding of pancreatic duct fluid and HCO3- secretion and result in information relevant to pancreatitis and Cystic Fibrosis. Public Health Relevance: The pancreas secretes digestive enzymes and fluid that contains a lot of HCO3- that wash the enzymes to the intestine. The HCO3- is needed to prevent premature activation of the enzymes so that they will not digest the pancreas itself. When HCO3- secretion is aberrant the pancreas digests itself as occur in the diseases Cystic Fibrosis and pancreatitis. This work is aimed to understand how the pancreas secretes HCO3- and why HCO3- secretion is aberrant in pancreatitis and Cystic Fibrosis.

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.