Plasma Membrane Calcium ATPase Activity Is Regulated by Actin Oligomers through Direct Interaction

As recently described by our group, plasma membrane calcium ATPase (PMCA) activity can be regulated by the actin cytoskeleton. In this study, we characterize the interaction of purified G-actin with isolated PMCA and examine the effect of G-actin during the first polymerization steps. As measured by surface plasmon resonance, G-actin directly interacts with PMCA with an apparent 1:1 stoichiometry in the presence of Ca²⁺ with an apparent affinity in the micromolar range. As assessed by the photoactivatable probe 1-O-hexadecanoyl-2-O-[9-[[[2-[¹²⁵I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine, the association of PMCA to actin produced a shift in the distribution of the conformers of the pump toward a calmodulin-activated conformation. G-actin stimulates Ca²⁺-ATPase activity of the enzyme when incubated under polymerizing conditions, displaying a cooperative behavior. The increase in the Ca²⁺-ATPase activity was related to an increase in the apparent affinity for Ca²⁺ and an increase in the phosphoenzyme levels at steady state. Although surface plasmon resonance experiments revealed only one binding site for G-actin, results clearly indicate that more than one molecule of G-actin was needed for a regulatory effect on the pump. Polymerization studies showed that the experimental conditions are compatible with the presence of actin in the first stages of assembly. Altogether, these observations suggest that the stimulatory effect is exerted by short oligomers of actin. The functional interaction between actin oligomers and PMCA represents a novel regulatory pathway by which the cortical actin cytoskeleton participates in the regulation of cytosolic Ca²⁺ homeostasis.     

Insulin Protects Pancreatic Acinar Cells from Palmitoleic Acid-induced Cellular Injury

Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired mitochondrial metabolism, cytosolic Ca²⁺ ([Ca²⁺]_i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca²⁺]_i are linked critically by the ATP-driven plasma membrane Ca²⁺-ATPase (PMCA) important for maintaining low resting [Ca²⁺]_i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca²⁺]_i was measured by fura-2 imaging. An in situ [Ca²⁺]_i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca²⁺ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca²⁺ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca²⁺ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced Ca²⁺ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002. These data provide the first evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.     

Determination of the Dissociation Constants for Ca2+ and Calmodulin from the Plasma Membrane Ca2+ Pump by a Lipid Probe That Senses Membrane Domain Changes

The purpose of this work was to obtain information about conformational changes of the plasma membrane Ca²⁺-pump (PMCA) in the membrane region upon interaction with Ca²⁺, calmodulin (CaM) and acidic phospholipids. To this end, we have quantified labeling of PMCA with the photoactivatable phosphatidylcholine analog [¹²⁵I]TID-PC/16, measuring the shift of conformation E₂ to the auto-inhibited conformation E₁I and to the activated E₁A state, titrating the effect of Ca²⁺ under different conditions. Using a similar approach, we also determined the CaM-PMCA dissociation constant. The results indicate that the PMCA possesses a high affinity site for Ca²⁺ regardless of the presence or absence of activators. Modulation of pump activity is exerted through the C-terminal domain, which induces an apparent auto-inhibited conformation for Ca²⁺ transport but does not modify the affinity for Ca²⁺ at the transmembrane domain. The C-terminal domain is affected by CaM and CaM-like treatments driving the auto-inhibited conformation E₁I to the activated E₁A conformation and thus modulating the transport of Ca²⁺. This is reflected in the different apparent constants for Ca²⁺ in the absence of CaM (calculated by Ca²⁺-ATPase activity) that sharply contrast with the lack of variation of the affinity for the Ca²⁺ site at equilibrium. This is the first time that equilibrium constants for the dissociation of Ca²⁺ and CaM ligands from PMCA complexes are measured through the change of transmembrane conformations of the pump. The data further suggest that the transmembrane domain of the PMCA undergoes major rearrangements resulting in altered lipid accessibility upon Ca²⁺ binding and activation.     

Glycolytic ATP Fuels the Plasma Membrane Calcium Pump Critical for Pancreatic Cancer Cell Survival

Pancreatic cancer is an aggressive cancer with poor prognosis and limited treatment options. Cancer cells rapidly proliferate and are resistant to cell death due, in part, to a shift from mitochondrial metabolism to glycolysis. We hypothesized that this shift is important in regulating cytosolic Ca²⁺ ([Ca²⁺]_i), as the ATP-dependent plasma membrane Ca²⁺ ATPase (PMCA) is critical for maintaining low [Ca²⁺]_i and thus cell survival. The present study aimed to determine the relative contribution of mitochondrial versus glycolytic ATP in fuelling the PMCA in human pancreatic cancer cells. We report that glycolytic inhibition induced profound ATP depletion, PMCA inhibition, [Ca²⁺]_i overload, and cell death in PANC1 and MIA PaCa-2 cells. Conversely, inhibition of mitochondrial metabolism had no effect, suggesting that glycolytic ATP is critical for [Ca²⁺]_i homeostasis and thus survival. Targeting the glycolytic regulation of the PMCA may, therefore, be an effective strategy for selectively killing pancreatic cancer while sparing healthy cells.     

Superinhibitory Phospholamban Mutants Compete with Ca2+ for Binding to SERCA2a by Stabilizing a Unique Nucleotide-dependent Conformational State

Three cross-linkable phospholamban (PLB) mutants of increasing inhibitory strength (N30C-PLB < N27A,N30C,L37A-PLB (PLB3) < N27A,N30C,L37A,V49G-PLB (PLB4)) were used to determine whether PLB decreases the Ca²⁺ affinity of SERCA2a by competing for Ca²⁺ binding. The functional effects of N30C-PLB, PLB3, and PLB4 on Ca²⁺-ATPase activity and E1∼P formation were correlated with their binding interactions with SERCA2a measured by chemical cross-linking. Successively higher Ca²⁺ concentrations were required to both activate the enzyme co-expressed with N30C-PLB, PLB3, and PLB4 and to dissociate N30C-PLB, PLB3, and PLB4 from SERCA2a, suggesting competition between PLB and Ca²⁺ for binding to SERCA2a. This was confirmed with the Ca²⁺ pump mutant, D351A, which is catalytically inactive but retains strong Ca²⁺ binding. Increasingly higher Ca²⁺ concentrations were also required to dissociate N30C-PLB, PLB3, and PLB4 from D351A, demonstrating directly that PLB antagonizes Ca²⁺ binding. Finally, the specific conformation of E2 (Ca²⁺-free state of SERCA2a) that binds PLB was investigated using the Ca²⁺-pump inhibitors thapsigargin and vanadate. Cross-linking assays conducted in the absence of Ca²⁺ showed that PLB bound preferentially to E2 with bound nucleotide, forming a remarkably stable complex that is highly resistant to both thapsigargin and vanadate. In the presence of ATP, N30C-PLB had an affinity for SERCA2a approaching that of vanadate (micromolar), whereas PLB3 and PLB4 had much higher affinities, severalfold greater than even thapsigargin (nanomolar or higher). We conclude that PLB decreases Ca²⁺ binding to SERCA2a by stabilizing a unique E2·ATP state that is unable to bind thapsigargin or vanadate.     

E2P-like states of plasma membrane Ca2+‑ATPase characterization of vanadate and fluoride-stabilized phosphoenzyme analogues

The plasma membrane Ca²⁺?ATPase (PMCA) belongs to the family of P-type ATPases, which share the formation of an acid-stable phosphorylated intermediate as part of their reaction cycle. The crystal structure of PMCA is currently lacking. Its abundance is approximately 0.1% of the total protein in the membrane, hampering efforts to produce suitable crystals for X-ray structure analysis. In this work we characterized the effect of beryllium fluoride (BeF_x), aluminium fluoride (AlF_x) and magnesium fluoride (MgF_x) on PMCA. These compounds are known inhibitors of P-type ATPases that stabilize E2P ground, E2·P phosphoryl transition and E2·P_i product states. Our results show that the phosphate analogues BeF_x, AlF_x and MgF_x inhibit PMCA Ca²⁺?ATPase activity, phosphatase activity and phosphorylation with high apparent affinity. Ca²⁺?ATPase inhibition by AlF_x and BeF_x depended on Mg²⁺ concentration indicating that this ion stabilizes the complex between these inhibitors and the enzyme. Low pH increases AlF_x and BeF_x but not MgF_x apparent affinity. Eosin fluorescent probe binds with high affinity to the nucleotide binding site of PMCA. The fluorescence of eosin decreases when fluoride complexes bind to PMCA indicating that the environment of the nucleotide binding site is less hydrophobic in E2P-like states. Finally, measuring the time course of E?→?E2P-like conformational change, we proposed a kinetic model for the binding of fluoride complexes and vanadate to PMCA.In summary, our results show that these fluoride complexes reveal different states of phosphorylated intermediates belonging to the mechanism of hydrolysis of ATP by the PMCA.     

Metabolic regulation of the PMCA: Role in cell death and survival

The plasma membrane Ca²⁺-ATPase (PMCA) is a ubiquitously expressed, ATP-driven Ca²⁺ pump that is critical for maintaining low resting cytosolic Ca²⁺ ([Ca²⁺]_i) in all eukaryotic cells. Since cytotoxic Ca²⁺ overload has such a central role in cell death, the PMCA represents an essential “linchpin” for the delicate balance between cell survival and cell death. In general, impaired PMCA activity and reduced PMCA expression leads to cytotoxic Ca²⁺ overload and Ca²⁺ dependent cell death, both apoptosis and necrosis, whereas maintenance of PMCA activity or PMCA overexpression is generally accepted as being cytoprotective. However, the PMCA has a paradoxical role in cell death depending on the cell type and cellular context. The PMCA can be differentially regulated by Ca²⁺-dependent proteolysis, can be maintained by a localised glycolytic ATP supply, even in the face of global ATP depletion, and can be profoundly affected by the specific phospholipid environment that it sits within the membrane. The major focus of this review is to highlight some of the controversies surrounding the paradoxical role of the PMCA in cell death and survival, challenging the conventional view of ATP-dependent regulation of the PMCA and how this might influence cell fate.     

Effects of Phenylalanine and its Metabolites on Cytoplasmic Free Calcium in Cortical Neurons

Classic phenylketonuria (PKU) is characterized by brain lesions. However, its underlying neurotoxic mechanisms remain unknown. Based on our previous studies, we hypothesized that calcium might participate in PKU-associated neuropathy. In cultured cortical neurons, cytoplasmic free calcium concentration ([Ca²⁺]_i) decreased dramatically when treatment with phenylalanine (Phe) and phenyllactic acid, while phenylacetic acid treatment immediately increased [Ca²⁺]_i, which began to decrease after 3 min. Moreover, [Ca²⁺]_i decreased dramatically after Phe treatment in the presence of EGTA suggesting that Phe might increase [Ca²⁺]_i efflux. Phe-induced [Ca²⁺]_i decrease was strongly inhibited by vanadate, a non-specific plasma membrane Ca²⁺-ATPase (PMCA) antagonist, suggesting that Phe might increase [Ca²⁺]_i efflux throught modulating PMCA. These findings were further supported by the facts that Phe could increase membrance ⁴⁵Ca-uptake capability and PMCA activity. In contrast, treatment of KBR7943 or thapsigargin, antagonists to Na/Ca Exchanger (NCX) and Sarco/Endoplasmic reticulum Ca²⁺-ATPase (SERCA), respectively, did not elicit any changes in [Ca²⁺]_i. Specific siRNA against PMCA had an effect similar to vanadate. Since the brain injury induced by phenylalaninemia was thought to be a chronic process, we cultured cortical neurons in the presence of Phe for 2 weeks and measured [Ca²⁺]_i, PMCA activity and ⁴⁵Ca-uptake capability at days 3, 7, 9 and 14, respectively. PMCA activity and ⁴⁵Ca-uptake capability decreased from day 9, at the same time [Ca²⁺]_i increase was observed. In conclusion, PMCA participate in regulating Phe-induced initial rapid decrease in [Ca²⁺]_i and subsequent long-term increase in [Ca²⁺]_i.     

A New Conformation in Sarcoplasmic Reticulum Calcium Pump and Plasma Membrane Ca2+ Pumps Revealed by a Photoactivatable Phospholipidic Probe

The purpose of this work was to obtain structural information about conformational changes in the membrane region of the sarcoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca²⁺ pumps. We have assessed changes in the overall exposure of these proteins to surrounding lipids by quantifying the extent of protein labeling by a photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[¹²⁵I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([¹²⁵I]TID-PC/16) under different conditions. We determined the following. 1) Incorporation of [¹²⁵I]TID-PC/16 to SERCA decreases 25% when labeling is performed in the presence of Ca²⁺. This decrease in labeling matches qualitatively the decrease in transmembrane surface exposed to the solvent calculated from crystallographic data for SERCA structures. 2) Labeling of PMCA incubated with Ca²⁺ and calmodulin decreases by approximately the same amount. However, incubation with Ca²⁺ alone increases labeling by more than 50%. Addition of C28, a peptide that prevents activation of PMCA by calmodulin, yields similar results. C28 has also been shown to inhibit ATPase SERCA activity. Interestingly, incubation of SERCA with C28 also increases [¹²⁵I]TID-PC/16 incorporation to the protein. These results suggest that in both proteins there are two different E₁ conformations as follows: one that is auto-inhibited and is in contact with a higher amount of lipids (Ca²⁺ + C28 for SERCA and Ca²⁺ alone for PMCA), and one in which the enzyme is fully active (Ca²⁺ for SERCA and Ca²⁺-calmodulin for PMCA) and that exhibits a more compact transmembrane arrangement. These results are the first evidence that there is an autoinhibited conformation in these P-type ATPases, which involves both the cytoplasmic regions and the transmembrane segments.Although membrane proteins constitute more than 20% of the total proteins, the structure of only few of them is known in detail. An important group of integral membrane proteins are ion-motive ATPases. These proteins belong to the family of P-type ATPases, which share in common the formation of an acid-stable phosphorylated intermediate as part of its reaction cycle. Crystallographic information is available for a few members of this family. There are several crystal structures of the Ca²⁺ pump of sarcoplasmic reticulum (SERCA)² revealing different conformations (1–5), and recently, crystal structures of the H⁺-ATPase (6) and of the Na,K-ATPase were reported as well (7).We are interested in obtaining structural information about the plasma membrane calcium pump (PMCA). This pump is an integral part of the Ca²⁺ signaling mechanism (8). It is highly regulated by calmodulin, which activates this protein by binding to an auto-inhibitory region and changing the conformation of the pump from an inhibited state to an activated one (8, 9). Crystallization of PMCA is particularly challenging because there is no natural source from which this protein can be obtained in large quantities. Moreover, the presence of several isoforms in the same tissue further complicates efforts to obtain a homogeneous sample suitable for crystallization.Information about the structure and assembly of the transmembrane domain of an integral membrane protein can also be obtained from the analysis of the lipid-protein interactions. In this work, we have used a hydrophobic photolabeling method to study the noncovalent interactions between PMCA and the surrounding phospholipids under different experimental conditions that lead to known conformations. We employed the photoactivatable phosphatidylcholine analog 1-palmitoyl-2-[9-[2′-[¹²⁵I]iodo-4′-(trifluoromethyldiazirinyl)-benzyloxycarbonyl]-nonaoyl]-sn-glycero-3-phosphocholine ([¹²⁵I]TID-PC/16) that has been previously used to analyze lipid-protein interfaces (10–12). This reagent is located in the phospholipidic milieu, and upon photolysis it reacts indiscriminately with its molecular neighbors. It is thus possible to directly analyze the interaction between a membrane protein and lipids belonging to its immediate environment (13–15). By measuring the amount of labeling of SERCA in conditions that promote conformations for which there are well resolved crystal structures, we were able to validate this photolabeling approach as a convenient tool for analyzing conformational changes within transmembrane regions. Furthermore, using this technique on PMCA and comparing the results obtained for SERCA, we were able to draw structural conclusions about these proteins under activated and inhibited states.     

The Plasma Membrane Calcium Pump in Pancreatic Cancer Cells Exhibiting the Warburg Effect Relies on Glycolytic ATP

Evidence suggests that the plasma membrane Ca²⁺-ATPase (PMCA), which is critical for maintaining a low intracellular Ca²⁺ concentration ([Ca²⁺]_i), utilizes glycolytically derived ATP in pancreatic ductal adenocarcinoma (PDAC) and that inhibition of glycolysis in PDAC cell lines results in ATP depletion, PMCA inhibition, and an irreversible [Ca²⁺]_i overload. We explored whether this is a specific weakness of highly glycolytic PDAC by shifting PDAC cell (MIA PaCa-2 and PANC-1) metabolism from a highly glycolytic phenotype toward mitochondrial metabolism and assessing the effects of mitochondrial versus glycolytic inhibitors on ATP depletion, PMCA inhibition, and [Ca²⁺]_i overload. The highly glycolytic phenotype of these cells was first reversed by depriving MIA PaCa-2 and PANC-1 cells of glucose and supplementing with α-ketoisocaproate or galactose. These culture conditions resulted in a significant decrease in both glycolytic flux and proliferation rate, and conferred resistance to ATP depletion by glycolytic inhibition while sensitizing cells to mitochondrial inhibition. Moreover, in direct contrast to cells exhibiting a high glycolytic rate, glycolytic inhibition had no effect on PMCA activity and resting [Ca²⁺]_i in α-ketoisocaproate- and galactose-cultured cells, suggesting that the glycolytic dependence of the PMCA is a specific vulnerability of PDAC cells exhibiting the Warburg phenotype.     

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

Ca²⁺ (sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA)) and Cu⁺ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca²⁺, demonstrated by the addition of ATP and Ca²⁺ to SERCA deprived of Ca²⁺ (E2) as compared with ATP to Ca²⁺-activated enzyme (E1·2Ca²⁺). Activation by Ca²⁺ is slower at low pH (2H⁺·E2 to E1·2Ca²⁺) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca²⁺ translocation. A “H⁺-gated pathway,” demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca²⁺ release by H⁺/Ca²⁺ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu⁺/H⁺ exchange. As opposed to SERCA after Ca²⁺ chelation, ATP7A/B does not undergo reverse phosphorylation with P_i after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.     

Hyperactivation of the Human Plasma Membrane Ca2+ Pump PMCA h4xb by Mutation of Glu99 to Lys

The transport of calcium to the extracellular space carried out by plasma membrane Ca²⁺ pumps (PMCAs) is essential for maintaining low Ca²⁺ concentrations in the cytosol of eukaryotic cells. The activity of PMCAs is controlled by autoinhibition. Autoinhibition is relieved by the binding of Ca²⁺-calmodulin to the calmodulin-binding autoinhibitory sequence, which in the human PMCA is located in the C-terminal segment and results in a PMCA of high maximal velocity of transport and high affinity for Ca²⁺. Autoinhibition involves the intramolecular interaction between the autoinhibitory domain and a not well defined region of the molecule near the catalytic site. Here we show that the fusion of GFP to the C terminus of the h4xb PMCA causes partial loss of autoinhibition by specifically increasing the V_max. Mutation of residue Glu⁹⁹ to Lys in the cytosolic portion of the M1 transmembrane helix at the other end of the molecule brought the V_max of the h4xb PMCA to near that of the calmodulin-activated enzyme without increasing the apparent affinity for Ca²⁺. Altogether, the results suggest that the autoinhibitory interaction of the extreme C-terminal segment of the h4 PMCA is disturbed by changes of negatively charged residues of the N-terminal region. This would be consistent with a recently proposed model of an autoinhibited form of the plant ACA8 pump, although some differences are noted.     

Population Shift Underlies Ca2+-induced Regulatory Transitions in the Sodium-Calcium Exchanger (NCX)

In eukaryotic Na⁺/Ca²⁺ exchangers (NCX) the Ca²⁺ binding CBD1 and CBD2 domains form a two-domain regulatory tandem (CBD12). An allosteric Ca²⁺ sensor (Ca3–Ca4 sites) is located on CBD1, whereas CBD2 contains a splice-variant segment. Recently, a Ca²⁺-driven interdomain switch has been described, albeit how it couples Ca²⁺ binding with signal propagation remains unclear. To resolve the dynamic features of Ca²⁺-induced conformational transitions we analyze here distinct splice variants and mutants of isolated CBD12 at varying temperatures by using small angle x-ray scattering (SAXS) and equilibrium ⁴⁵Ca²⁺ binding assays. The ensemble optimization method SAXS analysis demonstrates that the apo and Mg²⁺-bound forms of CBD12 are highly flexible, whereas Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift of conformational landscape to more rigidified states. Population shift occurs even under conditions in which no effect of Ca²⁺ is observed on the globally derived D_max (maximal interatomic distance), although under comparable conditions a normal [Ca²⁺]-dependent allosteric regulation occurs. Low affinity sites (Ca1–Ca2) of CBD1 do not contribute to Ca²⁺-induced population shift, but the occupancy of these sites by 1 mm Mg²⁺ shifts the Ca²⁺ affinity (K_d) at the neighboring Ca3–Ca4 sites from ∼ 50 nm to ∼ 200 nm and thus, keeps the primary Ca²⁺ sensor (Ca3–Ca4 sites) within a physiological range. Thus, Ca²⁺ binding to the Ca3–Ca4 sites results in a population shift, where more constraint conformational states become highly populated at dynamic equilibrium in the absence of global conformational transitions in CBD alignment.     

Reinterpreting the Action of ATP Analogs on KATP Channels

Neuroendocrine-type K_ATP channels, (SUR1/Kir6.2)₄, couple the transmembrane flux of K⁺, and thus membrane potential, with cellular metabolism in various cell types including insulin-secreting β-cells. Mutant channels with reduced activity are a cause of congenital hyperinsulinism, whereas hyperactive channels are a cause of neonatal diabetes. A current regulatory model proposes that ATP hydrolysis is required to switch SUR1 into post-hydrolytic conformations able to antagonize the inhibitory action of nucleotide binding at the Kir6.2 pore, thus coupling enzymatic and channel activities. Alterations in SUR1 ATPase activity are proposed to contribute to neonatal diabetes and type 2 diabetes risk. The regulatory model is partly based on the reduced ability of ATP analogs such as adenosine 5′-(β,γ-imino)triphosphate (AMP-PNP) and adenosine 5′-O-(thiotriphosphate) (ATPγS) to stimulate channel activity, presumably by reducing hydrolysis. This study uses a substitution at the catalytic glutamate, SUR1_E1507Q, with a significantly increased affinity for ATP, to probe the action of these ATP analogs on conformational switching. ATPγS, a slowly hydrolyzable analog, switches SUR1 conformations, albeit with reduced affinity. Nonhydrolyzable AMP-PNP and adenosine 5′-(β,γ-methylenetriphosphate) (AMP-PCP) alone fail to switch SUR1, but do reverse ATP-induced switching. AMP-PCP displaces 8-azido-[³²P]ATP from the noncanonical NBD1 of SUR1. This is consistent with structural data on an asymmetric bacterial ABC protein that shows that AMP-PNP binds selectively to the noncanonical NBD to prevent conformational switching. The results imply that MgAMP-PNP and MgAMP-PCP (AMP-PxP) fail to activate K_ATP channels because they do not support NBD dimerization and conformational switching, rather than by limiting enzymatic activity.     

ACTH Induces Cav3.2 Current and mRNA by cAMP-dependent and cAMP-independent Mechanisms

Bovine adrenal zona fasciculata (AZF) cells express Ca_v3.2 T-type Ca²⁺ channels that function pivotally in adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. The regulation of Ca_v3.2 expression in AZF cells by ACTH, cAMP analogs, and their metabolites was studied using Northern blot and patch clamp recording. Exposing AZF cells to ACTH for 3–6 days markedly enhanced the expression of Ca_v3.2 current. The increase in Ca_v3.2 current was preceded by an increase in corresponding CACNA1H mRNA. O-Nitrophenyl,sulfenyl-adrenocorticotropin, which produces a minimal increase in cAMP, also enhanced Ca_v3.2 current. cAMP analogs, including 8-bromoadenosine cAMP (600 μm) and 6-benzoyladenosine cAMP (300 μm) induced CACNA1H mRNA, but not Ca_v3.2 current. In contrast, 8-(4-chlorophenylthio) (8CPT)-cAMP (10–50 μm) enhanced CACNA1H mRNA and Ca_v3.2 current, whereas nonhydrolyzable Sp-8CPT-cAMP failed to increase either Ca_v3.2 current or mRNA. Metabolites of 8CPT-cAMP, including 8CPT-adenosine and 8CPT-adenine, increased Ca_v3.2 current and mRNA with a potency and effectiveness similar to the parent compound. The Epac activator 8CPT-2′-O-methyl-cAMP and its metabolites 8CPT-2′-OMe-5′-AMP and 8CPT-2′-O-methyl-adenosine increased CACNA1H mRNA and Ca_v3.2 current; Sp-8CPT-2′-O-methyl-cAMP increased neither Ca_v3.2 current nor mRNA. These results reveal an interesting dichotomy between ACTH and cAMP with regard to regulation of CACNA1H mRNA and Ca²⁺ current. Specifically, ACTH induces expression of CACNA1H mRNA and Ca_v3.2 current in AZF cells by mechanisms that depend at most only partly on cAMP. In contrast, cAMP enhances expression of CACNA1H mRNA but not the corresponding Ca²⁺ current. Surprisingly, chlorophenylthio-cAMP analogs stimulate the expression of Ca_v3.2 current indirectly through metabolites. ACTH and the metabolites may induce Ca_v3.2 expression by the same, unidentified mechanism.     

Resveratrol inhibits plasma membrane Ca2+-ATPase inducing an increase in cytoplasmic calcium

Plasma membrane Ca²⁺-ATPase (PMCA) plays a vital role in maintaining cytosolic calcium concentration ([Ca²⁺]_i). Given that many diseases have modified PMCA expression and activity, PMCA is an important potential target for therapeutic treatment. This study demonstrates that the non-toxic, naturally-occurring polyphenol resveratrol (RES) induces increases in [Ca²⁺]_i via PMCA inhibition in primary dermal fibroblasts and MDA-MB-231 breast cancer cells. Our results also illustrate that RES and the fluorescent intracellular calcium indicator Fura-2, are compatible for simultaneous use, in contrast to previous studies, which indicated that RES modulates the Fura-2 fluorescence independent of calcium concentration. Because RES has been identified as a PMCA inhibitor, further studies may be conducted to develop more specific PMCA inhibitors from RES derivatives for potential therapeutic use.     

In Vivo Epithelial Wound Repair Requires Mobilization of Endogenous Intracellular and Extracellular Calcium

We report that a localized intracellular and extracellular Ca²⁺ mobilization occurs at the site of microscopic epithelial damage in vivo and is required to mediate tissue repair. Intravital confocal/two-photon microscopy continuously imaged the surgically exposed stomach mucosa of anesthetized mice while photodamage of gastric epithelial surface cells created a microscopic lesion that healed within 15 min. Transgenic mice with an intracellular Ca²⁺-sensitive protein (yellow cameleon 3.0) report that intracellular Ca²⁺ selectively increases in restituting gastric epithelial cells adjacent to the damaged cells. Pretreatment with U-73122, indomethacin, 2-aminoethoxydiphenylborane, or verapamil inhibits repair of the damage and also inhibits the intracellular Ca²⁺ increase. Confocal imaging of Fura-Red dye in luminal superfusate shows a localized extracellular Ca²⁺ increase at the gastric surface adjacent to the damage that temporally follows intracellular Ca²⁺ mobilization. Indomethacin and verapamil also inhibit the luminal Ca²⁺ increase. Intracellular Ca²⁺ chelation (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid/acetoxymethyl ester, BAPTA/AM) fully inhibits intracellular and luminal Ca²⁺ increases, whereas luminal calcium chelation (N-(2-hydroxyetheyl)-ethylendiamin-N,N,N′-triacetic acid trisodium, HEDTA) blocks the increase of luminal Ca²⁺ and unevenly inhibits late-phase intracellular Ca²⁺ mobilization. Both modes of Ca²⁺ chelation slow gastric repair. In plasma membrane Ca-ATPase 1^+/− mice, but not plasma membrane Ca-ATPase 4^−/− mice, there is slowed epithelial repair and a diminished gastric surface Ca²⁺ increase. We conclude that endogenous Ca²⁺, mobilized by signaling pathways and transmembrane Ca²⁺ transport, causes increased Ca²⁺ levels at the epithelial damage site that are essential to gastric epithelial cell restitution in vivo.     

Stable Structural Analog of Ca2+-ATPase ADP-insensitive Phosphoenzyme with Occluded Ca2+ Formed by Elongation of A-domain/M1′-linker and Beryllium Fluoride Binding

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca²⁺ at the transport sites (E2PCa₂) of sarcoplasmic reticulum Ca²⁺-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca²⁺ deocclusion/release to the lumen; E1PCa₂ → E2PCa₂ → E2P + 2Ca²⁺. Stabilization was achieved by elongation of the Glu⁴⁰-Ser⁴⁸ loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly⁴⁶/Lys⁴⁷ and by binding of beryllium fluoride (BeF_x) to the phosphorylation site of the Ca²⁺-bound ATPase (E1Ca₂). The complex E2Ca₂·BeF₃⁻ was also produced by lumenal Ca²⁺ binding to E2·BeF₃⁻ (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 °C. Only BeF_x, but not AlF_x or MgF_x, produced the E2PCa₂ structural analog. Complex formation required binding of Mg²⁺, Mn²⁺, or Ca²⁺ at the catalytic Mg²⁺ site. Results reveal that the phosphorylation product E1PCa₂ and the E2P ground state (but not the transition states) become competent to produce the E2PCa₂ transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca²⁺ release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu⁴⁰-Ser⁴⁸ linker and of Mg²⁺ at the catalytic site in these processes.     

Critical Roles of Interdomain Interactions for Modulatory ATP Binding to Sarcoplasmic Reticulum Ca2+-ATPase

ATP has dual roles in the reaction cycle of sarcoplasmic reticulum Ca²⁺-ATPase. Upon binding to the Ca₂E1 state, ATP phosphorylates the enzyme, and by binding to other conformational states in a non-phosphorylating modulatory mode ATP stimulates the dephosphorylation and other partial reaction steps of the cycle, thereby ensuring a high rate of Ca²⁺ transport under physiological conditions. The present study elucidates the mechanism underlying the modulatory effect on dephosphorylation. In the intermediate states of dephosphorylation the A-domain residues Ser¹⁸⁶ and Asp²⁰³ interact with Glu⁴³⁹ (N-domain) and Arg⁶⁷⁸ (P-domain), respectively. Single mutations to these residues abolish the stimulation of dephosphorylation by ATP. The double mutation swapping Asp²⁰³ and Arg⁶⁷⁸ rescues ATP stimulation, whereas this is not the case for the double mutation swapping Ser¹⁸⁶ and Glu⁴³⁹. By taking advantage of the ability of wild type and mutant Ca²⁺-ATPases to form stable complexes with aluminum fluoride (E2·AlF) and beryllium fluoride (E2·BeF) as analogs of the E2·P phosphoryl transition state and E2P ground state, respectively, of the dephosphorylation reaction, the mutational effects on ATP binding to these intermediates are demonstrated. In the wild type Ca²⁺-ATPase, the ATP affinity of the E2·P phosphoryl transition state is higher than that of the E2P ground state, thus explaining the stimulation of dephosphorylation by nucleotide-induced transition state stabilization. We find that the Asp²⁰³-Arg⁶⁷⁸ and Ser¹⁸⁶-Glu⁴³⁹ interdomain bonds are critical, because they tighten the interaction with ATP in the E2·P phosphoryl transition state. Moreover, ATP binding and the Ser¹⁸⁶-Glu⁴³⁹ bond are mutually exclusive in the E2P ground state.     

Involvement of the Na+/Ca2+ exchanger isoform 1 (NCX1) in Neuronal Growth Factor (NGF)-induced Neuronal Differentiation through Ca2+-dependent Akt Phosphorylation

NGF induces neuronal differentiation by modulating [Ca²⁺]_i. However, the role of the three isoforms of the main Ca²⁺-extruding system, the Na⁺/Ca²⁺ exchanger (NCX), in NGF-induced differentiation remains unexplored. We investigated whether NCX1, NCX2, and NCX3 isoforms could play a relevant role in neuronal differentiation through the modulation of [Ca²⁺]_i and the Akt pathway. NGF caused progressive neurite elongation; a significant increase of the well known marker of growth cones, GAP-43; and an enhancement of endoplasmic reticulum (ER) Ca²⁺ content and of Akt phosphorylation through an early activation of ERK1/2. Interestingly, during NGF-induced differentiation, the NCX1 protein level increased, NCX3 decreased, and NCX2 remained unaffected. At the same time, NCX total activity increased. Moreover, NCX1 colocalized and coimmunoprecipitated with GAP-43, and NCX1 silencing prevented NGF-induced effects on GAP-43 expression, Akt phosphorylation, and neurite outgrowth. On the other hand, the overexpression of its neuronal splicing isoform, NCX1.4, even in the absence of NGF, induced an increase in Akt phosphorylation and GAP-43 protein expression. Interestingly, tetrodotoxin-sensitive Na⁺ currents and 1,3-benzenedicarboxylic acid, 4,4′-[1,4,10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis(5-methoxy-6,12-benzofurandiyl)]bis-, tetrakis[(acetyloxy)methyl] ester-detected [Na⁺]_i significantly increased in cells overexpressing NCX1.4 as well as ER Ca²⁺ content. This latter effect was prevented by tetrodotoxin. Furthermore, either the [Ca²⁺]_i chelator(1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid) (BAPTA-AM) or the PI3K inhibitor LY 294002 prevented Akt phosphorylation and GAP-43 protein expression rise in NCX1.4 overexpressing cells. Moreover, in primary cortical neurons, NCX1 silencing prevented Akt phosphorylation, GAP-43 and MAP2 overexpression, and neurite elongation. Collectively, these data show that NCX1 participates in neuronal differentiation through the modulation of ER Ca²⁺ content and PI3K signaling.