Crystal structure and mutational analysis of Ca2+-independent type II antifreeze protein from longsnout poacher, Brachyopsis rostratus

We recently found that longsnout poacher (Brachyosis rostratus) produces a Ca²⁺-independent type II antifreeze protein (lpAFP) and succeeded in expressing recombinant lpAFP using Phichia pastoris. Here, we report, for the first time, the X-ray crystal structure of lpAFP at 1.34 Å resolution. The lpAFP structure displayed a relatively planar surface, which encompasses two loop regions (Cys86-Lys89 and Asn91-Cys97) and a short β-strand (Trp109-Leu112) with three unstructured segments (Gly57-Ile58, Ala103-Ala104, and Pro113-His118). Electrostatic calculation of the protein surface showed that the relatively planar surface was divided roughly into a hydrophobic area (composed of the three unstructured segments lacking secondary structure) and a hydrophilic area (composed of the loops and β-strand). Site-directed mutation of Ile58 with Phe at the center of the hydrophobic area decreased activity significantly, whereas mutation of Leu112 with Phe at an intermediate area between the hydrophobic and hydrophilic areas retained complete activity. In the hydrophilic area, a peptide-swap mutant in the loops retained 60% activity despite simultaneous mutations of eight residues. We conclude that the epicenter of the ice-binding site of lpAFP is the hydrophobic region, which is centered by Ile58, in the relatively planar surface. We built an ice-binding model for lpAFP on the basis of a lattice match of ice and constrained water oxygen atoms surrounding the hydrophobic area in the lpAFP structure. The model in which lpAFP has been docked to a secondary prism (2-1-10) plane, which is different from the one determined for Ca²⁺-independent type II AFP from sea raven (11-21), appears to explain the results of the mutagenesis analysis.     

Integrated insights from simulation, experiment, and mutational analysis yield new details of LacI function

Protein structural change underlies many signal transduction processes. Although end-state structures are known for various allosteric proteins, intermediates are difficult to observe. Recently, targeted molecular dynamics simulation (TMD) was used to examine the conformational transition and predict relevant intermediates for wild-type lactose repressor (LacI). A catalog of involved residues suggests that the transition of this homodimer is asymmetric and that K84 is a prominent participant in the dynamic N-subdomain interface. Previous experiments indicated that hydrophobic substitutions at position 84 engender slowed, biphasic inducer binding kinetics, which might reflect the same phenomena observed in TMD. Here, we report biochemical confirmation that DNA and inducer binding remain allosterically linked in K84A and K84L, albeit with a differential smaller than that found in wild-type LacI. Other features of these mutant proteins are consistent with an allosteric conformational shift that approximates that of the wild type. As a consequence, these repressors can be utilized to explore an unanswered question about LacI function: How many inducers (one or two per dimer) are required to diminish operator affinity? The biphasic natures of the K84L and K84A inducer association rates allow direct correlation between the two distinct inducer binding events and operator release. Indeed, the kinetics of operator release for the K84A and K84L closely parallel those for the second inducer binding event. Together with implications from previous equilibrium results for wild-type and mutant proteins, these kinetic data demonstrate that binding of two inducers per dimeric DNA binding unit is required to release the operator in these variant LacI proteins.     

The crystal structure and mutational analysis of human NUDT9

Human ADP-ribose pyrophosphatase NUDT9 belongs to a superfamily of Nudix hydrolases that catabolize potentially toxic compounds in the cell. The enzyme hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5'-phosphate. NUDT9 shares 39% sequence identity with the C-terminal cytoplasmic domain of the ADPR-gated calcium channel TRPM2, which exhibits low but specific enzyme activity. We determined crystal structures of NUDT9 in the presence and in the absence of the reaction product ribose 5'-phosphate. On the basis of these structures and comparison with a bacterial homologue, a model of the substrate complex was built. The structure and activity of a double point mutant (R(229)E(230)F(231) to R(229)I(230)L(231)), which mimics the Nudix signature of the ion channel domain, was determined. Finally, the activities of a pair of additional mutated constructs were compared to the wild-type enzyme. The first corresponds to a minimal Nudix domain missing an N-terminal domain and C-terminal tail; the second disrupts two potential general bases in the active site. NUDT9 contains an N-terminal domain with a novel fold and a catalytic C-terminal Nudix domain. Unlike its closest functional homologue (homodimeric Escherichia coli ADPRase), it is active as a monomer, and the substrate is bound in a cleft between the domains. The structure of the RIL mutant provides structural basis for the reduced activity of the TRPM2 ion channel. The conformation and binding interactions of ADPR substrate are predicted to differ from those observed for E.coli ADPRase; mutation of structurally aligned acidic residues in their active sites produce significantly different effects on catalytic efficiency, indicating that their reaction pathways and mechanisms may have diverged.     

The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+ binding motif

The Na+/Ca2+ exchanger is a plasma membrane protein that regulates intracellular Ca2+ levels in cardiac myocytes. Transport activity is governed by Ca2+, and the primary Ca2+ sensor (CBD1) is located in a large cytoplasmic loop connecting two transmembrane helices. The binding of Ca2+ to the CBD1 sensory domain results in conformational changes that stimulate the exchanger to extrude Ca2+. Here, we present a crystal structure of CBD1 at 2.5A resolution, which reveals a novel Ca2+ binding site consisting of four Ca2+ ions arranged in a tight planar cluster. This intricate coordination pattern for a Ca2+ binding cluster is indicative of a highly sensitive Ca2+ sensor and may represent a general platform for Ca2+ sensing.     

Ca2+ dependence of the Ca2+-selective TRPV6 channel

Microfluorimetry and patch-clamp experiments were performed on TRPV6-expressing HEK cells to determine whether this Ca(2+)-sensing Ca(2+) channel is constitutively active. Intact cells loaded with fura-2 had an elevated intracellular free Ca(2+) concentration ([Ca(2+)](i)), which decreased to the same level such as in non-transfected cells if external Ca(2+) was chelated by EGTA. Whole cell recordings from non-transfected HEK cells and cells expressing human TRPV6 revealed the presence of a basal inward current in both types of cells when the internal solution contained 0.1 mm EGTA and 100 nm [Ca(2+)](i) or if the cytosolic Ca(2+) buffering remained undisturbed in perforated patch-clamp experiments. If recombinantly expressed TRPV6 forms open channels, one would expect Ca(2+)-induced current inhibition, because TRPV6 is negatively regulated by internal Ca(2+). However, dialyzing solutions with high [Ca(2+)] such as 1 microm into TRPV6-expressing cells did not block the basal inward current, which was not different from the recordings from non-transfected cells. In contrast, dialyzing 0.5 mm EGTA into TRPV6-expressing cells readily activated Ca(2+) inward currents, which were undetectable in non-transfected cells. Interestingly, monovalent cations permeated the TRPV6 channels under conditions where no Ca(2+) permeation was detectable, indicating that divalent cations block TRPV6 channels from the extracellular side. Like human TRPV6, the truncated human TRPV6(Delta695-725), which lacks the C-terminal domain required for Ca(2+)-calmodulin binding, does not form constitutive active channels, whereas the human TRPV6(D542A), carrying a point mutation in the presumed pore region, does not function as a channel. In summary, no constitutive open TRPV6 channels were detected in patch-clamp experiments from transfected HEK cells. However, channel activity is highly regulated by intracellular and extracellular divalent cations.     

The crystal structure at 2A resolution of the Ca2+ -binding protein S100P

S100P is a small calcium-binding protein of the S100 EF-hand-containing family of proteins. Elevated levels of its mRNA are reported to be associated with the progression to hormone independence and metastasis of prostate cancer and to be associated with loss of senescence in human breast epithelial cells in vitro. The first structure of human recombinant S100P in calcium-bound form is now reported at 2.0A resolution by X-ray diffraction. A flexible linker connects the two EF-hand motifs. The protein exists as a homodimer formed by non-covalent interactions between large hydrophobic areas on monomeric S100P. Experiments with an optical biosensor to study binding parameters of the S100P monomer interaction showed that the association rate constant was faster in the presence of calcium than in their absence, whereas the dissociation rate constant was independent of calcium. The K(d) values were 64(+/-24)nM and 2.5(+/-0.8) microM in the presence and in the absence of calcium ions, respectively. Dimerization of S100P is demonstrated in vivo using the yeast two-hybrid system. The effect of mutation of specific amino acids suggests that dimerization in vivo can be affected by amino acids on the dimer interface and in the hydrophobic core.     

Intraluminal Ca2+ dependence of Ca2+ and ryanodine-mediated regulation of skeletal muscle sarcoplasmic reticulum Ca2+ release.

The action of ryanodine upon sarcoplasmic reticulum (SR) Ca2+ handling is controversial with evidence for both activation and inhibition of SR Ca2+ release. In this study, the role of the intraluminal SR Ca2+ load was probed as a potential regulator of ryanodine-mediated effects upon SR Ca2+ release. Through dual-wavelength spectroscopy of Ca2+:antipyrylazo III difference absorbance, the intraluminal Ca2+ dependence of ryanodine and Ca(2+)-induced Ca2+ release (CICR) from skeletal SR vesicles was examined. Ryanodine addition after initiation of Ca2+ uptake (a) increased the intraluminal Ca2+ sensitivity of CICR and (b) stimulated spontaneous Ca2+ release with a delayed onset. These ryanodine effects were inversely proportional to the intraluminal Ca2+ load. Ryanodine also inhibited subsequent CICR after reaccumulation of Ca2+ released from the initial CICR. These results provide evidence that ryanodine inhibits transitions between low and high affinity Ca2+ binding states of an intraluminal Ca2+ compartment, possibly calsequestrin. Conformational transitions of calsequestrin may be reciprocally coupled to transitions between open and closed states of the Ca2+ release channel.     

A molecular dynamics study of the effect of Ca2+ removal on calmodulin structure

Calmodulin is a small (148 residues), ubiquitous, highly-conserved Ca(2+) binding protein serving as a modulator of many calcium-dependent processes. In this study, we followed, by means of molecular dynamics, the structural stability of the protein when one of its four bound Ca(2+) ions is removed, and compared it to a simulation of the fully Ca(2+) bound protein. We found that the removal of a single Ca(2+) ion from the N-lobe of the protein, which has a lower affinity for the ion, is sufficient to initiate a considerable structural rearrangement. Although the overall structure of the fully 4 Ca(2+) bound protein remained intact in the extended conformation, the Ca(2+)-removed protein changed its conformation into a compact state. The observation that the 3 Ca(2+) loaded protein assumes a compacted solution state is in accord with experimental observation that the NSCP protein, which binds only three Ca(2+) ions, is natively in a compact state. Examination of the folding dynamics reveals a cooperation between the C-lobe, N-lobe, and the interdomain helix that enable the conformation change. The forces driving this conformational change are discussed.     

电压门控钙通道钙依赖性易化和失活的分子机制

电压门控钙通道受钙依赖性易化和失活两种相互对立的反馈机制调节.不同浓度的钙离子,通过作为钙感受器的钙调蛋白的介导,主要与钙通道α1亚基羧基端的多个不连续片段发生复杂的相互作用,分别引发钙依赖性易化和失活.钙/钙调蛋白依赖性蛋白激酶Ⅱ及其它钙结合蛋白等也参与此调节过程.新近研究表明,钙通道的钙依赖性调节机制失衡与心律失常等的发病机制密切相关.     

The crystal structure and mutational binding analysis of the extracellular domain of the platelet-activating receptor CLEC-2

The human C-type lectin-like molecule CLEC-2 is expressed on the surface of platelets and signaling through CLEC-2 causes platelet activation and aggregation. CLEC-2 is a receptor for the platelet-aggregating snake venom protein rhodocytin. It is also a newly identified co-receptor for human immunodeficiency virus type 1 (HIV-1). An endogenous ligand has not yet been identified. We have solved the crystal structure of the extracellular domain of CLEC-2 to 1.6-A resolution, and identified the key structural features involved in ligand binding. A semi-helical loop region and flanking residues dominate the surface that is available for ligand binding. The precise distribution of hydrophobic and electrostatic features in this loop will determine the nature of any endogenous ligand with which it can interact. Major ligand-induced conformational change in CLEC-2 is unlikely as its overall fold is compact and robust. However, ligand binding could induce a tilt of a 3-10 helical portion of the long loop region. Mutational analysis and surface plasmon resonance binding studies support these observations. This study provides a framework for understanding the effects of rhodocytin venom binding on CLEC-2 and for understanding the nature of likely endogenous ligands and will provide a basis for rational design of drugs to block ligand binding.     

The bell-shaped Ca2+ dependence of the inositol 1,4, 5-trisphosphate-induced Ca2+ release is modulated by Ca2+/calmodulin.

Calmodulin inhibits inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor in both a Ca2+-dependent and a Ca2+-independent way. Because there are no functional data on the modulation of the IP3-induced Ca2+ release by calmodulin at various Ca2+ concentrations, we have studied how cytosolic Ca2+ and Sr2+ interfere with the effects of calmodulin on the IP3-induced Ca2+ release in permeabilized A7r5 cells. We now report that calmodulin inhibited Ca2+ release through the IP3 receptor with an IC50 of 4.6 microM if the cytosolic Ca2+ concentration was 0.3 microM or higher. This inhibition was particularly pronounced at low IP3 concentrations. In contrast, calmodulin did not affect IP3-induced Ca2+ release if the cytosolic Ca2+ concentration was below 0.3 microM. Calmodulin also inhibited Ca2+ release through the IP3 receptor in the presence of at least 10 microM Sr2+. We conclude that cytosolic Ca2+ or Sr2+ are absolutely required for the calmodulin-induced inhibition of the IP3-induced Ca2+ release and that this dependence represents the formation of the Ca2+/calmodulin or Sr2+/calmodulin complex.     

Monoclonal antibodies that coimmunoprecipitate the 1,4-dihydropyridine and phenylalkylamine receptors and reveal the Ca2+ channel structure

Monoclonal hybridoma cell lines secreting antibodies against the (+)-PN 200-110 and the (-)-demethoxyverapamil binding components of the voltage-dependent calcium channel from rabbit transverse-tubule membranes have been isolated. The specificity of these monoclonal antibodies was established by their ability to coimmunoprecipitate (+)-[3H]PN 200-110 and (-)-[3H]demethoxyverapamil receptors. Monoclonal antibodies described in this work cross-reacted with rat, mouse, chicken, and frog skeletal muscle Ca2+ channels but not with crayfish muscle Ca2+ channels. Cross-reactivity was also detected with membranes prepared from rabbit heart, brain, and intestinal smooth muscle. These antibodies were used in immunoprecipitation experiments with 125I-labeled detergent [3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin] solubilized membranes. They revealed a single immunoprecipitating component of molecular weight (Mr) 170,000 in nonreducing conditions. After disulfide bridge reduction the CHAPS-solubilized (+)-PN 200-110-(-)-demethoxyverapamil binding component gave rise to a large peptide of Mr 140,000 and to smaller polypeptides of Mr 30,000 and 26,000 whereas the digitonin-solubilized receptor appeared with subunits at Mr 170,000, 140,000, 30,000, and 26,000. All these results taken together are interpreted as showing that both the 1,4-dihydropyridine and the phenylalkylamine receptors are part of a single polypeptide chain of Mr 170,000.     

Phospholipase A2 from sheep erythrocyte membranes. Ca2+ dependence and localization.

The calcium dependence and the time course of phosphatidylethanolamine and phosphatidylcholine degradation by sheep erythrocyte membrane suspensions in presence of Triton X-100 were investigated. One enzyme with phospholipase A2 specificity was found to be responsible for both phosphatidyl-ethanolamine and phosphatidylcholine degradation. The localization of this enzyme in the membrane of the sheep erythrocyte was investigated by proteolytic treatment of sealed erythrocyte ghosts from the outside and of ghosts which had both sides of the membrane exposed to chymotrypsin. The inability of sealed ghosts to take up chymotrypsin was followed by flux measurements of [14C]dextran carboxyl previously trapped in the ghosts. No efflux of the marker was found during the proteolytic treatment. By comparing the residual phospholipase activities in the membranes from both ghost preparations, we concluded that the phospholipase is oriented to the exterior of the sheep erythrocyte.     

Ca2+-calmodulin-dependent facilitation and Ca2+ inactivation of Ca2+ release-activated Ca2+ channels

In non-excitable cells, one major route for Ca2+ influx is through store-operated Ca2+ channels in the plasma membrane. These channels are activated by the emptying of intracellular Ca2+ stores, and in some cell types store-operated influx occurs through Ca2+ release-activated Ca2+ (CRAC) channels. Here, we report that intracellular Ca2+ modulates CRAC channel activity through both positive and negative feedback steps in RBL-1 cells. Under conditions in which cytoplasmic Ca2+ concentration can fluctuate freely, we find that store-operated Ca2+ entry is impaired either following overexpression of a dominant negative calmodulin mutant or following whole-cell dialysis with a calmodulin inhibitory peptide. The peptide had no inhibitory effect when intracellular Ca2+ was buffered strongly at low levels. Hence, Ca2+-calmodulin is not required for the activation of CRAC channels per se but is an important regulator under physiological conditions. We also find that the plasma membrane Ca2+ATPase is the dominant Ca2+ efflux pathway in these cells. Although the activity of the Ca2+ pump is regulated by calmodulin, the store-operated Ca2+ entry is more sensitive to inhibition by the calmodulin mutant than by Ca2+ extrusion. Hence, these two plasmalemmal Ca2+ transport systems may differ in their sensitivities to endogenous calmodulin. Following the activation of Ca2+ entry, the rise in intracellular Ca2+ subsequently feeds back to further inhibit Ca2+ influx. This slow inactivation can be activated by a relatively brief Ca2+ influx (30-60 s); it reverses slowly and is not altered by overexpression of the calmodulin mutant. Hence, the same messenger, intracellular Ca2+, can both facilitate and inactivate Ca2+ entry through store-operated CRAC channels and through different mechanisms.     

Lipid structure and Ca2+-ATPase function

Effects of lipid structure on the function of the Ca²⁺-ATPase of skeletal muscle of sarcoplasmic reticulum are reviewed. Binding of phospholipids to the ATPase shows little specificity. Phosphatidylcholines with short (C14) or long (C24) fatty acyl chains have marked effects on the activity of the ATPase, including a change in the stoichiometry of Ca binding. Low ATPase activity in gel phase lipid follows from low rate of phosphorylation. Phosphatidylinositol 4-phosphate increases ATPase activity by increasing the rate of dephosphorylation of the phosphorylated ATPase. Stimulation is not seen with other anionic phospholipids; phosphatidic acid decreases ATPase activity in a Mg²⁺-dependent manner.Abbreviations di(C141)PC dimyristoleoylphosphatidycholine - di(C160)PC dipalmitoylphosphatidylcholine - di(C181)PC dioleoylphosphatidylcholine - di(Br₂C180)PC dibromostearoylphosphatidylcholine - di(C241)PC dinervonylphosphatidylcholine - di(C181)PA dioleoylphosphatidic acid - di(C181)PE dioleoylphosphatidylethanolamine - Ptdlns phosphatidylinositol - PtdIns-4P phos-phatidylinositol 4-phosphate     

Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca2+ release in renal epithelial LLC-PK1 cells

We have studied arginine vasopressin (AVP)-, thapsigargin- and inositol 1,4,5-trisphosphate (InsP₃)-mediated Ca²⁺ release in renal epithelial LLC-PK₁ cells. AVP-induced changes in the intracellular free calcium concentration ([Ca²⁺]_i) were studied in indo-1 loaded single cells by confocal laser cytometry. AVP-mediated Ca²⁺ mobilization was also observed in the absence of extracellular Ca²⁺, but was completely abolished after depletion of the intracellular Ca²⁺ stores by 2 μM thapsigargin. Using ⁴⁵Ca²⁺ fluxes in saponin-permeabilized cell monolayers, we have analysed how InsP₃ affected the Ca²⁺ content of nonmitochondrial Ca²⁺ pools in different loading and release conditions. Less than 10% of the Ca²⁺ was taken up in a thapsigargin-insensitive pool when loading was performed in a medium containing 0.1 μM Ca²⁺. The thapsigargin-insensitive compartment amounted to 35% in the presence of 110 μM Ca²⁺, but Ca²⁺ sequestered in this pool could not be released by InsP₃. The thapsigargin-sensitive Ca²⁺ pool, in contrast, was nearly completely InsP₃ sensitive. A submaximal [InsP₃], however, released only a fraction of the sequestered Ca²⁺. This fraction was dependent on the cytosolic as well as on the luminal [Ca²⁺]. The cytosolic free [Ca²⁺] affected the InsP₃-induced Ca²⁺ release in a biphasic way. Maximal sensitivity toward InsP₃ was found at a free cytosolic [Ca²⁺] between 0.1 and 0.5 μM, whereas higher cytosolic [Ca²⁺] decreased the InsP₃ sensitivity. Other divalent cations or La³⁺ did not provoke similar inhibitory effects on InsP₃-induced Ca²⁺ release. The luminal free [Ca²⁺] was manipulated by varying the time of incubation of Ca²⁺ -loaded cells in an EGTA-containing medium. Reduction of the Ca²⁺ content to one-third of its initial value resulted in a fivefold decrease in the InsP₃ sensitivity of the Ca²⁺ release. © 1993 Wiley-Liss, Inc.     

Relationships between Ca2+ release, Ca2+ cycling, and Ca2+-mediated permeability changes in mitochondria

Ruthenium red and/or EGTA prevent cyclic uptake and release of Ca2+ in mitochondria. These compounds inhibit but do not prevent the swelling of liver mitochondria induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. Ruthenium red and/or EGTA have complex effects on the release rate of Ca2+ and other cations induced by t-butyl hydroperoxide or N-ethylmaleimide. To determine the relationship between permeability changes and Ca2+ release in the absence of Ca2+ cycling, a novel method of data collection and analysis is developed which allows the relative time courses of Ca2+ release and Mg2+ release or swelling to be accurately and quantitatively compared. This method eliminates errors in time course comparisons which arise from the aging of mitochondrial preparations and allows data from different preparations to be directly contrasted. Using the method, it is shown that permeability changes caused by Ca2+-releasing agents are not secondary effects arising from Ca2+ cycling between uptake and release carriers. In the absence of Ca2+-cycling inhibitors, Ca2+ release induced by t-butyl hydroperoxide or N-ethylmaleimide is, in part, carrier-mediated. In the presence of EGTA and ruthenium red, Ca2+ release induced by either agent is mediated solely by the permeability pathway. No differences are apparent in the solute selectivity of the inner membrane permeability defect induced by Ca2+ plus t-butyl hydroperoxide or Ca2+ plus N-ethylmaleimide. A novel type of Ca2+ release from energized liver mitochondria is reported. This release is induced by EGTA, occurs in the absence of other releasing agents or nonspecific permeability changes, and is rapid (greater than or equal to 50 nmol/min/mg protein).     

Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs.

Monospecific antibodies directed against several U small nuclear ribonucleoprotein (U snRNP) particle proteins were affinity purified from a patient's anti-(U1,U2)RNP serum. These were used to demonstrate that: (i) proteins equivalent to the mammalian U2 snRNP-specific A' and B" proteins are present in Xenopus laevis oocytes; (ii) both proteins A' and B" have the same structural requirements for binding to U2 snRNA; (iii) proteins B, B' and D have the same structural requirement for binding to U2 snRNA; (iv) using very high specific activity RNA probes it is possible to detect a fraction of either U1 or U2 snRNA precipitable by antibodies directed against proteins specific for the other U snRNP, indicating an interaction between U1 and U2 snRNPs. The structural requirements of this interaction were studied for the U2 snRNP. All changes made to U2 snRNA or snRNP structure resulted in loss of the interaction with U1 snRNP.     

Sustained Ca2+ transfer across mitochondria is Essential for mitochondrial Ca2+ buffering,sore-operated Ca2+ entry,and Ca2+ store refilling

Mitochondria have been found to sequester and release Ca2+ during cell stimulation with inositol 1,4,5-triphosphate-generating agonists, thereby generating subplasmalemmal microdomains of low Ca2+ that sustain activity of capacitative Ca2+ entry (CCE). Procedures that prevent mitochondrial Ca2+ uptake inhibit local Ca2+ buffering and CCE, but it is not clear whether Ca2+ has to transit through or remains trapped in the mitochondria. Thus, we analyzed the contribution of mitochondrial Ca2+ efflux on the ability of mitochondria to buffer subplasmalemmal Ca2+, to maintain CCE, and to facilitate endoplasmic reticulum (ER) refilling in endothelial cells. Upon the addition of histamine, the initial mitochondrial Ca2+ transient, monitored with ratio-metric-pericam-mitochondria, was largely independent of extracellular Ca2+. However, subsequent removal of extracellular Ca2+ produced a reversible decrease in [Ca2+]mito, indicating that Ca2+ was continuously taken up and released by mitochondria, although [Ca2+]mito had returned to basal levels. Accordingly, inhibition of the mitochondrial Na+/Ca2+ exchanger with CGP 37157 increased [Ca2+]mito and abolished the ability of mitochondria to buffer subplasmalemmal Ca2+, resulting in an increased activity of BKCa channels and a decrease in CCE. Hence, CGP 37157 also reversibly inhibited ER refilling during cell stimulation. These effects of CGP 37157 were mimicked if mitochondrial Ca2+ uptake was prevented with oligomycin/antimycin A. Thus, during cell stimulation a continuous Ca2+ flux through mitochondria underlies the ability of mitochondria to generate subplasmalemmal microdomains of low Ca2+, to facilitate CCE, and to relay Ca2+ from the plasma membrane to the ER.     

A family of Ca2+-dependent activator proteins for secretion: comparative analysis of structure, expression, localization, and function

Ca2+-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.