Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

Ca²⁺-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca²⁺ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca²⁺-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca²⁺-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels.     

Critical Role of Lipid Scramblase TMEM16F in Phosphatidylserine Exposure and Repair of Plasma Membrane after Pore Formation

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Regulation of the Ca2+-activated chloride channel Anoctamin-1 (TMEM16A) by Ca2+-induced interaction with FKBP12 and calcineurin

Chloride fluxes through the calcium-gated chloride channel Anoctamin-1 (TMEM16A) control blood pressure, secretion of saliva, mucin, insulin, and melatonin, gastrointestinal motility, sperm capacitation and motility, and pain sensation. Calcium activates a myriad of regulatory proteins but how these proteins affect TMEM16A activity is unresolved. Here we show by co-immunoprecipitation that increasing intracellular calcium with ionomycin or by activating sphingosine-1-phosphate receptors, induces coupling of calcium/calmodulin-dependent phosphatase calcineurin and prolyl isomerase FK506-binding protein 12 (FKBP12) to TMEM16A in HEK-293 cells. Application of drugs that target either calcineurin (cyclosporine A) or FKBP12 (tacrolimus known as FK506 and sirolimus known as rapamycin) caused a decrease in TMEM16A activity. In addition, FK506 and BAPTA-AM prevented co-immunoprecipitation between FKBP12 and TMEM16A. FK506 rendered the channel insensitive to cyclosporine A without altering its apparent calcium sensitivity whereas zero intracellular calcium blocked the effect of FK506. Rapamycin decreased TMEM16A activity in cells pre-treated with cyclosporine A or FK506. These results suggest the formation of a TMEM16A-FKBP12-calcineurin complex that regulates channel function. We conclude that upon a cytosolic calcium increase the TMEM16A-FKPB12-calcineurin trimers are assembled. Such hetero-oligomerization enhances TMEM16A channel activity but is not mandatory for activation by calcium.     

Identification and functional characterization of TMEM16A, a Ca2+-activated Cl- channel activated by extracellular nucleotides, in biliary epithelium

Cl(-) channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP](i), an alternate Cl(-) secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca(2+)](i). The molecular identity of this Ca(2+)-activated Cl(-) channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca(2+)-activated Cl(-) secretion in response to extracellular nucleotides. Furthermore, Cl(-) currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl(-) currents. However, both large and small BECs express TMEM16A and exhibit Ca(2+)-activated Cl(-) efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (I(sc)). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl(-) channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.     

The EF-hand Ca2+ Binding Domain Is Not Required for Cytosolic Ca2+ Activation of the Cardiac Ryanodine Receptor

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca²⁺ is a central step in the process of Ca²⁺-induced Ca²⁺ release, but the molecular basis of RyR2 activation by cytosolic Ca²⁺ is poorly defined. It has been proposed recently that the putative Ca²⁺ binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca²⁺ sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca²⁺ activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca²⁺-dependent activation of [³H]ryanodine binding or the cytosolic Ca²⁺ activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca²⁺ activation of RyR2 and spontaneous Ca²⁺ release in HEK293 cells during store Ca²⁺ overload or store overload-induced Ca²⁺ release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca²⁺, it plays an important role in luminal Ca²⁺ activation and SOICR.     

Structure of the Ca2+-dependent PP2A heterotrimer and insights into Cdc6 dephosphorylation

The B″/PR72 family of protein phosphatase 2A (PP2A) is an important PP2A family involved in diverse cellular processes, and uniquely regulated by calcium binding to the regulatory subunit. The PR70 subunit in this family interacts with cell division control 6 (Cdc6), a cell cycle regulator important for control of DNA replication. Here, we report crystal structures of the isolated PR72 and the trimeric PR70 holoenzyme at a resolution of 2.1 and 2.4 Å, respectively, and in vitro characterization of Cdc6 dephosphorylation. The holoenzyme structure reveals that one of the PR70 calcium-binding motifs directly contacts the scaffold subunit, resulting in the most compact scaffold subunit conformation among all PP2A holoenzymes. PR70 also binds distinctively to the catalytic subunit near the active site, which is required for PR70 to enhance phosphatase activity toward Cdc6. Our studies provide a structural basis for unique regulation of B″/PR72 holoenzymes by calcium ions, and suggest the mechanisms for precise control of substrate specificity among PP2A holoenzymes.     

Structural and Biophysical Analysis of the CLCA1 VWA Domain Suggests Mode of TMEM16A Engagement

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Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2+ Signals Is Controlled by the Stoichiometry of MICU1/2 and MCU

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Comparison of the Effects Exerted by Luminal Ca2+ on the Sensitivity of the Cardiac Ryanodine Receptor to Caffeine and Cytosolic Ca2+

Ca²⁺ released from the sarcoplasmic reticulum (SR) via ryanodine receptor type 2 (RYR2) is the key determinant of cardiac contractility. Although activity of RYR2 channels is primary controlled by Ca²⁺ entry through the plasma membrane, there is growing evidence that Ca²⁺ in the lumen of the SR can also be effectively involved in the regulation of RYR2 channel function. In the present study, we investigated the effect of luminal Ca²⁺ on the response of RYR2 channels reconstituted into a planar lipid membrane to caffeine and Ca²⁺ added to the cytosolic side of the channel. We performed two sets of experiments when the channel was exposed to either luminal Ba²⁺ or Ca²⁺. The given ion served also as a charge carrier. Luminal Ca²⁺ effectively shifted the EC₅₀ for caffeine sensitivity to a lower concentration but did not modify the response of RYR2 channels to cytosolic Ca²⁺. Importantly, luminal Ca²⁺ exerted an effect on channel gating kinetics. Both the open and closed dwell times were considerably prolonged over the whole range (response to caffeine) or the partial range (response to cytosolic Ca²⁺) of open probability. Our results provide strong evidence that an alteration of the gating kinetics is the result of the interaction of luminal Ca²⁺ with the luminally located Ca²⁺ regulatory sites on the RYR2 channel complex.     

Structure of the Human Cation-Independent Mannose 6-Phosphate/IGF2 Receptor Domains 7–11 Uncovers the Mannose 6-Phosphate Binding Site of Domain 9

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Inhibition of Maitotoxin-Induced Ca2+ Influx in Rat Glioma C6 Cells by Brevetoxins and Synthetic Fragments of Maitotoxin

Abstract: ⁴⁵Ca²⁺ influx in rat glioma C6 cells induced by 0.3 n M maitotoxin (MTX) was markedly inhibited by brevetoxin A (PbTx1) and brevetoxin B (PbTx2), with EC₅₀ values of 16 and 13 µ M , respectively. This inhibition was observed immediately after addition of MTX when monitored with fura-2, which suggests that PbTx2 directly blocks the action of MTX. This blockade by PbTx2 was not affected by tetrodotoxin, which excludes the involvement of voltage-sensitive sodium channels. The depolarizing effects of these brevetoxins were also not a likely cause of this inhibition, because melittin, a channel-forming peptide, did not significantly block MTX-induced ⁴⁵Ca²⁺ influx. Instead, this inhibition was thought to be mediated by blockade of an MTX-binding site by the brevetoxins, based on the fact that these toxins, particularly PbTx2, closely mimic the partial structure of MTX. Synthetic fragments of MTX corresponding to the hydrophilic EF-GH rings (200 µ M ) and LM-NO rings (500 µ M ) of MTX significantly reduced MTX-elicited Ca²⁺ influx. The observation that the effects of MTX were inhibited by structural mimics of both its hydrophobic and hydrophilic portions implies that both portions of the MTX molecule recognize its target.     

Modulation of matrix Ca2+ content by the ADP/ATP carrier in brown adipose tissue mitochondria. Influence of membrane lipid composition

The role of the adenine nucleotide translocase on Ca²⁺ homeostasis in mitochondria from brown adipose tissue was examined. It was found that in mitochondria incubated with 50 M Ca²⁺, ADP was not needed to retain the cation, but it was required for strengthening the inhibitory effect of cyclosporin on membrane permeability transition as induced by menadione. In addition, carboxyatractyloside was unable to promote matrix Ca²⁺ release, even though it inhibits the ADP exchange reaction. However, when the Ca²⁺ concentration was increased to 150 M, carboxyatractyloside did induce Ca²⁺ release, and ADP favored Ca²⁺ retention. Determination of cardiolipin content in the inner membrane vesicles showed a greater concentration in brown adipose tissue mitochondria than that found in kidney mitochondria. It is suggested that the failure of the adenine nucleotide translocase to influence membrane permeability transition depends on the lipid composition of the inner membrane.     

The progressive effects of a fat enriched diet on ventricular myocyte contraction and intracellular Ca2+ in the C57BL/6J mouse

The C57BL/6J mouse has a genetic susceptibility to develop diabetes when fed with a high-fat, high-sucrose diet. The general characteristics of diet-induced diabetes in this model include progressive development of hyperinsulinaemia, hyperglycaemia, insulin resistance and obesity, features that are frequently observed in the clinical setting. This study investigated the progressive effects of a fat enriched (FE) diet on contraction and intracellular Ca²⁺ in ventricular myocytes from the C57BL/6J mouse. The characteristics of the mice fed with the FE diet compared to mice receiving control diet included progressive increase in the rate of body weight gain, increased fasting blood glucose and time-dependent differences in the disposal of blood glucose after a glucose challenge. The ultrastructure of cardiac myocytes and associated capillaries did not show any gross morphological alteration after 27 weeks of FE diet compared to controls.At 5 months the resting cell length (RCL) and the kinetics of shortening were not significantly altered in ventricular myocytes from mice receiving the FE diet compared to age-matched controls. At 5 and at 7 months the amplitude of shortening was increased in myocytes receiving the FED diet compared to controls. At 7 months the time to half (THALF) relaxation of myocyte contraction was shortened in myocytes from mice receiving the FE diet compared to controls. Mean THALF relaxation in myocytes from mice fed the FE diet was 32.0 ± 1.4 ms (n = 23) compared to 40.2 ± 2.0 ms (n = 27) in controls. Neither resting intracellular Ca²⁺ nor the kinetics or amplitude of the Ca²⁺ transient were altered by FE diet. Differences in myofilament sensitivity to Ca²⁺ might underlie the changes in contractility.     

Recycling of the Ca2+-activated K+ Channel,KCa2.3, Is Dependent upon RME-1, Rab35/EPI64C,and an N-terminal Domain

Regulation of the number of Ca²⁺-activated K⁺ channels at the endothelial cell surface contributes to control of the endothelium-derived hyperpolarizing factor response, although this process is poorly understood. To address the fate of plasma membrane-localized KCa2.3, we utilized an extracellular epitope-tagged channel in combination with fluorescence and biotinylation techniques in both human embryonic kidney cells and the human microvascular endothelial cell line, HMEC-1. KCa2.3 was internalized from the plasma membrane and degraded with a time constant of 18 h. Cell surface biotinylation demonstrated that KCa2.3 was rapidly endocytosed and recycled back to the plasma membrane. Consistent with recycling, expression of a dominant negative (DN) RME-1 or Rab35 as well as wild type EPI64C, the Rab35 GTPase-activating protein, resulted in accumulation of KCa2.3 in an intracellular compartment. Expression of DN RME-1, DN Rab35, or wild type EPI64C resulted in a decrease in steady-state plasma membrane expression. Knockdown of EPI64C increased cell surface expression of KCa2.3. Furthermore, the effect of EPI64C was dependent upon its GTPase-activating proteins activity. Co-immunoprecipitation studies confirmed an association between KCa2.3 and both Rab35 and RME-1. In contrast to KCa2.3, KCa3.1 was rapidly endocytosed and degraded in an RME-1 and Rab35-independent manner. A series of N-terminal deletions identified a 12-amino acid region, Gly²⁰⁶–Pro²¹⁷, as being required for the rapid recycling of KCa2.3. Deletion of Gly²⁰⁶–Pro²¹⁷ had no effect on the association of KCa2.3 with Rab35 but significantly decreased the association with RME-1. These represent the first studies elucidating the mechanisms by which KCa2.3 is maintained at the plasma membrane.     

F1FO ATP Synthase Is Expressed at the Surface of Embryonic Rat Heart‐Derived H9c2 Cells and Is Affected by Cardiac‐Like Differentiation

Taking advantage from the peculiar features of the embryonic rat heart‐derived myoblast cell line H9c2, the present study is the first to provide evidence for the expression of F₁F_O ATP synthase and of ATPase Inhibitory Factor 1 (IF₁) on the surface of cells of cardiac origin, together documenting that they were affected through cardiac‐like differentiation. Subunits of both the catalytic F₁ sector of the complex (ATP synthase‐β) and of the peripheral stalk, responsible for the correct F₁‐F_O assembly/coupling, (OSCP, b, F6) were detected by immunofluorescence, together with IF₁. The expression of ATP synthase‐β, ATP synthase‐b and F6 were similar for parental and differentiated H9c2, while the levels of OSCP increased noticeably in differentiated cells, where the results of in situ Proximity Ligation Assay were consistent with OSCP interaction within ecto‐F₁F_O complexes. An opposite trend was shown by IF₁ whose ectopic expression appeared greater in the parental H9c2. Here, evidence for the IF₁ interaction with ecto‐F₁F_O complexes was provided. Functional analyses corroborate both sets of data. i) An F₁F_O ATP synthase contribution to the exATP production by differentiated cells suggests an augmented expression of holo‐F₁F_O ATP synthase on plasma membrane, in line with the increase of OSCP expression and interaction considered as a requirement for favoring the F₁‐F_O coupling. ii) The absence of exATP generation by the enzyme, and the finding that exATP hydrolysis was largely oligomycin‐insensitive, are in line in parental cells with the deficit of OSCP and suggest the occurrence of sub‐assemblies together evoking more regulation by IF₁. J. Cell. Biochem. 9999: 1–13, 2015. © 2015 Wiley Periodicals, Inc. J. Cell. Biochem. 117: 470–482, 2016. © 2015 Wiley Periodicals, Inc.     

Altered Domain Structure of the Prion Protein Caused by Cu2+ Binding and Functionally Relevant Mutations: Analysis by Cross-Linking,MS/MS,and NMR

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Structure-based drug design studies of the interactions of ent-kaurane diterpenes derived from Wedelia paludosa with the Plasmodium falciparum sarco/endoplasmic reticulum Ca2+-ATPase PfATP6

Malaria is responsible for more deaths around the world than any other parasitic disease. Due to the emergence of strains that are resistant to the current chemotherapeutic antimalarial arsenal, the search for new antimalarial drugs remains urgent though hampered by a lack of knowledge regarding the molecular mechanisms of artemisinin resistance. Semisynthetic compounds derived from diterpenes from the medicinal plant Wedelia paludosa were tested in silico against the Plasmodium falciparum Ca²⁺-ATPase, PfATP6. This protein was constructed by comparative modelling using the three-dimensional structure of a homologous protein, 1IWO, as a scaffold. Compound 21 showed the best docking scores, indicating a better interaction with PfATP6 than that of thapsigargin, the natural inhibitor. Inhibition of PfATP6 by diterpene compounds could promote a change in calcium homeostasis, leading to parasite death. These data suggest PfATP6 as a potential target for the antimalarial ent-kaurane diterpenes.