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Ca release from the endoplasmic reticulum is an important component of Ca signal transduction that controls numerous physiological processes in eukaryotic cells. Release of Ca from the endoplasmic reticulum is coupled to the activation of store-operated Ca entry into cells. Store-operated Ca entry provides Ca for replenishing depleted endoplasmic reticulum Ca stores and a Ca signal that regulates Ca-dependent intracellular biochemical events. Central to connecting discharge of endoplasmic reticulum Ca stores following G protein-coupled receptor activation with the induction of store-operated Ca entry are stromal interaction molecules (STIM1 and STIM2). These highly homologous endoplasmic reticulum transmembrane proteins function as sensors of the Ca concentration within the endoplasmic reticulum lumen and activators of Ca release-activated Ca channels. Emerging evidence indicates that in addition to their role in Ca release-activated Ca channel gating and store-operated Ca entry, STIM1 and STIM2 regulate other cellular signaling events. Recent studies have shown that disruption of STIM expression and function is associated with the pathogenesis of several diseases including autoimmune disorders, cancer, cardiovascular disease, and myopathies. Here, we provide an overview of the latest developments in the molecular physiology and pathophysiology of STIM1 and STIM2. Impact statement Intracellular Ca signaling is a fundamentally important regulator of cell physiology. Recent studies have revealed that Ca-binding stromal interaction molecules (Stim1 and Stim2) expressed in the membrane of the endoplasmic reticulum (ER) are essential components of eukaryote Ca signal transduction that control the activity of ion channels and other signaling effectors present in the plasma membrane. This review summarizes the most recent information on the molecular physiology and pathophysiology of stromal interaction molecules. We anticipate that the work presented in our review will provide new insights into molecular interactions that participate in interorganelle signaling crosstalk, cell function, and the pathogenesis of human diseases.
Excessive glucocorticoid exposure has been shown to be deleterious for pancreatic β-cell function and insulin release. However, glucocorticoids at physiological levels are essential for many homeostatic processes, including glycemic control. We show that corticosterone and cortisol and their less active precursors 11-dehydrocorticosterone (11-DHC) and cortisone suppress voltage-dependent Ca channel function and Ca fluxes in rodent as well as in human β-cells. However, insulin secretion, maximal ATP/ADP responses to glucose, and β-cell identity were all unaffected. Further examination revealed the upregulation of parallel amplifying cAMP signals and an increase in the number of membrane-docked insulin secretory granules. Effects of 11-DHC could be prevented by lipotoxicity and were associated with paracrine regulation of glucocorticoid activity because global deletion of 11β-hydroxysteroid dehydrogenase type 1 normalized Ca and cAMP responses. Thus, we have identified an enzymatically amplified feedback loop whereby glucocorticoids boost cAMP to maintain insulin secretion in the face of perturbed ionic signals. Failure of this protective mechanism may contribute to diabetes in states of glucocorticoid excess, such as Cushing syndrome, which are associated with frank dyslipidemia.
© 2017 by the American Diabetes Association.
Amyloid beta (Aβ) peptides impair multiple cellular pathways and play a causative role in Alzheimer's disease (AD) pathology, but how the brain proteome is remodeled by this process is unknown. To identify protein networks associated with AD-like pathology, we performed global quantitative proteomic analysis in three mouse models at young and old ages. Our analysis revealed a robust increase in Apolipoprotein E (ApoE) levels in nearly all brain regions with increased Aβ levels. Taken together with prior findings on ApoE driving Aβ accumulation, this analysis points to a pathological dysregulation of the ApoE-Aβ axis. We also found dysregulation of protein networks involved in excitatory synaptic transmission. Analysis of the AMPA receptor (AMPAR) complex revealed specific loss of TARPγ-2, a key AMPAR-trafficking protein. Expression of TARPγ-2 in hAPP transgenic mice restored AMPA currents. This proteomic database represents a resource for the identification of protein alterations responsible for AD.
Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Neuronal excitation can induce new mRNA transcription, a phenomenon called excitation-transcription (E-T) coupling. Among several pathways implicated in E-T coupling, activation of voltage-gated L-type Ca channels (LTCCs) in the plasma membrane can initiate a signaling pathway that ultimately increases nuclear CREB phosphorylation and, in most cases, expression of immediate early genes. Initiation of this long-range pathway has been shown to require recruitment of Ca-sensitive enzymes to a nanodomain in the immediate vicinity of the LTCC by an unknown mechanism. Here, we show that activated Ca/calmodulin-dependent protein kinase II (CaMKII) strongly interacts with a novel binding motif in the N-terminal domain of Ca1 LTCC α1 subunits that is not conserved in Ca2 or Ca3 voltage-gated Ca channel subunits. Mutations in the Ca1.3 α1 subunit N-terminal domain or in the CaMKII catalytic domain that largely prevent the interaction also disrupt CaMKII association with intact LTCC complexes isolated by immunoprecipitation. Furthermore, these same mutations interfere with E-T coupling in cultured hippocampal neurons. Taken together, our findings define a novel molecular interaction with the neuronal LTCC that is required for the initiation of a long-range signal to the nucleus that is critical for learning and memory.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
KEY POINTS - The AMPA-type ionotropic glutamate receptors (AMPARs) mediate the majority of excitatory synaptic transmission and their function impacts learning, cognition and behaviour. The gating of AMPARs occurs in milliseconds, precisely controlled by a variety of auxiliary subunits that are expressed differentially in the brain, but the difference in mechanisms underlying AMPAR gating modulation by auxiliary subunits remains elusive and is investigated. The elements of the AMPAR that are functionally recruited by auxiliary subunits, stargazin and cornichon 3, are located not only in the extracellular domains but also in the lipid-accessible surface of the AMPAR. We reveal that the two auxiliary subunits require a shared surface on the transmembrane domain of the AMPAR for their function, but the gating is influenced by this surface in opposing directions for each auxiliary subunit. Our results provide new insights into the mechanistic difference of AMPAR modulation by auxiliary subunits and a conceptual framework for functional engineering of the complex.
ABSTRACT - During excitatory synaptic transmission, various structurally unrelated transmembrane auxiliary subunits control the function of AMPA receptors (AMPARs), but the underlying mechanisms remain unclear. We identified lipid-exposed residues in the transmembrane domain (TMD) of the GluA2 subunit of AMPARs that are critical for the function of AMPAR auxiliary subunits, stargazin (Stg) and cornichon 3 (CNIH3). These residues are essential for stabilizing the AMPAR-CNIH3 complex in detergents and overlap with the contacts made between GluA2 TMD and Stg in the cryoEM structures. Mutating these residues had opposite effects on gating modulation and complex stability when Stg- and CNIH3-bound AMPARs were compared. Specifically, in detergent the GluA2-A793F formed an unstable complex with CNIIH3 but in the membrane the GluA2-A793F-CNIH3 complex expressed a gain of function. In contrast, the GluA2-A793F-Stg complex was stable, but had diminished gating modulation. GluA2-C528L destabilized the AMPAR-CNIH3 complex but stabilized the AMPAR-Stg complex, with overall loss of function in gating modulation. Furthermore, loss-of-function mutations in this TMD region cancelled the effects of a gain-of-function Stg carrying mutation in its extracellular loop, demonstrating that both the extracellular and the TMD elements contribute independently to gating modulation. The elements of AMPAR functionally recruited by auxiliary subunits are, therefore, located not only in the extracellular domains but also in the lipid accessible surface of the AMPAR. The TMD surface we defined is a potential target for auxiliary subunit-specific compounds, because engineering of this hotspot induces opposing functional outcomes by Stg and CNIH3. The collection of mutant-phenotype mapping provides a framework for engineering AMPAR gating using auxiliary subunits.
© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.
Hypertrophic cardiomyopathy (HCM) is a highly heterogeneous disease displaying considerable interfamilial and intrafamilial phenotypic variation, including disease severity, age of onset, and disease progression. This poorly understood variance raises the possibility of genetic modifier effects, particularly in MYBPC3-associated HCM.In a large consanguineous Chinese HCM family, we identified 8 members harboring the MYBPC3 c.3624delC (p.Lys1209Serfs) disease-causing mutation, but with very disparate phenotypes. Genotyping ruled out the modifying effect of previously described variants in renin-angiotensin-aldosterone system. Afterwards, we screened for modifying variants in all known causing genes and closely related genes for cardiomyopathy and channelopathy by performing targeted next-generation sequencing. For first time, we showed that a c.1598C>T (p.Ser533Leu) mutation in voltage-dependent l-type calcium channel subunit beta-2 (CACNB2) was present in all severely affected HCM patients, but not in those moderately affected or genotype-positive phenotype-negative patients. This CACNB2 p.Ser533Leu mutation is extremely conserved in evolution, and was not found in 550 healthy controls.Our results suggest that CACNB2 is a possible candidate genetic modifier of MYBPC3-associated familial HCM, but more genetic evidence and functional experiments are needed to confirm.
Dravet syndrome, an early onset epileptic encephalopathy, is most often caused by de novo mutation of the neuronal voltage-gated sodium channel gene SCN1A. Mouse models with deletion of Scn1a recapitulate Dravet syndrome phenotypes, including spontaneous generalized tonic-clonic seizures, susceptibility to seizures induced by elevated body temperature, and elevated risk of sudden unexpected death in epilepsy. Importantly, the epilepsy phenotype of Dravet mouse models is highly strain-dependent, suggesting a strong influence of genetic modifiers. We previously identified Cacna1g, encoding the Cav3.1 subunit of the T-type calcium channel family, as an epilepsy modifier in the Scn2a transgenic epilepsy mouse model. In this study, we asked whether transgenic alteration of Cacna1g expression modifies severity of the Scn1a Dravet phenotype. Scn1a mice with decreased Cacna1g expression showed partial amelioration of disease phenotypes with improved survival and reduced spontaneous seizure frequency. However, reduced Cacna1g expression did not alter susceptibility to hyperthermia-induced seizures. Transgenic elevation of Cacna1g expression had no effect on the Scn1a epilepsy phenotype. These results provide support for Cacna1g as a genetic modifier in a mouse model of Dravet syndrome and suggest that Cav3.1 may be a potential molecular target for therapeutic intervention in patients.
Wiley Periodicals, Inc. © 2017 International League Against Epilepsy.
BACKGROUND - The widely used macrolide antibiotic azithromycin increases risk of cardiovascular and sudden cardiac death, although the underlying mechanisms are unclear. Case reports, including the one we document here, demonstrate that azithromycin can cause rapid, polymorphic ventricular tachycardia in the absence of QT prolongation, indicating a novel proarrhythmic syndrome. We investigated the electrophysiological effects of azithromycin in vivo and in vitro using mice, cardiomyocytes, and human ion channels heterologously expressed in human embryonic kidney (HEK 293) and Chinese hamster ovary (CHO) cells.
METHODS AND RESULTS - In conscious telemetered mice, acute intraperitoneal and oral administration of azithromycin caused effects consistent with multi-ion channel block, with significant sinus slowing and increased PR, QRS, QT, and QTc intervals, as seen with azithromycin overdose. Similarly, in HL-1 cardiomyocytes, the drug slowed sinus automaticity, reduced phase 0 upstroke slope, and prolonged action potential duration. Acute exposure to azithromycin reduced peak SCN5A currents in HEK cells (IC=110±3 μmol/L) and Na current in mouse ventricular myocytes. However, with chronic (24 hour) exposure, azithromycin caused a ≈2-fold increase in both peak and late SCN5A currents, with findings confirmed for I in cardiomyocytes. Mild block occurred for K currents representing I (CHO cells expressing hERG; IC=219±21 μmol/L) and I (CHO cells expressing KCNQ1+KCNE1; IC=184±12 μmol/L), whereas azithromycin suppressed L-type Ca currents (rabbit ventricular myocytes, IC=66.5±4 μmol/L) and I (HEK cells expressing Kir2.1, IC=44±3 μmol/L).
CONCLUSIONS - Chronic exposure to azithromycin increases cardiac Na current to promote intracellular Na loading, providing a potential mechanistic basis for the novel form of proarrhythmia seen with this macrolide antibiotic.
© 2017 American Heart Association, Inc.
Voltage-gated Ca1.2 and Ca1.3 (L-type) Ca channels regulate neuronal excitability, synaptic plasticity, and learning and memory. Densin-180 (densin) is an excitatory synaptic protein that promotes Ca-dependent facilitation of voltage-gated Ca1.3 Ca channels in transfected cells. Mice lacking densin (densin KO) exhibit defects in synaptic plasticity, spatial memory, and increased anxiety-related behaviors-phenotypes that more closely match those in mice lacking Ca1.2 than Ca1.3. Therefore, we investigated the functional impact of densin on Ca1.2. We report that densin is an essential regulator of Ca1.2 in neurons, but has distinct modulatory effects compared with its regulation of Ca1.3. Densin binds to the N-terminal domain of Ca1.2, but not that of Ca1.3, and increases Ca1.2 currents in transfected cells and in neurons. In transfected cells, densin accelerates the forward trafficking of Ca1.2 channels without affecting their endocytosis. Consistent with a role for densin in increasing the number of postsynaptic Ca1.2 channels, overexpression of densin increases the clustering of Ca1.2 in dendrites of hippocampal neurons in culture. Compared with wild-type mice, the cell surface levels of Ca1.2 in the brain, as well as Ca1.2 current density and signaling to the nucleus, are reduced in neurons from densin KO mice. We conclude that densin is an essential regulator of neuronal Ca1 channels and ensures efficient Ca1.2 Ca signaling at excitatory synapses. The number and localization of voltage-gated Ca Ca channels are crucial determinants of neuronal excitability and synaptic transmission. We report that the protein densin-180 is highly enriched at excitatory synapses in the brain and enhances the cell surface trafficking and postsynaptic localization of Ca1.2 L-type Ca channels in neurons. This interaction promotes coupling of Ca1.2 channels to activity-dependent gene transcription. Our results reveal a mechanism that may contribute to the roles of Ca1.2 in regulating cognition and mood.
Copyright © 2017 the authors 0270-6474/17/374679-13$15.00/0.
AMPA receptors (AMPAR) are ligand gated ion channels critical for synaptic transmission and plasticity. Their dysfunction is implicated in a variety of psychiatric and neurological diseases ranging from major depressive disorder to amyotrophic lateral sclerosis. Attempting to potentiate or depress AMPAR activity is an inherently difficult balancing act between effective treatments and debilitating side effects. A newly explored strategy to target subsets of AMPARs in the central nervous system is to identify compounds that affect specific AMPAR-auxiliary subunit complexes. This exploits diverse spatio-temporal expression patterns of known AMPAR auxiliary subunits, providing means for designing brain region-selective compounds. Here we report a high-throughput screening-based pipeline that can identify compounds that are selective for GluA2-CNIH3 and GluA2-stargazin complexes. These compounds will help us build upon the growing library of AMPAR-auxiliary subunit specific inhibitors, which have thus far all been targeted to TARP γ-8. We used a cell-based assay combined with a voltage-sensitive dye (VSD) to identify changes in glutamate-gated cation flow across the membranes of HEK cells co-expressing GluA2 and an auxiliary subunit. We then used a calcium flux assay to further validate hits picked from the VSD assay. VU0612951 and VU0627849 are candidate compounds from the initial screen that were identified as negative and positive allosteric modulators (NAM and PAM), respectively. They both have lower IC50/EC50s on complexes containing stargazin and CNIH3 than GSG1L or the AMPAR alone. We have also identified a candidate compound, VU0539491, that has NAM activity in GluA2(R)-CNIH3 and GluA2(Q) complexes and PAM activity in GluA2(Q)-GSG1L complexes.