Calcium-calmodulin dependent protein kinase II (CaMKII): a main signal responsible for early reperfusion arrhythmias.

To explore whether CaMKII-dependent phosphorylation events mediate reperfusion arrhythmias, Langendorff perfused hearts were submitted to global ischemia/reperfusion. Epicardial monophasic or transmembrane action potentials and contractility were recorded. In rat hearts, reperfusion significantly increased the number of premature beats (PBs) relative to pre-ischemic values. This arrhythmic pattern was associated with a significant increase in CaMKII-dependent phosphorylation of Ser2814 on Ca(2+)-release channels (RyR2) and Thr17 on phospholamban (PLN) at the sarcoplasmic reticulum (SR). These phenomena could be prevented by the CaMKII-inhibitor KN-93. In transgenic mice with targeted inhibition of CaMKII at the SR membranes (SR-AIP), PBs were significantly decreased from 31±6 to 5±1 beats/3min with a virtually complete disappearance of early-afterdepolarizations (EADs). In mice with genetic mutation of the CaMKII phosphorylation site on RyR2 (RyR2-S2814A), PBs decreased by 51.0±14.7%. In contrast, the number of PBs upon reperfusion did not change in transgenic mice with ablation of both PLN phosphorylation sites (PLN-DM). The experiments in SR-AIP mice, in which the CaMKII inhibitor peptide is anchored in the SR membrane but also inhibits CaMKII regulation of L-type Ca(2+) channels, indicated a critical role of CaMKII-dependent phosphorylation of SR proteins and/or L-type Ca(2+) channels in reperfusion arrhythmias. The experiments in RyR2-S2814A further indicate that up to 60% of PBs related to CaMKII are dependent on the phosphorylation of RyR2-Ser2814 site and could be ascribed to delayed-afterdepolarizations (DADs). Moreover, phosphorylation of PLN-Thr17 and L-type Ca(2+) channels might contribute to reperfusion-induced PBs, by increasing SR Ca(2+) content and Ca(2+) influx.

Increased intracellular Ca2+ and SR Ca2+ load contribute to arrhythmias after acidosis in rat heart. Role of Ca2+/calmodulin-dependent protein kinase II.

Returning to normal pH after acidosis, similar to reperfusion after ischemia, is prone to arrhythmias. The type and mechanisms of these arrhythmias have never been explored and were the aim of the present work. Langendorff-perfused rat/mice hearts and rat-isolated myocytes were subjected to respiratory acidosis and then returned to normal pH. Monophasic action potentials and left ventricular developed pressure were recorded. The removal of acidosis provoked ectopic beats that were blunted by 1 muM of the CaMKII inhibitor KN-93, 1 muM thapsigargin, to inhibit sarcoplasmic reticulum (SR) Ca(2+) uptake, and 30 nM ryanodine or 45 muM dantrolene, to inhibit SR Ca(2+) release and were not observed in a transgenic mouse model with inhibition of CaMKII targeted to the SR. Acidosis increased the phosphorylation of Thr(17) site of phospholamban (PT-PLN) and SR Ca(2+) load. Both effects were precluded by KN-93. The return to normal pH was associated with an increase in SR Ca(2+) leak, when compared with that of control or with acidosis at the same SR Ca(2+) content. Ca(2+) leak occurred without changes in the phosphorylation of ryanodine receptors type 2 (RyR2) and was blunted by KN-93. Experiments in planar lipid bilayers confirmed the reversible inhibitory effect of acidosis on RyR2. Ectopic activity was triggered by membrane depolarizations (delayed afterdepolarizations), primarily occurring in epicardium and were prevented by KN-93. The results reveal that arrhythmias after acidosis are dependent on CaMKII activation and are associated with an increase in SR Ca(2+) load, which appears to be mainly due to the increase in PT-PLN.

Secretion of annexin V from cultured cells requires a signal peptide.

Annexin V is an intracellular protein that lacks a hydrophobic signal peptide. However, there are several studies reporting the extracellular presence of annexin V. In this study, we designed transgenes of annexin V with or without an attached secretory signal peptide and investigated the secretion of the transgene products in COS-7 cells. The signal peptide, targeted annexin V to the endoplasmic reticulum (ER), the Golgi and culture media of transfected cells. In contrast, without the signal peptide, annexin V was present only in the cytoplasm and was not detected in the medium. To confirm our results we also evaluated the presence of extracellular annexin V in two cultured cell lines: BeWo, a choriocarcinoma cell model of placental trophoblasts, and human umbilical vein endothelial cells (HUVEC). Our results showed that annexin V was immunolocalized on the surfaces of both cells but could not be detected in the culture medium of either cell type. Our results suggest that the secretion of annexin V required the recombinant addition of a hydrophobic signal peptide and that the limited quantities of endogenous cell surface annexin V on BeWo and HUVEC cells is most likely derived from adjacent damaged cells.

Annexin V--heparin oligosaccharide complex suggests heparan sulfate--mediated assembly on cell surfaces.

BACKGROUND: Annexin V, an abundant anticoagulant protein, has been proposed to exert its effects by self-assembling into highly ordered arrays on phospholipid membranes to form a protective anti-thrombotic shield at the cell surface. The protein exhibits very high-affinity calcium-dependent interactions with acidic phospholipid membranes, as well as specific binding to glycosaminoglycans (GAGs) such as heparin and heparan sulfate, a major component of cell surface proteoglycans. At present, there is no structural information to elucidate this interaction or the role it may play in annexin V function at the cell surface. RESULTS: We report the 1.9 A crystal structure of annexin V in complex with heparin-derived tetrasaccharides. This structure represents the first of a heparin oligosaccharide binding to a protein where calcium ions are essential for the interaction. Two distinct GAG binding sites are situated on opposite protein surfaces. Basic residues at each site were identified from the structure and site-directed mutants were prepared. The heparin binding properties of these mutants were measured by surface plasmon resonance. The results confirm the roles of these mutated residues in heparin binding, and the kinetic and thermodynamic data define the functionally distinct character of each distal binding surface. CONCLUSION: The annexin V molecule, as it self-assembles into an organized array on the membrane surface, can bind the heparan sulfate components of cell surface proteoglycans. A novel model is presented in which proteoglycan heparan sulfate could assist in the localization of annexin V to the cell surface membrane and/or stabilization of the entire molecular assembly to promote anticoagulation.

Phosphorylation mutants elucidate the mechanism of annexin IV-mediated membrane aggregation.

Site-directed mutagenesis, electron microscopy, and X-ray crystallography were used to probe the structural basis of annexin IV-induced membrane aggregation and the inhibition of this property by protein kinase C phosphorylation. Site-directed mutants that either mimic (Thr6Asp, T6D) or prevent (Thr6Ala, T6A) phosphorylation of threonine 6 were produced for these studies and compared with wild-type annexin IV. In vitro assays showed that unmodified wild-type annexin IV and the T6A mutant, but not PKC-phosphorylated wild-type or the T6D mutant, promote vesicle aggregation. Electron crystallographic data of wild-type and T6D annexin IV revealed that, similar to annexin V, the annexin IV proteins form 2D trimer-based ordered arrays on phospholipid monolayers. Cryo-electron microscopic images of junctions formed between lipid vesicles in the presence of wild-type annexin IV indicated a separation distance corresponding to the thickness of two layers of membrane-bound annexin IV. In this orientation, a single layer of WT annexin IV, attached to the outer leaflet of one vesicle, would undergo face-to-face self-association with the annexin layer of a second vesicle. The 2.0-A resolution crystal structure of the T6D mutant showed that the mutation causes release of the N-terminal tail from the protein core. This change would preclude the face-to-face annexin self-association required to aggregate vesicles. The data suggest that reversible complex formation through phosphorylation and dephosphorylation could occur in vivo and play a role in the regulation of vesicle trafficking following changes in physiological states.

Targeted neutralization of calcineurin, by expression of an inhibitor peptide under the control of a cholinergic specific promoter in PC12 cells, promotes neurite outgrowth in the presence of NGF.

We have characterized a region of the mouse vesicular acetylcholine transporter(VAChT)/choline acetyltransferase (ChAT) gene locus that serves as a cholinergic-specific promoter for the expression of both VAChT and ChAT genes, as well as a reporter gene (LacZ) in vivo. We have used this promoter to direct the expression of an inhibitor peptide, derived from the calcineurin (CalN) autoregulatory domain, to directly neutralize the function of CalN to define the role of this Ca2+/Calmodulin regulated phosphatase in neurite outgrowth. Targeted inhibition of CalN promotes neurite outgrowth in PC12 cells in the presence of NGF, as early as 24 h after transfection. Inhibition of CalN-mediated enhancement of neurite outgrowth in PC12 cells reaches a maximum effect within the first 4 to 6 days after transfection, and does not cause adverse effects when highly expressed for up to 12 days. Cyclosporin A, a nontargeted CalN inhibitor, increases the number of neurites in mock transfected cells by 1.5 fold, while in transfected PC12 cells, the expression of the CalN inhibitor peptide increases the neurite number by 1.8 fold. These data demonstrate that CalN is an important regulator of the neurotrophic response in cholinergic cells and may prove valuable in developing treatment strategies to promote recovery from neurological injury.

Mutation of highly conserved arginine residues disrupts the structure and function of annexin V.

BACKGROUND: Annexins are a family of structurally related proteins that bind to phospholipid membranes in a Ca(2+)-dependent manner. Annexins are characterized by highly conserved canonical domains of approximately 70 amino acids. Annexin V contains four such domains. Each of these domains has a highly conserved arginine (R). METHODS: To evaluate the role of the conserved arginines in the molecular structure of annexin V, negatively charged amino acids were substituted for arginines at positions R43, R115, R199, and R274 using site-directed mutagenesis. RESULTS: Mutants R199D and R274E were rapidly degraded when expressed in bacteria, and were not further characterized. R43E exhibited an electrophoretic mobility similar to the wild-type protein, while R115E migrated significantly in a slower fashion, suggesting a less compact conformation. R43E and R115E exhibited much greater susceptibility to proteolytic digestion than the wild type. While Ca(2+)-dependence for phospholipid binding was similar in both mutants (half-maximal 50-80 microM Ca2+), R43E and R115E exhibited a 6- and 2-fold decrease in phospholipid affinity, respectively. Consistent with the different phospholipid affinities of the annexins, a phospholipid-dependent clotting reaction, the activated partial thromboplastin time (aPTT), was significantly prolonged by the wild-type protein and mutants R115E and R115A. The aPTT was unaffected by R43E. CONCLUSIONS: Our data suggest that mutation of these highly conserved arginine residues in each of the four canonical domains of annexin have differential effects on the phospholipid binding, tertiary structure, and proteolytic susceptibility of annexin V. The site I mutation, R43E, produced a large decrease in phospholipid affinity associated with an increase in proteolytic susceptibility. The site II mutation, R115E, produced a small change in phospholipid binding but a significant modification of electrophoretic mobility. Our data suggest that highly conserved arginine residues are required to stabilize the tertiary structure of annexin V by establishing hydrogen bonds and ionic bridges.

Phospholipid membranes form specific nonbilayer molecular arrangements that are antigenic.

Hexagonal phase (H(II))-preferring lipids such as phosphatidate, cardiolipin, and phosphatidylserine form nonbilayer molecular arrangements in lipid bilayers. While their presence in biological membranes has not been established, in vitro studies suggest that alterations in membrane properties modify their function. In this study, antiphospholipid monoclonal antibodies were developed against nonbilayer structures. One of the monoclonal antibodies identifies nonplanar surfaces in liposomes and in membranes of cultured cells. These results are the first evidence that natural membranes maintain a fragile balance between bilayer and nonbilayer lipid arrangements. Therefore, these antibodies can be used to evaluate the role of H(II)-preferring lipids in the modulation of membrane activities. Our studies demonstrated that nonplanar surfaces are highly immunogenic. Although these structures are normally transient, their formation can be stabilized by temperature variations, drugs, antibiotics, apolar peptides, and divalent cations. Our studies demonstrated that abnormal exposure of nonbilayer arrangements may induce autoimmune responses as found in the antiphospholipid syndrome.

Annexin V is critical in the maintenance of murine placental integrity.

OBJECTIVES: Recurrent fetal loss can be a consequence of placental thrombosis, frequently occurring in autoimmune disorders such as antiphospholipid syndrome. A potent anticoagulant, annexin V, is abundant in placental tissues. We investigated the role of annexin V in maintaining fetal viability. STUDY DESIGN: Sites of annexin V activity in placenta were found and neutralized, and the physiologic consequences on fetal development were evaluated. To find extracellular binding sites for annexin V on placental membrane, 2 approaches were taken. An epitope-tagged recombinant annexin V was infused into pregnant BALB/c mice. Endogenous annexin V was evaluated by immunohistochemical techniques. To define a role for annexin V during pregnancy, annexin V was neutralized by tail-vein infusion of affinity-purified anti-annexin V antibodies immediately before mating, 16 hours before the vaginal plugs were observed. Fetal viability, number, and size were evaluated at days 11 or 15 after conception. RESULTS: Endogenous annexin V is enriched along the apical surfaces of trophoblasts. Animals infused with epitope-tagged annexin V had confirmed presence of extracellular binding sites for annexin V exclusively along these surfaces. In mice infused with anti-annexin V antibodies, various degrees of fetal absorption were observed. Thrombosis and necrosis were present in the fetal component of placentas from partially absorbed embryos. Focal necrosis and fibrosis were present in the decidua of placentas from embryos that were significantly smaller than the normal embryos in the same uterus. CONCLUSIONS: Apical surfaces of syncytiotrophoblasts in the placenta possess annexin V binding sites. The binding of annexin V to these coagulation-promoting surfaces is crucial for the maintenance of blood flow through the placenta and consequently for fetal viability. Infusion of anti-annexin V antibodies decreased the availability of annexin V to bind to the trophoblast surfaces and caused placental thrombosis, necrosis, and fetal loss. Our study suggests that anti-annexin V autoantibodies may contribute to recurrent pregnancy failure resulting from placental thrombosis, as found in patients with certain autoimmune diseases.

Identification and transgenic analysis of a murine promoter that targets cholinergic neuron expression.

Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using beta-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.

Mutational and crystallographic analyses of interfacial residues in annexin V suggest direct interactions with phospholipid membrane components.

Annexin V belongs to a family of eukaryotic calcium-dependent membrane-binding proteins. The calcium-binding sites at the annexin-membrane interface have been investigated in some detail; however, little is known about the functional roles of highly conserved interfacial residues that do not coordinate calcium themselves. In the present study, the importance of tryptophan 185, and threonine or serine at positions 72, 144, 228, and 303, in rat annexin V is investigated by site-directed mutagenesis, X-ray crystallography, and functional assays. The high-resolution crystal structures of the mutants show that the mutations do not cause structural perturbations of the annexin molecule itself or disappearance of bound calcium ions from calcium-binding sites. The assays indicate that relative to wild-type annexin V, loss of the methyl substituent at position 72 (Thr72-->Ser) has no effect while loss of the hydroxyl group (Thr72-->Ala or Thr72-->Lys) causes reduction of membrane binding. Multiple lysine substitutions (e.g., Thr72,Ser144,Ser228,Ser303-->Lys) have a greater adverse effect than the single lysine mutation, suggesting that in annexin V the introduction of potentially favorable electrostatic interactions between the lysine side chains and the net negatively charged membrane surface is not sufficient to overcome the loss of the hydroxyl side chains. Replacement of the unique tryptophan, Trp185, by alanine similarly decreases membrane binding affinity. Taken together, the data suggest that the side chains mutated in this study contribute to phospholipid binding and participate directly in intermolecular contacts with phospholipid membrane components.

Altered cardiac annexin mRNA and protein levels in the left ventricle of patients with end-stage heart failure.

Annexins are a unique family of membrane-associated, Ca2+ and phospholipid-binding proteins found in various tissues. Among the 12 isoforms, Annexin II, V and VI exist in heart tissue in the highest amounts. Annexin VI has been shown to affect intracellular Ca2+ cycling and contractility in isolated cardiomyocytes. Annexin V is present in both cardiomyocytes and non-myocyte cell types in the heart and may play a role in the regulation of cellular ion fluxes, organization and secretion, while the cardiac effects of annexin II are unclear. To identify changes in annexin II, V and VI isoforms that might occur in human heart failure, we measured mRNA and protein levels of these three annexins in transplanted left ventricular tissue of 12 patients with end-stage congestive heart failure due to coronary artery disease (CAD, n=6) or idiopathic dilated cardiomyopathy (DCM, n=6) who underwent cardiac transplantation. Normal heart tissue (C, n=6) was used as a control. Northern blot analyses showed a significant decrease (61%) in annexin VI mRNA levels in heart failure patients compared with controls (1.08+/-0.16 v 2.79+/-0.20 A.U.C. unit, determined by laser densitometry, mean+/-s.e.). In contrast, we found a 67% increase (2. 32+/-0.27 v 3.88+/-0.29) in annexin II mRNA levels and a two-fold increase (1.00+/-0.24 v 2.21+/-0.29) in annexin V mRNA levels in cardiomyopathic hearts as compared to normal hearts. Western blot analyses demonstrated a corresponding decrease (46.1%) in annexin VI protein levels in the heart failure group as compared to controls (2. 63+/-0.22 v 4.88+/-0.52), while annexin II protein levels showed a significant 40.7% increase in patients with heart failure compared to those in normal hearts (5.08+/-0.67 v 3.61+/-0.32). Annexin V protein levels were also significantly increased (45%) in heart failure patients compared with normal (2.14+/-0.19 v 1.48+/-0.11). No difference in either annexins II, V or VI mRNA and protein levels were found between CAD and DCM patients. We conclude that human end-stage heart failure is associated with a down regulation of annexin VI and up regulation of annexin II and V proteins. Coordinate changes were observed in steady-state mRNA levels. These results suggest that these annexin isoforms may contribute to the regulation of intracellular Ca2+ homeostasis in the cardiomyopathic heart.

Temporal inhibition of calmodulin in the nucleus.

Calmodulin (CaM) acts as a primary mediator of calcium signaling by interacting with target proteins. We have previously shown that nuclear CaM is critical for cell cycle progression using a transgene containing four repeats of a CaM inhibitor peptide and nuclear targeting signals (J. Wang et al., J. Biol. Chem. 270 (1995) 30245 30248; Biochim. Biophys. Acta 1313 (1996) 223-228). To evaluate the role of CaM in the nucleus specifically during S phase of the cell cycle, a motif which stabilizes the mRNA only during S phase was included in the transgene. The CaM inhibitor mRNA transcript contains a self-annealing stem-loop derived from histone H2B at the 3' end. This structure provides stability of the mRNA only during S phase, thereby restricting CaM inhibitor expression to S phase. The inhibitor accumulates in the nucleus, particularly in the nucleoli. Flow cytometric analysis demonstrated that the CaM inhibitor is expressed in S and G2. Transfected cells show growth inhibition and a reduction in DNA synthesis. The CaM inhibitor peptide is a versatile reagent that allows spatial as well as temporal dissection of calmodulin function.

Annexins.

The annexins are a family of proteins that bind anionic phospholipid surfaces in a Ca(2+)-dependent manner (general reviews include Raynal & Pollard 1994, Swairjo & Seaton 1994, Seaton 1996, Mollenhauer, 1997). Due to this functional property, individual annexins have been discovered independently by numerous laboratories with diverse experimental goals. Ca2+ characteristically causes the annexins to shift from a soluble to membrane associated state. This shift is believed to be the mechanism that underlies annexin cellular function.

Electrophorus electricus as a model system for the study of membrane excitability.

The stunning sensations produced by electric fish, particularly the electric eel, Electrophorus electricus, have fascinated scientists for centuries. Within the last 50 years, however, electric cells of Electrophorus have provided a unique model system that is both specialized and appropriate for the study of excitable cell membrane electrophysiology and biochemistry. Electric tissue generates whole animal electrical discharges by means of membrane potentials that are remarkably similar to those of mammalian neurons, myocytes and secretory cells. Electrocytes express ion channels, ATPases and signal transduction proteins common to these other excitable cells. Action potentials of electrocytes represent the specialized end function of electric tissue whereas other excitable cells use membrane potential changes to trigger sophisticated cellular processes, such as myofilament cross-bridging for contraction, or exocytosis for secretion. Because electric tissue lacks these functions and the proteins associated with them, it provides a highly specialized membrane model system. This review examines the basic mechanisms involved in the generation of the electrical discharge of the electric eel and the membrane proteins involved. The valuable contributions that electric tissue continues to make toward the understanding of excitable cell physiology and biochemistry are summarized, particularly those studies using electrocytes as a model system for the study of the regulation of membrane excitability by second messengers and signal transduction pathways.

A major second messenger mediator of Electrophorus electricus electric tissue is CaM kinase II.

Electric tissue of the electric eel, Electrophorus electricus, has been used extensively as a model system for the study of excitable membrane biochemistry and electrophysiology. Membrane receptors, ion channels, and ATPases utilized by electrocytes are conserved in mammalian neurons and myocytes. In this study, we show that Ca2+ predominates as the major mediator of electric tissue phosphorylation relative to cyclic AMP and cyclic GMP-induced phosphorylation. Mastoparan, a calmodulin inhibitor peptide, and a peptide corresponding to the pseudosubstrate region of mammalian calmodulin-dependent protein kinase II (CaMKII (281-302)) attenuated Ca(2+)-dependent phosphorylation in a dose-dependent manner. These experiments demonstrated that calmodulin-dependent protein kinase II activity predominates in electric tissue. The Electrophorus kinase was purified by a novel affinity chromatography procedure utilizing Ca2+/calmodulin-dependent binding to the CaMKII (281-302) peptide coupled to Sepharose. The purified 51 kDa calmodulin-dependent protein kinase II demonstrated extensive autophosphorylation and exhibited a 3- to 4-fold increase in Ca(2+)-independent activity following autophosphorylation. Immunofluorescent localization experiments demonstrated calmodulin to be abundant in electrocytes, particularly subjacent to the plasma membrane. Calmodulin-dependent protein kinase II had a punctate distribution indicating that it may be compartmentalized by association with vesicles or the cytoskeleton. As the primary mediator of phosphorylation within electric tissue, CaM kinase II may be critical for the regulation of the specialized electrophysiological function of electrocytes.

Regulation of gene expression by hypoxia: a molecular approach.

Oxygen is a strict requirement for cell function. The cellular mechanisms by which organisms detect and respond to changes in oxygen tension remain a major unanswered question in pulmonary physiology. Part of the difficulty in addressing this question is due to the limited scope of experiments that can be performed in vivo. In the past few years, several laboratories have begun to make progress in this area, using a variety of cell culture model systems and sophisticated genetic manipulations. Here, we review the current state of knowledge of regulation of gene expression by hypoxia, and describe novel experimental approaches that promise to broaden our understanding of how cells and whole organisms respond to alterations in O2 tension.

Molecular characterization of the mouse vesicular acetylcholine transporter gene.

The organization of the mouse choline acetyltransferase (ChAT) gene has been previously analyzed. Here we show that the first intron of the mouse ChAT gene contains an uninterrupted open reading frame. It is in the same transcriptional orientation as ChAT and encodes the vesicular acetylcholine (ACh) transporter (VAChT), the protein responsible for the translocation of cytoplasmic ACh into synaptic vesicles. The sequence of this transporter is very similar to the VAChT from rat and human (99% and 95% identity, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed expression of mouse VAChT mRNA in spinal cord, brain (excluding the cerebellum) and brain stem, but not in peripheral tissues such as liver and kidney. Transgenic mouse analysis revealed that the 5'-flanking region of the mouse ChAT gene encompasses regulatory elements that allowed elevated expression of VAChT in the cholinergic system of transgenic mice.

Annexins II and V inhibit cell migration.

Cell motility is a crucial component involved in wound healing, development, and tumor metastasis. This study investigated whether extracellular annexins, members of a calcium- and phospholipid-binding family of proteins, play a role in the migration of Lewis lung carcinoma cells. Using assays for wound closure and migration through 8-micron pores, it was found that annexins II and V significantly (> 40%) inhibited migration of these highly metastatic cells. Additionally, anti-annexin II antibodies enhanced migration of these same cells in the wound closure assay, while an irrelevant antibody (anti-calmodulin) showed no effect. These effects may be due to annexin-membrane binding and inhibition of phospholipid movement that is necessary for the formation of membrane protrusions.

Annexin VI modulates Ca2+ and K+ conductances of spinal cord and dorsal root ganglion neurons.

Annexin VI is a member of a Ca(2+)-dependent phospholipid-binding protein family that participates in the transduction of the intracellular Ca2+ signal. We have identified annexin VI as one of the major annexins expressed differentially by sensory neurons of dorsal root ganglia (DRG) and by neurons of spinal cord (SC) of the rat and the mouse. This annexin shows a preferential localization at the plasma membrane of the soma and cellular processes, particularly in motoneurons of the SC. This finding suggests an active role of annexin VI in the Ca(2+)-dependent regulation of plasma membrane functions. To test this possibility, the neuronal function of annexin VI was evaluated by whole cell electrophysiology of mouse embryo SC and DRG neurons. An antibody was developed that has the property of neutralizing annexin VI-phospholipid interactions. The intracellular perfusion of individual neurons in culture, either from SC or DRG, with monospecific affinity-purified anti-annexin VI antibodies resulted in an increase in the magnitude of the K+ current and in an increase in the Ca2+ current in sensory neurons. Our results suggest that the endogenous annexin VI regulates the Ca2+ conductance, which indirectly modifies Ca(2+)-dependent ionic conductances in SC and DRG neurons.