A peptide that mimics the C-terminal sequence of SNAP-25 inhibits secretory vesicle docking in chromaffin cells

Excitation-secretion uncoupling peptides (ESUPs) are inhibitors of Ca2+-dependent exocytosis in neural and endocrine cells. Their mechanism of action, however, remains elusive. We report that ESUP-A, a 20-mer peptide patterned after the C terminus of SNAP-25 (synaptosomal associated protein of 25 kDa) and containing the cleavage sequence for botulinum neurotoxin A (BoNT A), abrogates the slow, ATP-dependent component of the exocytotic pathway, without affecting the fast, ATP-independent, Ca2+-mediated fusion event. Ultrastructural analysis indicates that ESUP-A induces a drastic accumulation of dense-core vesicles near the plasma membrane, mimicking the effect of BoNT A. Together, these findings argue in favor of the notion that ESUP-A inhibits ATP-primed exocytosis by blocking vesicle docking. Identification of blocking peptides which mimic sequences that bind to complementary partner domains on interacting proteins of the exocytotic machinery provides new pharmacological tools to dissect the molecular and mechanistic details of neurosecretion. Our findings may assist in developing ESUPs as substitute drugs to BoNTs for the treatment of spasmodic disorders.This work was supported by grants from the Spanish Direccion General de Investigacion Cientifica y Tecnica PM-0110 (to S. V.), Grant DAMD 17-93-C-3100 from the U. S. Army Medical Research and Development Command (to M. M.), and a Postdoctoral Fellowship from the Dystonia Medical Research Foundation (to J. M. C.).Peer reviewe

Effects of pH on the kinetics of the interaction between anthracyclines and lipid bilayers

In the present study we have analyzed the kinetics of the initial steps (first 10 seconds) of the interaction between the anthracycline daunomycin (DNM) and artificial lipid vesicles bearing opposite surface charge. The process can be monitored by measuring the fluorescence increase of the drug accompanying its association with the lipid bilayers. The results indicated that DNM consistently interacts to a larger extent with the liposomes having negative surface charge than with those having positive surface charge, suggesting the involvement of electrostatic components in the interaction. In contrast, DNM associates with the vesicles bearing positive surface charge 2 – 3 times faster (in terms of the apparent rate constants describing the process of interaction) than with those having negative surface charge, an observation probably related to the more fluid physical state of the former. Regarding the rate of access of DNM to the vesicles, rather than depending on the surface charge of the vesicles, this is critically affected by the ionization state of the drug, i. e. by the pH. Thus, the rate at which the interaction proceeds is increased nearly 15-fold when the pH of the medium increases from 7.0 to 8.3, regardless of the surface charge of the liposomes. On this basis, and taking into account the fact that the anthracyclines enter the cells by passive diffusion, possible effects of pH on the transport of these drugs through the membranes of tumor cells are discussed.This work has been partly supported by grants from the DGICYT (grants PB93-0934 and PM95-0108). M.L. is recipient of postdoctoral fellowship from the “Programa de Formación de Personal Investigador” and “Fundación de Investigaciones Biomedicas” (Bancaja).Peer reviewe

A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca2+-dependent exocytosis in chromaffin cells

SNAP-25, a synaptosomal associated membrane protein of 25 kDa, participates in the presynaptic process of vesicle-plasma membrane fusion that results in neurotransmitter release at central nervous system synapses. SNAP-25 occurs in neuroendocrine cells and, in analogy to its role in neurons, has been implicated in catecholamine secretion, yet the nature of the underlying mechanism remains obscure. Here we use an anti-SNAP-25 monoclonal antibody to show that SNAP-25 is localized at the cytosolic surface of the plasma membrane of chromaffin cells. This antibody inhibited the Ca2+-evoked catecholamine release from digitonin-permeabilized chromaffin cells in a time- and dose-dependent manner. Remarkably, a 20-mer synthetic peptide representing the sequence of the C-terminal domain of SNAP-25 blocked Ca2+-dependent catecholamine release with an IC50 = 20 μM. The inhibitory activity of the peptide was sequence-specific as evidenced by the inertness of a control peptide with the same amino acid composition but random order. The C-terminal segment of SNAP-25, therefore, plays a key role in regulating Ca2+-dependent exocytosis, presumably mediated via interactions with other protein components of the fusion complex.This work was supported by a grant from the Spanish DGICYT (PM 91-0022-C02-02)and a grant from the Department of the Army Medical Research (DAMD 17-93-C-3100)to M.M.J.M.C. is a post-doctoral fellow of MEC (Spain).Peer reviewe

Botulinum toxin type A inhibits Ca2+-dependent transport of acetylcholine in reconstituted giant liposomes made from presynaptic membranes from cholinergic nerve terminals

Giant liposomes were made from a mixture of asolectin phospholipid vesicles and presynaptic plasma membranes isolated from Torpedo cholinergic nerve; endings. Acetylcholine filled giant liposomes were able to release neurotransmitter upon stimulation by the Ca2+ ionophore A23187 and Ca2+. Botulinum neurotoxin type A inhibited this Ca2+-dependent acetylcholine transport. Additionally, Botulinum toxin type A decreased membrane fluidity of liposomes. These results suggest that Botulinum toxin can interact directly with components of the presynaptic plasma membrane and inhibit acetylcholine translocation. Furthermore, since the reconstituted liposomes do not have synaptic vesicle components, the observed effects may account for the action of Botulinum toxin on the non-quantal release of acetylcholine from motor nerve terminals.This work is supported by grants from DGICYT (Ministerio de Educacion, Spain) to C.S., J.M. and J.M.G.R. Elena Lopez-Alonso is a fellow of Generalitat de Valencia.Peer reviewe