Neuron-specific antigen HPC-1 from bovine brain reveals strong homology to epimorphin, an essential factor involved in epithelial morphogenesis: identification of a novel protein family.

We have already cloned the cDNA for the HPC-1 antigen, a neuron-specific protein antigen from the rat brain. Here we report the molecular cloning of the bovine HPC-1 antigen homologue, and much strong sequence conservation between rat and bovine. By searching the recent protein data base, it was found that the HPC-1 antigen revealed unusual similarity to epimorphin which was mesenchymal factor related to the morphogenesis of primitive epidermal tissues in embryonic stages. We also found that the HPC-1 antigen was identical to p35A (syntaxin) which bound both to a synaptic vesicle protein and to N-type calcium channel. Although the relationship of the physiological functions, structures and topologies along cellular membrane between the HPC-1 antigen and epimorphin have not been consistent yet, these two proteins belong to a novel protein family.     

Contact-dependent regulation of N-type calcium channel subunits during synaptogenesis

The developmental regulation of the N-type calcium channel during synaptogenesis was studied using cultured rat hippocampal neurons to elucidate the roles of extrinsic versus intrinsic cues in the expression and distribution of this channel. Prior to synapse formation, α_1B and β₃ subunits of the N-type calcium channel were distributed diffusely throughout neurites, growth cones, and somata. As synaptogenesis proceeded, the subunit distributions became punctate and colocalized with the synaptic vesicle protein synaptotagmin. Isolated neurons were also examined to test for the requirement of extrinsic cues that control N-type calcium channel expression and distribution. These neurons expressed N-type calcium channel subunits, but their distributions remained diffuse. Functional ω-conotoxin GVIA-sensitive channels were expressed in isolated neurons, although the distribution of α_1B subunits was diffuse. The distribution of the α_1B subunit and synaptotagmin only became punctate when neuron-neuron contact was allowed. Thus, the expression of functional N-type calcium channels is the result of an intrinsic program while extrinsic regulatory cues mediated by neuron-neuron contact are required to control their distribution during synaptogenesis. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 198–208, 1998     

Interactions between Presynaptic Calcium Channels and Proteins Implicated in Synaptic Vesicle Trafficking and Exocytosis

Monoclonal antibodies were generated by immunizing mice with chick brain synaptic membranes and screening for immunoprecipitation of solubilized conotoxin GVIA receptors (N-type calcium channels). Antibodies against two synaptic proteins (p35--syntaxin 1 and p58--synaptotagmin) were produced and used to purify and characterize a ternary complex containing N-type channels associated with these two proteins. These results provided the first evidence for a specific interaction between presynaptic calcium channels and SNARE proteins involved in synaptic vesicle docking and calcium-dependent exocytosis. Immunoprecipitation experiments supported the conclusion that syntaxin 1/SNAP-25/VAMP/synaptotagmin I or II complexes associate with N-type, P/Q-type, but not L-type calcium channels from rat brain nerve terminals. Immunofluorescent confocal microscopy at the frog neuromuscular junction was consistent with the co-localization of syntaxin 1, SNAP-25, and calcium channels, all of which are predominantly expressed at active zones of the presynaptic plasma membrane facing post-synaptic folds rich in acetylcholine receptors. The interaction of proteins implicated in calcium-dependent exocytosis with presynaptic calcium channels may locate the sensor(s) that trigger vesicle fusion within a microdomain of calcium entry.     

Interactions between proteins implicated in exocytosis and voltage-gated calcium channels

Neurotransmitter release from synaptic vesicles is triggered by voltage-gated calcium influx through P/Q-type or N-type calcium channels. Purification of N-type channels from rat brain synaptosomes initially suggested molecular interactions between calcium channels and two key proteins implicated in exocytosis: synaptotagmin I and syntaxin 1. Co-immunoprecipitation experiments were consistent with the hypothesis that both N- and P/Q-type calcium channels, but not L-type channels, are associated with the 7S complex containing syntaxin 1, SNAP-25, VAMP and synaptotagmin I or II. Immunofluorescence confocal microscopy at the frog neuromuscular junction confirmed that calcium channels, syntaxin 1 and SNAP-25 are co-localized at active zones of the presynaptic plasma membrane where transmitter release occurs. Experiments with recombinant proteins were performed to map synaptic protein interaction sites on the alpha 1A subunit, which forms the pore of the P/Q-type calcium channel. In vitro-translated 35S-synaptotagmin I bound to a site located on the cytoplasmic loop linking homologous domains II and III of the alpha 1A subunit. This direct link would target synaptotagmin, a putative calcium sensor for exocytosis, to a microdomain of calcium influx close to the channel mouth. Cysteine string proteins (CSPs) contain a J-domain characteristic of molecular chaperones that cooperate with Hsp70. They are located on synaptic vesicles and thought to be involved in modulating the activity of presynaptic calcium channels. CSPs were found to bind to the same domain of the calcium channel as synaptotagmin, and also to associate with VAMP. CSPs may act as molecular chaperones in association with Hsp70 to direct assembly or dissociation of multiprotein complexes at the calcium channel.     

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.     

Calcium channel beta subunits differentially regulate the inhibition of N-type channels by individual Gbeta isoforms

The direct inhibition of N- and P/Q-type calcium channels by G protein betagamma subunits is considered a key mechanism for regulating presynaptic calcium levels. We have recently reported that a number of features associated with this G protein inhibition are dependent on the G protein beta subunit isoform (Arnot, M. I., Stotz, S. C., Jarvis, S. E., Zamponi, G. W. (2000) J. Physiol. (Lond.) 527, 203-212; Cooper, C. B., Arnot, M. I., Feng, Z.-P., Jarvis, S. E., Hamid, J., Zamponi, G. W. (2000) J. Biol. Chem. 275, 40777-40781). Here, we have examined the abilities of different types of ancillary calcium channel beta subunits to modulate the inhibition of alpha(1B) N-type calcium channels by the five known different Gbeta subunit subtypes. Our data reveal that the degree of inhibition by a particular Gbeta subunit is strongly dependent on the specific calcium channel beta subunit, with N-type channels containing the beta(4) subunit being less susceptible to Gbetagamma-induced inhibition. The calcium channel beta(2a) subunit uniquely slows the kinetics of recovery from G protein inhibition, in addition to mediating a dramatic enhancement of the G protein-induced kinetic slowing. For Gbeta(3)-mediated inhibition, the latter effect is reduced following site-directed mutagenesis of two palmitoylation sites in the beta(2a) N-terminal region, suggesting that the unique membrane tethering of this subunit serves to modulate G protein inhibition of N-type calcium channels. Taken together, our data suggest that the nature of the calcium channel beta subunit present is an important determinant of G protein inhibition of N-type channels, thereby providing a possible mechanism by which the cellular/subcellular expression pattern of the four calcium channel beta subunits may regulate the G protein sensitivity of N-type channels expressed at different loci throughout the brain and possibly within a neuron.     

Antigens Associated with N- and L-Type Calcium Channels in Lambert-Eaton Myasthenic Syndrome

Abstract: In Lambert-Eaton myasthenic syndrome neurotransmitter release is reduced by an autoimmune response directed against the calcium channel complex of the nerve terminal. Autoantibodies were detected by immunoprecipitation assays using solubilized receptors labeled with ligands selective for N-type (¹²⁵I-ω conotoxin GVIA) and L-type ([³H]PN200-110) calcium channels. Sera with a high antibody titer (>3 n M ) against rat brain N-type channels contained autoantibodies that immunoprecipitated neuronal and muscle L-type channels. These IgG fractions stained a 55-kDa protein in immunoblots of purified skeletal muscle dihydropyridine receptor, suggesting that they contain autoantibodies against the β subunit of the calcium channel. A distinct antibody population in the same fractions reacted with a nerve terminal 65-kDa protein that is unrelated to the β subunit and displays properties similar to those of synaptotagmin.     

Synaptotagmin VI participates in the acrosome reaction of human spermatozoa.

Acrosomal exocytosis is a calcium-dependent secretion event causing the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. The synaptotagmins are a family of calcium-binding proteins that participate in the exocytosis of synaptic vesicles. The ubiquitous synaptotagmin VI isoform was found in human sperm cells by Western blot analysis. Immunocytochemistry at the optical and electron microscopy levels localized the protein to the outer acrosomal membrane. Calcium-triggered acrosomal exocytosis in permeabilized sperm cells was abrogated by a specific anti-synaptotagmin VI antibody, indicating that the protein is required for the process. Moreover, a recombinant fusion protein between glutathione S-transferase and the two calcium and phospholipid binding domains of synaptotagmin VI completely inhibited calcium-triggered exocytosis. Interestingly, phorbol ester-dependent in vitro phosphorylation of this recombinant protein abolished its inhibitory effect. We previously showed that, in permeabilized spermatozoa, addition of active Rab3A triggers acrosomal exocytosis at very low calcium concentration. Rab3A-promoted exocytosis was inhibited by the cytosolic domain of synaptotagmin VI and by the anti-synaptotagmin VI antibody, indicating that synaptotagmin is also necessary for Rab-mediated acrosomal content release. In conclusion, the results strongly indicate that synaptotagmin VI is a key component of the secretory machinery involved in acrosomal exocytosis.     

Omega-conotoxin CVID inhibits a pharmacologically distinct voltage-sensitive calcium channel associated with transmitter release from preganglionic nerve terminals

Neurotransmitter release from preganglionic parasympathetic neurons is resistant to inhibition by selective antagonists of L-, N-, P/Q-, R-, and T-type calcium channels. In this study, the effects of different omega-conotoxins from genus Conus were investigated on current flow-through cloned voltage-sensitive calcium channels expressed in Xenopus oocytes and nerve-evoked transmitter release from the intact preganglionic cholinergic nerves innervating the rat submandibular ganglia. Our results indicate that omega-conotoxin CVID from Conus catus inhibits a pharmacologically distinct voltage-sensitive calcium channel involved in neurotransmitter release, whereas omega-conotoxin MVIIA had no effect. omega-Conotoxin CVID and MVIIA inhibited depolarization-activated Ba(2+) currents recorded from oocytes expressing N-type but not L- or R-type calcium channels. High affinity inhibition of the CVID-sensitive calcium channel was enhanced when position 10 of the omega-conotoxin was occupied by the smaller residue lysine as found in CVID instead of an arginine as found in MVIIA. Given that relatively small differences in the sequence of the N-type calcium channel alpha(1B) subunit can influence omega-conotoxin access (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), it is likely that the calcium channel in preganglionic nerve terminals targeted by CVID is a N-type (Ca(v)2.2) calcium channel variant.     

Isolation of a full-length mouse cDNA clone coding for an immunologically distinct p53 molecule.

Transfection of a cloned p53 gene into a p53 nonproducer Abelson murine leukemia virus-transformed cell line, L12, reconstituted p53 expression. The protein expressed in these cells was indistinguishable from that naturally expressed in p53 producer tumor cells. Conversely, p53 protein expressed in L12-derived clones that were established by transfection with a full-length p53 cDNA clone (pM8) exhibited a discrete immunological form. Immunoprecipitation of p53 with a panel of monoclonal anti-p53 antibodies showed that L12-derived clones that were transfected with the genomic p53 clone contained the same antigenic determinants as those found in the p53 protein expressed in tumor cells. These p53 proteins bound all monoclonal antibody types as well as the polyclonal anti-p53 tested. However, L12-derived clones established by transfection of the p53 cDNA clone (pM8) expressed a p53 protein that bound the RA3-2C2 and PAb200.47 anti-p53 monoclonal antibodies as well as polyclonal anti-p53 serum but totally lacked the antigenic receptor for the PAb122 and PAb421 monoclonal antibodies. The p53 proteins expressed by either genomic or cDNA p53 clones exhibited the same apparent molecular sizes and identical partial peptide maps. We suggest that transfection of the p53 gene induced expression of the entire group of the possible mRNA species, whereas cloned p53 cDNA (pM8) represented a single mRNA molecule that codes for a discrete species of p53 protein.     

The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites

A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5- bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flugge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flugge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296- 304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.     

A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase.

Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.3 lymphoma cell line, and a human hypothalamus cDNA library, demonstrating that these PCR primers can be used to amplify the cDNA of multiple PDE isozymes. One unique mouse PDE clone was found to encode a polypeptide identical with the corresponding portion of the bovine brain 63-kDa calmodulin-dependent PDE as reported in the companion article (Bentley, J. K., Kadlecek, A., Sherbert, C. H., Seger, D., Sonnenburg, W. K., Charbonneau, H., Novack, J. P., and Beavo, J. A. (1992) J. Biol. Chem. 267, 18676-18682). This mouse clone was used as a probe to screen a rat brain cDNA library for a full-length clone. The conceptual translation of the nucleotide sequence of the resulting rat clone has an open reading frame of 535 amino acids and maintains a high degree of homology with the bovine 63-kDa calmodulin-dependent PDE, indicating that this protein is likely to be the rat homolog of the 63-kDa calmodulin-dependent PDE. Expression of the full-length clone in Escherichia coli yielded a cGMP hydrolyzing activity that was stimulated severalfold by calmodulin. Northern blot analysis demonstrated that the mRNA encoding this PDE is highly expressed in rat brain and also in the S49.1 T-lymphocyte cell line. These data demonstrate that the PCR method described is a viable strategy to isolate cDNA clones of known and novel members of different families of PDE isozymes. Molecular cloning of these PDEs will provide valuable tools for investigating the roles of these isozymes in regulation of intracellular concentrations of the cyclic nucleotides.     

Functional and physical coupling of voltage-sensitive calcium channels with exocytotic proteins: ramifications for the secretion mechanism

The secretion of neurotransmitters is a rapid Ca(2+)-regulated process that brings about vesicle fusion with the plasma membrane. This rapid process (< 100 microseconds) involves multiple proteins located at the plasma and vesicular membranes. Because of their homology to proteins participating in constitutive secretion and protein trafficking, they have been characterized extensively. The sequential events that lead these proteins to vesicle docking and fusion are still unclear. We will review recent studies that demonstrate the operative role played by voltage-sensitive Ca(2+) channels and discuss the relevance for the process of evoked transmitter release. The regulation of Ca(2+) influx by syntaxin, synaptosome-associated protein of 25 kDa (SNAP-25) and synaptotagmin, and the reciprocity of these proteins in controlling the kinetic properties of the channel will be discussed. Calcium channel and synaptic proteins expressed in Xenopus oocytes demonstrate a strong functional interaction, which could be pertinent to the mechanism of secretion. First, the voltage-sensitive Ca(2+) channels are negatively modulated by syntaxin: this inhibition is reversed by synaptotagmin. Second, the modulation of N-type Ca(2+) channel activation kinetics strongly suggests that the vesicle could be docked at the plasma membrane through direct interaction with synaptotagmin. Finally, these interactions provide evidence for the assembly of the voltage-sensitive Ca(2+) channel with syntaxin 1A, SNAP-25 and synaptotagmin into an excitosome complex: a putative fusion complex with a potential role in the final stages of secretion. Studies suggest that cross-talk between the synaptic proteins and the channel in a tightly organized complex may enable a rapid secretory response to an incoming signal such as membrane depolarization.     

Cloning and characterization of the annexin II receptor on human marrow stromal cells

Annexin II is a heterotetramer, consisting of two 11-kDa (p11) and two 36-kDa (p36) subunits, that is produced by osteoclasts and stimulates osteoclast formation. However, its receptor is unknown. We showed that annexin II binds to normal primary human marrow stromal cells and the Paget's marrow-derived PSV10 stromal cell line to induce osteoclast formation. 125I-Labeled annexin II binding assays with PSV10 cells demonstrated that there was a single class of annexin II receptors with a Kd of 5.79 nm and Bmax of 2.13 x 10(5) receptors/cell. Annexin III or annexin V did not bind this receptor. Using 125I-labeled annexin II binding to screen NIH3T3 transfected with a human marrow cDNA expression library, we identified a putative annexin II receptor clone, which encoded a novel 26-kDa type I membrane receptor protein when expressed in HEK 293 cells. HEK 293 cells transformed with the cloned annexin II receptor cDNA showed a similar binding affinity to annexin II as that observed in PSV10 cells. Chemical cross-linking experiments with biotinylated annexin II and intact PSV10 cells identified a 55-kDa band on Western blot analysis that reacted with both an anti-p11 antibody and streptavidin but not anti-p36 antibody. A rabbit polyclonal antibody raised against the putative recombinant annexin II receptor also recognized the same 26-kDa protein band detected in PSV10 cells. Importantly, the annexin II receptor antibody dose-dependently blocked the stimulatory effects of annexin II on human osteoclast formation, demonstrating that the receptor mediates the effects of annexin II on osteoclast formation.     

Cloning and expression of guanylin. Its existence in various mammalian tissues.

Guanylin (PNTCEICAYAACTGC) is a peptide recently isolated from the intestine, the actions of which appear to be mimicked by bacterial heat-stable enterotoxins (Currie, M. G., Fok, K. F., Kato, J., Moore, R. J., Hamra, F. K., Duffin, K. L., and Smith, C. E. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 947-951). A cDNA clone encoding the peptide was isolated from a rat intestinal cDNA library using a degenerate oligonucleotide probe. The mRNA (approximately 0.8-0.9 kilobase) encoding the peptide contained an open reading frame of 115 amino acids, including an amino-terminal signal peptide. The carboxyl-terminal region of the predicted polypeptide contained a sequence identical to guanylin, but the 15-amino acid peptide likely represents an artifact of previous acetic acid extraction methods, since an aspartate residue precedes the amino-terminal proline. A lysine-lysine dipeptide bond is one likely processing site of pro-guanylin and would generate a 60-amino acid mature peptide. Other potential cleavage sites exist at single lysine and arginine residues, which could result in peptides ranging from 22 to 56 amino acids. Transfection of COS-7 cells with the guanylin cDNA resulted in the expression of a secreted protein of M(r) 10,000. The expressed proguanylin failed to elevate cyclic GMP concentrations in human colonic T84 cells, but acetic acid treatment of pro-guanylin activated it and resulted in large elevations of cyclic GMP. Guanylin mRNA was prevalent in rat intestine but was also found in low abundance in adrenal gland, kidney, and uterus/oviduct. Guanylyl cyclase C, the apparent guanylin receptor, was found in abundant amounts in the intestine by Northern analysis, and by the polymerase chain reaction or cDNA cloning it was also found in adrenal gland, airway epithelial cells, brain, and olfactory and tracheal mucosa. Therefore, the ligand and apparent receptor (guanylyl cyclase C) both originate from mammalian genes, and are expressed in various mammalian tissues.     

Cross-talk between G-protein and protein kinase C modulation of N-type calcium channels is dependent on the G-protein beta subunit isoform

The modulation of N-type calcium current by protein kinases and G-proteins is a factor in the fine tuning of neurotransmitter release. We have previously shown that phosphorylation of threonine 422 in the alpha(1B) calcium channel domain I-II linker region resulted in a dramatic reduction in somatostatin receptor-mediated G-protein inhibition of the channels and that the I-II linker consequently serves as an integration center for cross-talk between protein kinase C (PKC) and G-proteins (Hamid, J., Nelson, D., Spaetgens, R., Dubel, S. J., Snutch, T. P., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 6195-6202). Here we show that opioid receptor-mediated inhibition of N-type channels is affected to a lesser extent compared with that seen with somatostatin receptors, hinting at the possibility that PKC/G-protein cross-talk might be dependent on the G-protein subtype. To address this issue, we have examined the effects of four different types of G-protein beta subunits on both wild type and mutant alpha(1B) calcium channels in which residue 422 has been replaced by glutamate to mimic PKC-dependent phosphorylation and on channels that have been directly phosphorylated by protein kinase C. Our data show that phosphorylation or mutation of residue 422 antagonizes the effect of Gbeta(1) on channel activity, whereas Gbeta(2), Gbeta(3), and Gbeta(4) are not affected. Our data therefore suggest that the observed cross-talk between G-proteins and protein kinase C modulation of N-type channels is a selective feature of the Gbeta(1) subunit.     

Putting the clamps on membrane fusion: how complexin sets the stage for calcium-mediated exocytosis

Three recent papers have addressed a long-standing question in exocytosis: how does a sudden calcium influx trigger a coordinated synchronous release in regulated exocytosis [Giraudo, C.G., Eng, W.S., Melia, T.J. and Rothman, J.E. (2006) A clamping mechanism involved in SNARE-dependent exocytosis. Science 313, 676-680; Schaub, J.R., Lu, X., Doneske, B., Shin, Y.K. and McNew, J.A. (2006) Hemifusion arrest by complexin is relieved by Ca(2+)-synaptotagmin I. Nat. Struct. Mol. Biol. 13, 748-750; Tang, J., Maximov, A., Shin, O.H., Dai, H., Rizo, J. and Sudhof, T.C. (2006) A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175-1187]? Using diverse approaches that include cell-free reconstitution of the membrane fusion machinery and in vivo manipulation of fusogenic proteins, these groups have established that the complexin proteins are fusion clamps. By arresting vesicle secretion just prior to fusion, complexin primes select vesicles for a fast, synchronous response to calcium.     

Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA

The Conus magus peptide toxin omega-conotoxin MVIIA is considered an irreversible, specific blocker of N-type calcium channels, and is now in clinical trials as an intrathecal analgesic. Here, we have examined the action of MVIIA on mutant and wild type calcium channels transiently expressed in tsA-201 cells. Although we have shown previously that mutations in a putative external EF-hand motif in the domain IIIS5-H5 region alters block by both omega-conotoxin GVIA and MVIIA (Feng, Z. P., Hamid, J., Doering, C., Bosey, G. M., Snutch, T. P., and Zamponi, G. W. (2001) J. Biol. Chem. 276, 15728-15735), the introduction of five point mutations known to affect GVIA blocking (and located downstream of the EF-hand) affected MVIIA block to a smaller degree compared with GVIA. These data suggest that despite some overlap, MVIIA and GVIA block does not share identical channel structural determinants. At higher concentrations (approximately 3 microm), MVIIA reversibly blocked L-, P/Q-, and R-type, but not T-type channels, indicating that the overall architecture of the MVIIA site is conserved in all types of high voltage-activated calcium channels. A kinetic analysis of the MVIIA effects on the N-type channel showed that MVIIA blocked resting, open, and inactivated channels. Although the development of MVIIA block did not appear to be voltage-, nor frequency-dependent, the degree of recovery from block strongly depended on the potential applied during washout. Interestingly, the degree of washout was highly variable and appeared to weakly depend on the holding potential applied during toxin application. We propose a model in which N-type calcium channels can form both reversible and irreversible complexes with MVIIA.     

The identification and characterization of an epidermal growth factor-stimulated phosphorylation of a specific low molecular weight GTP-binding protein in a reconstituted phospholipid vesicle system

The abilities of different GTP-binding proteins to serve as phosphosubstrates for the epidermal growth factor (EGF) receptor/tyrosine kinase have been examined in reconstituted phospholipid vesicle systems. During the course of these studies we discovered that a low molecular mass, high affinity GTP-binding protein from bovine brain (designated as the 22-kDa protein) served as an excellent phosphosubstrate for the tyrosine-agarose-purified human placental EGF receptor. The EGF-stimulated phosphorylation of the purified 22-kDa protein occurs on tyrosine residues, with stoichiometries approaching 2 mol of 32Pi incorporated/mol of [35S]guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S)-binding sites. The EGF-stimulated phosphorylation of the brain 22-kDa protein requires its reconstitution into phospholipid vesicles. No phosphorylation of this GTP-binding protein is detected if it is simply mixed with the purified EGF receptor in detergent solution or if detergent is added back to lipid vesicles containing the EGF receptor and the 22-kDa protein. The EGF-stimulated phosphorylation of this GTP-binding protein is also markedly attenuated by guanine nucleotides, i.e. GTP, GTP gamma S, or GDP, suggesting that maximal phosphorylation occurs when the GTP-binding protein is in a guanine nucleotide-depleted state. Purified preparations of the 22-kDa phosphosubstrate do not cross-react with antibodies against the ras proteins. However, they do cross-react against two different peptide antibodies generated against specific sequences of the human platelet (and placental) GTP-binding protein originally designated Gp (Evans, T., Brown, M. L., Fraser, E. D., and Northrup, J. K. (1986) J. Biol. Chem. 261, 7052-7059) and more recently named G25K (Polakis, P. G., Synderman, R., and Evans, T. (1989) Biochem. Biophys. Res. Commun. 160, 25-32). When highly purified preparations of the human platelet Gp (G25K) protein are reconstituted with the purified EGF receptor into phospholipid vesicles, an EGF-stimulated phosphorylation of the platelet GTP-binding protein occurs with a stoichiometry approaching 2 mol of 32Pi incorporated/mol of [35S]GTP gamma S-binding sites. As is the case for the brain 22-kDa protein, the EGF-stimulated phosphorylation of the platelet GTP-binding protein is attenuated by guanine nucleotides. Overall, these results suggest that the brain 22-kDa phosphosubstrate for the EGF receptor is very similar, if not identical, to the Gp (G25K) protein. Although guanine nucleotide binding to the brain 22-kDa protein or to the platelet. GTP-binding protein inhibits phosphorylation, the phosphorylated GTP-binding proteins appear to bind [35S]GTP gamma S slightly better than their nonphosphorylated counterparts.