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.
organelles; TRPMLs; TPCs; calcium; membrane traffic; fusion; fission
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.
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.
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.
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.
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).
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.
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.
Ca2+ influx; inflammation; cell death; therapeutics
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.
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.
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+.
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.
Apical membrane trafficking; Mucolipin-1; Vesicles; Stomach
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.
STIM1; Orai1; TRPC1; polarized; recruitment; epithelial cells
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.
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.
IRBIT; IP3 receptors; NBCe1-B; CFTR; NHE3; Fip1
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.
Orai; TRPC; channels; STIM1; gating
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.
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.
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.
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.
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.
Exocrine Function Test; CFTR; Aquaporin Water Channel; CD133
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.
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.
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.
TRPML3; Ca2+ channel; recycles; membrane trafficking; autophagy
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.
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.
Here, we determine the role of deletion of Trpc3 in mice on Ca2+ signaling, exocytosis, intracellular trypsin activation and pancreatitis.
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.
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.
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.
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.
Store-operated Ca2+ influx is mediated by store002Doperated Ca2+ channels (SOCs) and is a central component of receptor-evoked Ca2+ signals1. The Orai channels mediate SOCs2–4 and STIM1 is the ER-resident Ca2+ sensor that gates the channels5, 6. How STIM1 gates and regulates the Orai channels is unknown. Here, we report the molecular basis for gating of Orais by STIM1. All Orai channels are fully activated by the conserved STIM1(344–442), which we termed SOAR (the STIM1 Orai Activating Region). SOAR acts in combination with STIM1(450–485) to regulate the strength of interaction with Orai1. Orai1 activated by SOAR recapitulates all the entire kinetic properties of Orai1 activated by STIM1. Mutations of STIM1 within SOAR prevent activation of Orai1 without preventing co-clustering of STIM1 and Orai1 in response to Ca2+ store depletion, indicating that STIM1-Orai1 co-clustering is not sufficient for Orai1 activation. An intact C-terminus α-helicial region of Orai is required for activation by SOAR. Deleting most of Orai1 N terminus impaired Orai1 activation by STIM1, but (Δ1–73)Orai1 interacts with and is fully activated by SOAR. Accordingly, the characteristic inward rectification of Orai is mediated by an interaction between the polybasic STIM1(672–685) and a proline-rich region in the N terminus of Orai1. Hence, the essential properties of Orai1 function can be rationalized by interactions with discrete regions of STIM1.
Lysosomal storage diseases (LSDs) are a class of genetic disorders in which proteins responsible for digestion or absorption of endocytosed material do not function or do not localize properly. The resulting cellular “indigestion” causes buildup of intracellular storage inclusions that contain unprocessed lipids and proteins that form macromolecular complexes. The buildup of storage material is associated with degenerative processes that are observed in all LSDs, albeit the correlation between the amount of storage inclusions and the severity of the degenerative processes is not always evident. The latter suggests that a specific mechanism set in motion by aberrant lysosomal function drives the degenerative processes in LSDs. It is becoming increasingly clear that in addition to their function in degrading endocytosed material, lysosomes are essential housekeeping organelles responsible for maintaining healthy population of intracellular organelles, in particular mitochondria. The present review surveys the current knowledge on the lysosomal-mitochondrial axis and its possible role as a contributing factor to mitochondrial Ca2+ homeostasis and to cell death in LSDs.
Stromal interacting molecule 1 (STIM1) is a Ca2+ sensor that conveys the Ca2+ load of the endoplasmic reticulum to store-operated channels (SOCs) at the plasma membrane. Here, we report that STIM1 binds TRPC1, TRPC4 and TRPC5 and determines their function as SOCs. Inhibition of STIM1 function inhibits activation of TRPC5 by receptor stimulation, but not by La3+, suggesting that STIM1 is obligatory for activation of TRPC channels by agonists, but STIM1 is not essential for channel function. Through a distinct mechanism, STIM1 also regulates TRPC3 and TRPC6. STIM1 does not bind TRPC3 and TRPC6, and regulates their function indirectly by mediating the heteromultimerization of TRPC3 with TRPC1 and TRPC6 with TRPC4. TRPC7 is not regulated by STIM1. We propose a new definition of SOCs, as channels that are regulated by STIM1 and require the store depletion-mediated clustering of STIM1. By this definition, all TRPC channels, except TRPC7, function as SOCs.
Fluid and HCO3– secretion are vital functions of secretory epithelia. In most epithelia, this entails HCO3– entry at the basolateral membrane, mediated by the Na+-HCO3– cotransporter, pNBC1, and exit at the luminal membrane, mediated by a CFTR-SLC26 transporters complex. Here we report that the protein IRBIT (inositol-1,4,5-trisphosphate [IP3] receptors binding protein released with IP3), a previously identified activator of pNBC1, activates both the basolateral pNBC1 and the luminal CFTR to coordinate fluid and HCO3– secretion by the pancreatic duct. We used video microscopy and ion selective microelectrodes to measure fluid secretion and Cl– and HCO3– concentrations in cultured murine sealed intralobular pancreatic ducts. Short interference RNA–mediated knockdown of IRBIT markedly inhibited ductal pNBC1 and CFTR activities, luminal Cl– absorption and HCO3– secretion, and the associated fluid secretion. Single-channel measurements suggested that IRBIT regulated CFTR by reducing channel mean close time. Furthermore, expression of IRBIT constructs in HEK cells revealed that activation of pNBC1 required only the IRBIT PEST domain, while activation of CFTR required multiple IRBIT domains, suggesting that IRBIT activates these transporters by different mechanisms. These findings define IRBIT as a key coordinator of epithelial fluid and HCO3– secretion and may have implications to all CFTR-expressing epithelia and to cystic fibrosis.
Homers are scaffolding proteins that bind Ca2+ signaling proteins in cellular microdomains. The Homers participate in targeting and localization of Ca2+ signaling proteins in signaling complexes. However, recent work showed that the Homers are not passive scaffolding proteins, but rather they regulate the activity of several proteins within the Ca2+ signaling complex in an isoform specific manner. Homer2 increases the GAP activity of RGS proteins and PLCβ that accelerate the GTPase activity of Gα subunits. Homer1 gates the activity of TRPC channels, controls the rates of their translocation and retrieval from the plasma membrane and mediates the conformational coupling between TRPC channels and IP3Rs. Homer1 stimulates the activity of the cardiac and neuronal L-type Ca2+ channels Cav1.2 and Cav1.3. Homer1 also mediates the communication between the cardiac and smooth muscle ryanodine receptor RyR2 and Cav1.2 to regulate E–C coupling. In many cases the Homers function as a buffer to reduce the intensity of Ca2+ signaling and create a negative bias that can be reversed by the immediate early gene form of Homer 1. Hence, the Homers should be viewed as the buffers of Ca2+ signaling that ensure a high spatial and temporal fidelity of the Ca2+ signaling and activation of downstream effects.
Our understanding of magnesium (Mg2+) regulation has recently been catapulted forward by the discovery of several disease loci for monogenic disorders of Mg2+ homeostasis. In this issue of the JCI, Groenestege et al. report that their study of a rare inherited Mg2+ wasting disorder in consanguineous kindred shows that EGF acts as an autocrine/paracrine magnesiotropic hormone (see the related article beginning on page 2260). EGF stimulates Mg2+ reabsorption in the renal distal convoluted tubule (DCT) via engagement of its receptor on the basolateral membrane of DCT cells and activation of the Mg2+ channel TRPM6 (transient receptor potential cation channel, subfamily M, member 6) in the apical membrane. These authors show that a point mutation in pro-EGF retains EGF secretion to the apical but not the basolateral membrane, disrupting this cascade and causing renal Mg2+ wasting. This work is another seminal example of the power of the study of monogenic disorders in the quest to understand human physiology.