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1.  The α2B adrenergic receptor is mutant in cortical myoclonus and epilepsy 
Annals of neurology  2014;75(1):77-87.
Objective
Autosomal dominant cortical myoclonus and epilepsy (ADCME) is characterized by distal, fairly rhythmic myoclonus and epilepsy with variable severity. We have previously mapped the disease locus on chromosome 2p11.1-q12.2 by genome-wide linkage analysis. Additional pedigrees affected by similar forms of epilepsy have been associated to chromosome 8q, 5p and 3q, but none of the causing genes has been identified. We aim at identifying the mutant gene responsible for this epileptic form.
Methods
Genes included in the ADCME critical region were prioritized and directly sequenced. Co-immunoprecipitation, immunofluorescence and electrophysiology approaches on transfected human cells have been utilized for testing the functional significance of the identified mutation.
Results
Here we show that mutation in the α2-adrenergic receptor subtype B (α2B-AR) associates to ADCME by identifying a novel in-frame insertion/deletion in two Italian families. The mutation alters several conserved residues of the third intracellular (3i) loop, neither hampering the α2B-AR plasma membrane localization nor the arrestin-mediated internalization capacity, but altering the binding with the scaffolding protein spinophilin upon neurotransmitter activation. Spinophilin, in turn, regulates interaction of GPCRs with Regulators of G proteins Signaling proteins. Accordingly, the mutant α2B-AR increases the epinephrine-stimulated calcium signaling.
Interpretation
The identified mutation is responsible for ADCME, as the loss of α2B-AR/spinophilin interaction causes a gain of function effect. This work implicates for the first time the α-adrenergic system in human epilepsy and opens new ways for understanding the molecular pathway of epileptogenesis, widening the spectrum of possible therapeutic targets.
doi:10.1002/ana.24028
PMCID: PMC3932827  PMID: 24114805
2.  Transient Receptor Potential Canonical Type 3 Channels Control the Vascular Contractility of Mouse Mesenteric Arteries 
PLoS ONE  2014;9(10):e110413.
Transient receptor potential canonical type 3 (TRPC3) channels are non-selective cation channels and regulate intracellular Ca2+ concentration. We examined the role of TRPC3 channels in agonist-, membrane depolarization (high K+)-, and mechanical (pressure)-induced vasoconstriction and vasorelaxation in mouse mesenteric arteries. Vasoconstriction and vasorelaxation of endothelial cells intact mesenteric arteries were measured in TRPC3 wild-type (WT) and knockout (KO) mice. Calcium concentration ([Ca2+]) was measured in isolated arteries from TRPC3 WT and KO mice as well as in the mouse endothelial cell line bEnd.3. Nitric oxide (NO) production and nitrate/nitrite concentrations were also measured in TRPC3 WT and KO mice. Phenylephrine-induced vasoconstriction was reduced in TRPC3 KO mice when compared to that of WT mice, but neither high K+- nor pressure-induced vasoconstriction was altered in TRPC3 KO mice. Acetylcholine-induced vasorelaxation was inhibited in TRPC3 KO mice and by the selective TRPC3 blocker pyrazole-3. Acetylcholine blocked the phenylephrine-induced increase in Ca2+ ratio and then relaxation in TRPC3 WT mice but had little effect on those outcomes in KO mice. Acetylcholine evoked a Ca2+ increase in endothelial cells, which was inhibited by pyrazole-3. Acetylcholine induced increased NO release in TRPC3 WT mice, but not in KO mice. Acetylcholine also increased the nitrate/nitrite concentration in TRPC3 WT mice, but not in KO mice. The present study directly demonstrated that the TRPC3 channel is involved in agonist-induced vasoconstriction and plays important role in NO-mediated vasorelaxation of intact mesenteric arteries.
doi:10.1371/journal.pone.0110413
PMCID: PMC4195735  PMID: 25310225
3.  Irbit Mediates Synergy Between Ca2+ and cAMP Signaling Pathways During Epithelial Transport in Mice 
Gastroenterology  2013;145(1):232-241.
Background & Aims
The cAMP and Ca2+ signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts.
Methods
We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6−/− mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit−/− mice). We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity by measuring Cl− current. Protein interactions were determined by immunoprecipitation analyses.
Results
Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca2+ and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit−/− or Slc26a6−/− mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation.
Conclusions
Irbit promotes synergy between the Ca2+ and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in CF, pancreatitis, or Sjögren’s syndrome.
doi:10.1053/j.gastro.2013.03.047
PMCID: PMC3696401  PMID: 23542070
signal transduction; ion and water secretion; fluid; electrolyte
4.  Aberrant CFTR-dependent HCO3- transport inmutations associated with cystic fibrosis 
Nature  2001;410(6824):94-97.
Cystic fibrosis (CF) is a disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Initially, Cl− conductance in the sweat duct was discovered to be impaired in CF1, a finding that has been extended to all CFTR-expressing cells2–4. Subsequent cloning of the gene5,6 showed that CFTR functions as a cyclic-AMP-regulated Cl− channel7; and some CF-causing mutations inhibit CFTR Cl− channel activity2–4,8. The identification of additional CF-causing mutants with normal Cl− channel activity indicates, however, that other CFTR-dependent processes contribute to the disease. Indeed, CFTR regulates other transporters3,4, including Cl−-coupled HCO3- transport9,10. Alkaline fluids are secreted by normal tissues, whereas acidic fluids are secreted by mutant CFTR-expressing tissues11, indicating the importance of this activity. HCO3- and pH affect mucin viscosity12,13 and bacterial binding14,15. We have examined Cl−-coupled HCO3- transport by CFTR mutants that retain substantial or normal Cl− channel activity. Here we show that mutants reported to be associated with CF with pancreatic insufficiency do not support HCO3- transport, and those associated with pancreatic sufficiency show reduced HCO3- transport. Our findings demonstrate the importance of HCO3- transport in the function of secretory epithelia and in CF.
doi:10.1038/35065099
PMCID: PMC3943212  PMID: 11242048
5.  Gating of CFTR by the STAS domain of SLC26 transporters 
Nature cell biology  2004;6(4):343-350.
Chloride absorption and bicarbonate secretion are vital functions of epithelia1–6, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR7, which activates electrogenic chloride-bicarbonate exchange by SLC26T8. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NPo) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.
doi:10.1038/ncb1115
PMCID: PMC3943213  PMID: 15048129
6.  The STIM1 CTID domain determines access of SARAF to SOAR to regulate Orai1 channel function 
The Journal of Cell Biology  2013;202(1):71-79.
Two distinct lobes in the C-terminal inhibitory domain in STIM1 determine access of the inhibitor SARAF to the activating SOAR domain to regulate the slow Ca2+-dependent inactivation of Orai1.
Ca2+ influx by store-operated Ca2+ channels (SOCs) mediates all Ca2+-dependent cell functions, but excess Ca2+ influx is highly toxic. The molecular components of SOC are the pore-forming Orai1 channel and the endoplasmic reticulum Ca2+ sensor STIM1. Slow Ca2+-dependent inactivation (SCDI) of Orai1 guards against cell damage, but its molecular mechanism is unknown. Here, we used homology modeling to identify a conserved STIM1(448–530) C-terminal inhibitory domain (CTID), whose deletion resulted in spontaneous clustering of STIM1 and full activation of Orai1 in the absence of store depletion. CTID regulated SCDI by determining access to and interaction of the STIM1 inhibitor SARAF with STIM1 Orai1 activation region (SOAR), the STIM1 domain that activates Orai1. CTID had two lobes, STIM1(448–490) and STIM1(490–530), with distinct roles in mediating access of SARAF to SOAR. The STIM1(448–490) lobe restricted, whereas the STIM1(490–530) lobe directed, SARAF to SOAR. The two lobes cooperated to determine the features of SCDI. These findings highlight the central role of STIM1 in SCDI and provide a molecular mechanism for SCDI of Orai1.
doi:10.1083/jcb.201301148
PMCID: PMC3704993  PMID: 23816623
7.  The intracellular Ca2+ channels of membrane traffic 
Channels  2012;6(5):344-351.
Regulation of organellar fusion and fission by Ca2+ has emerged as a central paradigm in intracellular membrane traffic. Originally formulated for Ca2+-driven SNARE-mediated exocytosis in the presynaptic terminals, it was later expanded to explain membrane traffic in other exocytic events within the endo-lysosomal system. The list of processes and conditions that depend on the intracellular membrane traffic includes aging, antigen and lipid processing, growth factor signaling and enzyme secretion. Characterization of the ion channels that regulate intracellular membrane fusion and fission promises novel pharmacological approaches in these processes when their function becomes aberrant. The recent identification of Ca2+ permeability through the intracellular ion channels comprising the mucolipin (TRPMLs) and the two-pore channels (TPCs) families pinpoints the candidates for the Ca2+ channel that drive intracellular membrane traffic. The present review summarizes the recent developments and the current questions relevant to this topic.
doi:10.4161/chan.21723
PMCID: PMC3508773  PMID: 22907062
organelles; TRPMLs; TPCs; calcium; membrane traffic; fusion; fission
8.  The WNK/SPAK and IRBIT/PP1 Pathways in Epithelial Fluid and Electrolyte Transport 
Physiology (Bethesda, Md.)  2012;27(5):291-299.
Fluid and electrolyte homeostasis is a fundamental physiological function required for survival and is associated with a plethora of diseases when aberrant. Systemic fluid and electrolyte composition is regulated by the kidney, and all secretory epithelia generate biological fluids with defined electrolyte composition by vectorial transport of ions and the obligatory water. A major regulatory pathway that immerged in the last several years is regulation of ion transporters by the WNK/SPAK kinases and IRBIT/PP1 pathways. The IRBIT/PP1 pathway functions to reverse the effects of the WNK/SPAK kinases pathway, as was demonstrated for NBCe1-B and CFTR. Since many transporters involved in fluid and electrolyte homeostasis are affected by PP1 and/or calcineurin, it is possible that WNK/SPAK and IRBIT/PP1 form a common regulatory pathway to tune the activity of fluid and electrolyte transport in response to physiological demands.
doi:10.1152/physiol.00028.2012
PMCID: PMC3686318  PMID: 23026752
9.  Molecular Mechanism of Pancreatic and Salivary Glands Fluid and HCO3− Secretion 
Physiological reviews  2012;92(1):39-74.
Fluid and HCO3− secretion is a vital function of all epithelia and is required for the survival of the tissue. Aberrant fluid and HCO3− secretion is associated with many epithelial diseases, such as cystic fibrosis, pancreatitis, Sjögren’s syndrome and other epithelial inflammatory and autoimmune diseases. Significant progress has been made over the last 20 years in our understanding of epithelial fluid and HCO3− secretion, in particular by secretory glands. Fluid and HCO3− secretion by secretory glands is a two step process. Acinar cells secrete isotonic fluid in which the major salt is NaCl. Subsequently, the duct modifies the volume and electrolyte composition of the fluid to absorb the Cl− and secrete HCO3−. The relative volume secreted by acinar and duct cells and modification of electrolyte composition of the secreted fluids varies among secretory glands to meet their physiological functions. In the pancreas, acinar cells secrete small amount of NaCl-rich fluid, while the duct absorbs the Cl− and secretes HCO3− and the bulk of the fluid in the pancreatic juice. Fluid secretion appears to be driven by active HCO3− secretion. In the salivary glands, acinar cells secrete the bulk of the fluid in the saliva that contains high concentrations of Na+ and Cl− and fluid secretion is mediated by active Cl− secretion. The salivary glands duct absorbs both the Na+ and Cl− and secretes K+ and HCO3−. In this review, we focus on the molecular mechanism of fluid and HCO3− secretion by the pancreas and salivary glands, to highlight the similarities of the fundamental mechanisms of acinar and duct cell functions, and point the differences to meet glands specific secretions.
doi:10.1152/physrev.00011.2011
PMCID: PMC3667394  PMID: 22298651
10.  TRPML: TRansPorters of Metals in Lysosomes essential for cell survival? 
Cell calcium  2011;50(3):288-294.
Key aspects of lysosomal function are affected by the ionic content of the lysosomal lumen and, therefore, by the ion permeability in the lysosomal membrane. Such functions include regulation of lysosomal acidification, a critical process in delivery and activation of the lysosomal enzymes, release of metals from lysosomes into the cytoplasm and the Ca2+-dependent component of membrane fusion events in the endocytic pathway. While the basic mechanisms of lysosomal acidification have been largely defined, the lysosomal metal transport system is not well understood. TRPML1 is a lysosomal ion channel whose malfunction is implicated in the lysosomal storage disease Mucolipidosis Type IV. Recent evidence suggests that TRPML1 is involved in Fe2+, Ca2+ and Zn2+ transport across the lysosomal membrane, ascribing novel physiological roles to this ion channel, and perhaps to its relatives TRPML2 and TRPML3 and illuminating poorly understood aspects of lysosomal function. Further, alterations in metal transport by the TRPMLs due to mutations or environmental factors may contribute to their role in the disease phenotype and cell death.
doi:10.1016/j.ceca.2011.04.009
PMCID: PMC3164942  PMID: 21621258
11.  Genetic and Pharmacological Inhibition of the Ca2+ Influx Channel TRPC3 Protects Secretory Epithelia from Ca2+-Dependent Toxicity 
Gastroenterology  2011;140(7):2107-2115.e4.
Background & Aims
Excessive Ca2+ influx mediates many cytotoxic processes, including those associated with autoimmune inflammatory diseases such as acute pancreatitis and Sjögren's syndrome. TRPC3 is a major Ca2+ influx channel in pancreatic and salivary gland cells. We investigated whether genetic or pharmacological inhibition of TRPC3 protects pancreas and salivary glands from Ca2+-dependent damage.
Methods
We developed a Ca2+-dependent model of cell damage for salivary gland acini. Acute pancreatitis was induced by injection of cerulein into wild-type and Trpc3−/− mice. Mice were also given the Trpc3-selective inhibitor pyrazole 3 (Pyr3).
Results
Salivary glands and pancreas of Trpc3−/− mice were protected from Ca2+-mediated cell toxicity. Analysis of Ca2+ signaling in wild-type and Trpc3−/− acini showed that Pyr3 is highly specific inhibitor of Tprc3; it protected salivary glands and pancreas cells from Ca2+-mediated toxicity by inhibiting the Trpc3-mediated component of Ca2+ influx.
Conclusions
TRPC3-mediated Ca2+ influx mediates damage to pancreas and salivary glands. Pharmacological inhibition of TRPC3 with the highly selective TRPC3 inhibitor Pyr3 might be developed for treatment of patients with acute pancreatitis and Sjögren's syndrome.
doi:10.1053/j.gastro.2011.02.052
PMCID: PMC3109139  PMID: 21354153
Ca2+ influx; inflammation; cell death; therapeutics
12.  A role for the Ca2+ channel TRPML1 in gastric acid secretion, based on analysis of knockout mice 
Gastroenterology  2010;140(3):857-867.e1.
Background & Aims
Mutations in TRPML1, a lysosomal Ca2+-permeable TRP channel, lead to mucolipidosis type IV (MLIV), a neurodegenerative lysosomal storage disease. An unusual feature of MLIV is constitutive achlorhydria. We produced Trpml1−/− (null) mice to investigate the requirement for this protein in gastric acid secretion.
Methods
Trpml1-null mice were generated by gene targeting. The expression of Trpml1 and its role in acid secretion by gastric parietal cells were analyzed using biochemical, histological, and ultrastructural approaches.
Results
Trpml1 is expressed by parietal cells and localizes predominantly to the lysosomes; it was dynamically palmitoylated and dephosphorylated in vivo following histamine stimulation of acid secretion. Trpml1-null mice had significant impairments in basal and histamine-stimulated gastric acid secretion and markedly reduced levels of the gastric proton pump. Histological and ultrastructural analyses revealed that Trpml1−/− parietal cells were enlarged, had multivesicular and multi-lamellated lysosomes, and maintained an abnormal intracellular canalicular membrane. The intralysosomal Ca2+ content and receptor-mediated Ca2+ signaling were, however, unaffected in Trpml1−/− gastric glands, indicating that Trpml1 does not function in the regulation of lysosomal Ca2+.
Conclusions
Loss of Trpml1 causes reduced levels and mislocalization of the gastric proton pump and alters the secretory canaliculi, causing hypochlorhydria and hypergastrinemia. The lysosomal enlargement and defective intracellular canaliculi formation observed in Trpml1−/− parietal cells indicate that Trpml1 functions in the formation and trafficking of the tubulovesicles. This study provides direct evidence for the regulation of gastric acid secretion by a TRP channel; TRPML1 is an important protein in parietal cell apical-membrane trafficking.
doi:10.1053/j.gastro.2010.11.040
PMCID: PMC3057336  PMID: 21111738
Apical membrane trafficking; Mucolipin-1; Vesicles; Stomach
13.  Polarized but differential localization and recruitment of STIM1/Orai1 and STIM1/TRPC channels in secretory cells 
Traffic (Copenhagen, Denmark)  2010;12(2):232-245.
Polarized Ca2+ signals in secretory epithelial cells are determined by compartmentalized localization of Ca2+ signaling proteins at the apical pole. Recently the ER Ca2+ sensor STIM1 and the Orai channels were shown to play a critical role in store-dependent Ca2+ influx. STIM1 also gates the TRPC channels. Here, we asked how cell stimulation affects the localization, recruitment and function of the native proteins in polarized cells. Inhibition of Orai1, STIM1, or deletion of TRPC1 reduces Ca2+ influx and frequency of Ca2+ oscillations. Orai1 localization is restricted to the apical pole of the lateral membrane. Surprisingly, cell stimulation does not lead to robust clustering of native Orai1, as is observed with expressed Orai1. Unexpectedly, cell stimulation causes polarized recruitment of native STIM1 to both the apical and lateral regions, thus to regions with and without Orai1. Accordingly, STIM1 and Orai1 show only 40% co-localization. Consequently, STIM1 shows higher co-localization with the basolateral membrane marker E-cadherin than does Orai1, while Orai1 showed higher co-localization with the tight junction protein ZO1. 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. These findings reveal that in addition to Orai1, STIM1 likely regulates other Ca2+ permeable channels, such as the TRPCs. Both channels contribute to the frequency of [Ca2+] oscillations and thus impact critical cellular functions.
doi:10.1111/j.1600-0854.2010.01138.x
PMCID: PMC3021582  PMID: 21054717
STIM1; Orai1; TRPC1; polarized; recruitment; epithelial cells
14.  Determinants of coupled transport and uncoupled current by the electrogenic SLC26 transporters 
The Journal of General Physiology  2011;137(2):239-251.
Members of the SLC26 family of anion transporters mediate the transport of diverse molecules ranging from halides to carboxylic acids and can function as coupled transporters or as channels. A unique feature of the two members of the family, Slc26a3 and Slc26a6, is that they can function as both obligate coupled and mediate an uncoupled current, in a channel-like mode, depending on the transported anion. To identify potential features that control the two modes of transport, we performed in silico modeling of Slc26a6, which suggested that the closest potential fold similarity of the Slc26a6 transmembrane domains is to the CLC transporters, despite their minimal sequence identity. Examining the predicted Slc26a6 fold identified a highly conserved glutamate (Glu−; Slc26a6(E357)) with the predicted spatial orientation similar to that of the CLC-ec1 E148, which determines coupled or uncoupled transport by CLC-ec1. This raised the question of whether the conserved Glu− in Slc26a6(E357) and Slc26a3(E367) have a role in the unique transport modes by these transporters. Reversing the Glu− charge in Slc26a3 and Slc26a6 resulted in the inhibition of all modes of transport. However, most notably, neutralizing the charge in Slc26a6(E357A) eliminated all forms of coupled transport without affecting the uncoupled current. The Slc26a3(E367A) mutation markedly reduced the coupled transport and converted the stoichiometry of the residual exchange from 2Cl−/1HCO3− to 1Cl−/1HCO3−, while completely sparing the current. These findings suggest the possibility that similar structural motif may determine multiple functional modes of these transporters.
doi:10.1085/jgp.201010531
PMCID: PMC3032377  PMID: 21282402
15.  IRBIT: It Is Everywhere 
Neurochemical research  2010;36(7):1166-1174.
IRBIT (IP3Rs binding protein released with IP3) is a protein originally identified by the Mikoshiba group as an inhibitor of IP3 receptors function. Subsequently it was found to have multiple functions and regulate the activity of diverse proteins, including regulation of HCO2− transporters to coordinate epithelial HCO3− secretion and to determine localization of the Fip1 subunit of the CPSF complex to regulate mRNA processing. This review highlights the remarkably divers functions of IRBIT that are likely only a fraction of all the potential functions of this protein.
doi:10.1007/s11064-010-0353-6
PMCID: PMC3112469  PMID: 21152975
IRBIT; IP3 receptors; NBCe1-B; CFTR; NHE3; Fip1
16.  An Endoplasmic Reticulum/Plasma Membrane Junction: STIM1/Orai1/TRPCs 
FEBS letters  2009;584(10):2022-2027.
Summary
Ca2+ entering cells through store-operated channels (SOCs) affects most cell functions, and excess SOC is associated with pathologies. The molecular makeup of SOCs and their mechanisms of gating were clarified with the discovery of the Orais and STIM1. Another form of SOCs are the TRPCs. STIM1 gates both Orai and TRPC channels but does so by different mechanisms. Although the STIM1 SOAR domain mediates the binding of STIM1 to both channel types, SOAR is sufficient to open the Orais but the STIM1 polylysine domain mediates opening of the TRPC channels. This short review discusses recent findings on how STIM1 gates and regulates the Orais and TRPCs, and how the STIM1/Orai1/TRPCs complexes may function in vivo to mediate SOC activity.
doi:10.1016/j.febslet.2009.11.078
PMCID: PMC2866752  PMID: 19944100
Orai; TRPC; channels; STIM1; gating
17.  Opening the TRPML Gates 
Chemistry & biology  2010;17(3):209-210.
The intracellular TRPML channels have multiple biological roles, but the physiological stimuli that open them remained unknown. In a previous issue of Chemistry & Biology, Grimm et al. report a high-throughput chemical screen that identified a plethora of selective activators of TRPML3 that should open the way to fully characterize these channels and their physiological roles.
doi:10.1016/j.chembiol.2010.02.009
PMCID: PMC2857704  PMID: 20338511
18.  Corticosteroids Correct Aberrant CFTR Localization in the Duct and Regenerate Acinar Cells in Autoimmune Pancreatitis 
Gastroenterology  2010;138(5):1988-1996.
Background & Aims
Corticosteroids are now widely accepted as a treatment for autoimmune pancreatitis (AIP). However, the molecular mechanism by which steroid treatment improves AIP remains largely unknown. The aim of this study was to elucidate cellular mechanisms by which corticosteroids improve both pancreatic exocrine function and histopathology in AIP.
Methods
Pancreatic exocrine function was evaluated by the secretin-stimulated function test and pancreatic biopsy specimens were processed for histologic analysis at the time of diagnosis and 3 months after initiation of steroid treatment. Expression and localization of proteins was assayed by immunohistochemistry. Analysis of immunoglobulin (Ig)G4-positive plasma cells was used to verify inflammation in AIP.
Results
The number of IgG4-positive plasma cells in pancreatic sections was decreased by steroid treatment, indicating reduced inflammation. Fluid, bicarbonate (HCO3−), and digestive enzyme secretions all were impaired in most patients with AIP. Corticosteroids improved both HCO3− and digestive enzyme secretion. A large fraction of the cystic fibrosis transmembrane conductance regulator (CFTR), which plays a central role in pancreatic duct HCO3− secretion, was mislocalized to the cytoplasm of duct cells before treatment. Corticosteroids corrected the localization of CFTR to the apical membrane, accounting for the improved HCO3− secretion. Steroid treatment resulted in regeneration of acinar cells, accounting for restored digestive enzyme secretion.
Conclusions
Corticosteroids reduce inflammation and restore both digestive enzyme and HCO3− secretion in patients with AIP by regenerating acinar cells and correcting CFTR localization in pancreatic duct cells. Mislocalization of CFTR may explain aberrant HCO3− secretion in other forms of pancreatitis.
doi:10.1053/j.gastro.2010.01.001
PMCID: PMC3061436  PMID: 20080093
Exocrine Function Test; CFTR; Aquaporin Water Channel; CD133
19.  IRBIT governs epithelial secretion in mice by antagonizing the WNK/SPAK kinase pathway 
Fluid and HCO3– secretion are fundamental functions of epithelia and determine bodily fluid volume and ionic composition, among other things. Secretion of ductal fluid and HCO3– in secretory glands is fueled by Na+/HCO3– cotransport mediated by basolateral solute carrier family 4 member 4 (NBCe1-B) and by Cl–/HCO3– exchange mediated by luminal solute carrier family 26, member 6 (Slc26a6) and CFTR. However, the mechanisms governing ductal secretion are not known. Here, we have shown that pancreatic ductal secretion in mice is suppressed by silencing of the NBCe1-B/CFTR activator inositol-1,4,5-trisphosphate (IP3) receptor–binding protein released with IP3 (IRBIT) and by inhibition of protein phosphatase 1 (PP1). In contrast, silencing the with-no-lysine (WNK) kinases and Ste20-related proline/alanine-rich kinase (SPAK) 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. Silencing of SPAK and IRBIT in the same ducts rescued ductal secretion due to silencing of IRBIT alone. 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.
doi:10.1172/JCI43475
PMCID: PMC3049373  PMID: 21317537
20.  Aberrant Ca2+ handling in lysosomal storage disorders 
Cell calcium  2010;47(2):103-111.
Lysosomal storage diseases (LSDs) are caused by inability of cells to process the material captured during endocytosis. While they are essentially diseases of cellular “indigestion”, LSDs affect large number of cellular activities and, as such, they teach us about the integrative function of the cell, as well as about the gaps in our knowledge of the endocytic pathway and membrane transport. The present review summarizes recent findings on Ca2+ handling in LSDs and attempts to identify the key questions on alterations inCa2+ signaling and membrane transport in this group of diseases, answers to which may lie in delineating the cellular pathogeneses of LSDs.
doi:10.1016/j.ceca.2009.12.007
PMCID: PMC2838446  PMID: 20053447
21.  The Ca2+ Channel TRPML3 Regulates Membrane Trafficking and Autophagy 
Traffic (Copenhagen, Denmark)  2009;10(8):1157-1167.
TRPML3 is an inward rectifying Ca2+ channel that is regulated by extracytosolic H+. Although gain-of-function mutation in TRPML3 causes the varitint-waddler phenotype, the role of TRPML3 in cellular physiology is not known. Here, 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 knock-down 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 Ca2+ in the vicinity of cellular organelles that is necessary to regulate these cellular events.
doi:10.1111/j.1600-0854.2009.00924.x
PMCID: PMC2993507  PMID: 19522758
TRPML3; Ca2+ channel; recycles; membrane trafficking; autophagy
22.  Peptidyl-prolyl isomerase FKBP52 controls chemotropic guidance of neuronal growth cones via regulation of TRPC1 channel opening 
Neuron  2009;64(4):471-483.
Summary
Immunophilins, including FK506-binding proteins (FKBPs), are protein chaperones with peptidyl-prolyl isomerase (PPIase) activity. Initially identified as pharmacological receptors for immunosuppressants to regulate immune responses via isomerase independent mechanisms, FKBPs are most highly expressed in the nervous system where their physiological function as isomerases remains unknown. We demonstrate that FKBP12 and FKBP52 catalyze cis/trans isomerization of regions of TRPC1 implicated in controlling channel opening. FKBP52 mediates stimulus-dependent TRPC1 gating through isomerization, which is required for chemotropic turning of neuronal growth cones to netrin-1 and myelin-associated glycoprotein and for netrin-1/DCC-dependent midline axon guidance of commissural interneurons in the developing spinal cord. By contrast, FKBP12 mediates spontaneous opening of TRPC1 through isomerization and is not required for growth cone responses to netrin-1. Our study demonstrates a novel physiological function of proline isomerases in chemotropic nerve guidance through TRPC1 gating and may have significant implication in clinical applications of immunophilin-related therapeutic drugs.
doi:10.1016/j.neuron.2009.09.025
PMCID: PMC2786904  PMID: 19945390
23.  Deletion of TRPC3 in mice reduces Store-Operated Ca2+ influx and the severity of acute pancreatitis 
Gastroenterology  2009;137(4):1509-1517.
Background and Aims
Receptor–stimulated Ca2+ influx is a critical component of the Ca2+ signal and mediates all cellular functions regulated by Ca2+. However, excessive Ca2+ influx is highly toxic resulting in cell death, which is the nodal point in all forms of pancreatitis. Ca2+ influx is mediated by store-operated channels (SOCs). The identity and function of the native SOCs in most cells is unknown.
Methods
Here, we determine the role of deletion of Trpc3 in mice on Ca2+ signaling, exocytosis, intracellular trypsin activation and pancreatitis.
Results
Deletion of TRPC3 reduced the receptor-stimulated and SOCs-mediated Ca2+ influx by about 50%, indicating that TRPC3 functions as SOC in vivo. The reduced Ca2+ influx in TRPC3−/− acini resulted in reduced frequency of the physiological Ca2+ oscillations and of the pathological sustained [Ca2+]i increase caused by supramaximal stimulation and by the toxins bile acids and palmitoleic acid ethyl ester. Consequently, deletion of TRPC3 shifted the dose response for receptor-stimulated exocytosis, and prevented the pathological inhibition of digestive enzyme secretion at supramaximal agonist concentrations. Accordingly, deletion of TRPC3 markedly reduced intracellular trypsin activation and excessive actin depolymerization in vitro and the severity of pancreatitis in vivo.
Conclusions
These findings establish the native TRPC3 as a SOC in vivo and a role for TRPC3-mediated Ca2+ influx in the pathogenesis of acute pancreatitis and suggest that TRPC3 should be considered a target for prevention of the pancreatic damage in acute pancreatitis.
doi:10.1053/j.gastro.2009.07.042
PMCID: PMC2757493  PMID: 19622358
24.  STIM1 gates TRPC channels but not Orai1 by electrostatic interaction 
Molecular cell  2008;32(3):439-448.
Summary
The receptor-evoked Ca2+ signal includes activation of the store-operated channels (SOCs) TRPC and Orai channels. Although both are gated by STIM1, it is not known how STIM1 gates the channels and whether STIM1 gates the TRPCs and Orais by the same mechanism. Here, we report the molecular mechanism by which STIM1 gates TRPC1, which involves interaction between two conserved, negatively charged aspartates in TRPC1(639DD640) with the positively charged STIM1(684KK685) in STIM1 polybasic domain. Charge swapping and functional analysis revealed that exact orientation of the charges on TRPC1 and STIM1 are required, but all positive-negative charge combinations on TRPC1 and STIM1, except STIM1(684EE685)+TRPC1(639RR640), are functional as long as they are reciprocal, indicating that STIM1 gates TRPC1 by intermolecular electrostatic interaction. Similar gating was observe with TRPC3(697DD698). STIM1 gates Orai1 by a different mechanism since the polybasic and S/P domains of STIM1 are not required for activation of Orai1 by STIM1.
doi:10.1016/j.molcel.2008.09.020
PMCID: PMC2586614  PMID: 18995841
25.  TRPC channels as STIM1-regulated store-operated channels 
Cell calcium  2007;42(2):205-211.
Receptor-activated Ca2+ influx is mediated largely by store-operated channels (SOCs). TRPC channels mediate a significant portion of the receptor-activated Ca2+ influx. However, whether any of the TRPC channels function as a SOC remains controversial. Our understanding of the regulation of TRPC channels and their function as SOCs is being reshaped with the discovery of the role of STIM1 in the regulation of Ca2+ influx channels. The findings that STIM1 is an ER resident Ca2+ binding protein that regulates SOCs allow an expanded and molecular definition of SOCs. SOCs can be considered as channels that are regulated by STIM1 and require the clustering of STIM1 in response to depletion of the ER Ca2+ stores and its translocation towards the plasma membrane. TRPC1 and other TRPC channels fulfill these criteria. STIM1 binds to TRPC1, TRPC2, TRPC4 and TRPC5 but not to TRPC3, TRPC6 and TRPC7, and STIM1 regulates TRPC1 channel activity. Structure-function analysis reveals that the C-terminus of STIM1 contains the binding and gating function of STIM1. The ERM domain of STIM1 binds to TRPC channels and a lysine-rich region participates in the gating of SOCs and TRPC1. Knock-down of STIM1 by siRNA and prevention of its translocation to the plasma membrane inhibit the activity of native SOCs and TRPC1. These findings support the conclusion that TRPC1 is a SOC. Similar studies with other TRPC channels should further clarify their regulation by STIM1 and function as SOCs.
doi:10.1016/j.ceca.2007.03.004
PMCID: PMC2764400  PMID: 17517433

Results 1-25 (38)