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A V Ferrer-Montiel

Publications and source records attributed to A V Ferrer-Montiel.

At least 19 recordsLinked to original sources
PubMed2002

Tyrosine phosphorylation of the inactivating peptide of the shaker B potassium channel: a structural-functional correlate.

A synthetic peptide patterned after the sequence of the inactivating "ball" domain of the Shaker B K(+) channel restores fast (N-type) inactivation in mutant deletion channels lacking their constitutive ball domains, as well as in K(+) channels that do not normally inactivate. We now report on the effect of phosphorylation at a single tyrosine in position 8 of the inactivating peptide both on its ability to restore fast channel inactivation in deletion mutant channels and on the conformation adopted by the phosphorylated peptide when challenged by anionic lipid vesicles, a model target mimicking features of the inactivation site in the channel protein. We find that the inactivating peptide phosphorylated at Y8 behaves functionally as well as structurally as the noninactivating mutant carrying the mutation L7E. Moreover, it is observed that the inactivating peptide can be phosphorylated by the Src tyrosine kinase either as a free peptide in solution or when forming part of the membrane-bound protein channel as the constitutive inactivating domain. These findings suggest that tyrosine phosphorylation-dephosphorylation of this inactivating ball domain could be of physiological relevance to rapidly interconvert fast-inactivating channels into delayed rectifiers and vice versa.

Amino Acid Sequence↗
PubMed2000

Arginine-rich peptides are blockers of VR-1 channels with analgesic activity.

Vanilloid receptors (VRs) play a fundamental role in the transduction of peripheral tissue injury and/or inflammation responses. Molecules that antagonize VR channel activity may act as selective and potent analgesics. We report that synthetic arginine-rich hexapeptides block heterologously expressed VR-1 channels with submicromolar efficacy in a weak voltage-dependent manner, consistent with a binding site located near/at the entryway of the aqueous pore. Dynorphins, natural arginine-rich peptides, also blocked VR-1 activity with micromolar affinity. Notably, synthetic and natural arginine-rich peptides attenuated the ocular irritation produced by topical capsaicin application onto the eyes of experimental animals. Taken together, our results imply that arginine-rich peptides are VR-1 channel blockers with analgesic activity. These findings may expand the development of novel analgesics by targeting receptor sites distinct from the capsaicin binding site.

Amino Acid Sequence↗
PubMed1999

Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger.

Depletion of Ca2+ stores in Xenopus oocytes activated entry of Ca2+ across the plasma membrane, which was measured as a current I(soc) in subsequently formed cell-attached patches. I(soc) survived excision into inside-out configuration. If cell-attached patches were formed before store depletion, I(soc) was activated outside but not inside the patches. I(soc) was potentiated by microinjection of Clostridium C3 transferase, which inhibits Rho GTPase, whereas I(soc) was inhibited by expression of wild-type or constitutively active Rho. Activation of I(soc) was also inhibited by botulinum neurotoxin A and dominant-negative mutants of SNAP-25 but was unaffected by brefeldin A. These results suggest that oocyte I(soc) is dependent not on aqueous diffusible messengers but on SNAP-25, probably via exocytosis of membrane channels or regulatory molecules.

ADP Ribose Transferases↗
PubMed1999

Peptides that mimic the carboxy-terminal domain of SNAP-25 block acetylcholine release at an Aplysia synapse.

Botulinum neurotoxin serotypes A and E (BoNT/A and BoNT/E) block neurotransmitter release, presumably by cleaving SNAP-25, a protein involved in docking of synaptic vesicles with the presynaptic plasma membrane. Three excitation-secretion uncoupling peptides (ESUPs), which mimic the carboxy-terminal domain of SNAP-25 and span or adjoin the cleavage sites for BoNT/A and BoNT/E, also inhibit transmitter release from permeabilized bovine chromaffin cells. In this study, these peptides were tested for effects on acetylcholine (ACh) release at an identified cholinergic synapse in isolated buccal ganglia of Aplysia californica. The presynaptic neuron was stimulated electrically to elicit action potentials. The postsynaptic neuron was voltage-clamped, and evoked inhibitory postsynaptic currents (IPSCs) were recorded. The ESUPs were pressure-injected into the presynaptic neuron, and their effects on the amplitude of the IPSCs were studied. Acetylcholine release from presynaptic cells, as measured by IPSC amplitudes, was gradually inhibited by the ESUPs. All three peptides caused ca. 40% reduction in IPSC amplitude in 2 h. Random-sequence peptides of the same amino acid composition had no effect. Injection of BoNT/E, in contrast, caused ca. 50% reduction in IPSC amplitude in 30 min and almost complete inhibition in 2 h. These results are the first demonstration that ESUPs block neuronal cholinergic synaptic transmission. They are consistent with the concept that ESUPs compete with the intact SNAP-25 for binding with other fusion proteins, thus inhibiting stimulus-evoked exocytosis of neurotransmitter.

Acetylcholine↗
PubMed1998

The 26-mer peptide released from SNAP-25 cleavage by botulinum neurotoxin E inhibits vesicle docking.

Botulinum neurotoxin E (BoNT E) cleaves SNAP-25 at the C-terminal domain releasing a 26-mer peptide. This peptide product may act as an excitation-secretion uncoupling peptide (ESUP) to inhibit vesicle fusion and thus contribute to the efficacy of BoNT E in disabling neurosecretion. We have addressed this question using a synthetic 26-mer peptide which mimics the amino acid sequence of the naturally released peptide, and is hereafter denoted as ESUP E. This synthetic peptide is a potent inhibitor of Ca2+-evoked exocytosis in permeabilized chromaffin cells and reduces neurotransmitter release from identified cholinergic synapses in in vitro buccal ganglia of Aplysia californica. In chromaffin cells, both ESUP E and BoNT E abrogate the slow component of secretion without affecting the fast, Ca2+-mediated fusion event. Analysis of immunoprecipitates of the synaptic ternary complex involving SNAP-25, VAMP and syntaxin demonstrates that ESUP E interferes with the assembly of the docking complex. Thus, the efficacy of BoNTs as inhibitors of neurosecretion may arise from the synergistic action of cleaving the substrate and releasing peptide products that disable the fusion process by blocking specific steps of the exocytotic cascade.

Amino Acid Sequence↗
PubMed1998

Structural determinants of the blocker binding site in glutamate and NMDA receptor channels.

Glutamate receptor channels of the NMDA-type (N-methyl-D-aspartate) and non-NMDA-type (GluR) differ in their pore properties. The N-site in the M2 transmembrane segment of NMDA receptors (NMDAR), or the corresponding Q/R-site in GluRs, is a pivotal structural determinant of their permeation and blockade characteristics. Substitutions at a second site in M2, the L-site (L577) in GluR1, drastically alter the receptor selectivity to divalent cations. Here we report that M2 mutants carrying an asparagine or a threonine residue at the Q-site of GluR1, along with a tryptophan residue at the L-site, form homomeric GluR1 channels that are highly sensitive to structurally diverse, uncompetitive NMDA antagonists such as arylcyclohexylamines, dibenzocycloheptenimines, and to morphinian and adamantane derivatives. Analysis of the voltage dependence of channel blockade locates the blocker binding site approximately 0.65 partway into the transmembrane electric field in both GluR1 mutants and NMDAR channels. Our results suggest that the homomeric GluR1 double mutants, L577W/Q582N and L577W/Q582T, fairly approximate the pore properties of the heteromeric NMDA receptor and support the structural kinship of their permeation pathways.

Amino Acid Substitution↗
PubMed1998

Selected peptides targeted to the NMDA receptor channel protect neurons from excitotoxic death.

Excitotoxic neuronal death, associated with neurodegeneration and stroke, is triggered primarily by massive Ca2+ influx arising from overactivation of glutamate receptor channels of the N-methyl-D-aspartate (NMDA) subtype. To search for channel blockers, synthetic combinatorial libraries were assayed for block of agonist-evoked currents by the human NR1-NR2A NMDA receptor subunits expressed in amphibian oocytes. A set of arginine-rich hexapeptides selectively blocked the NMDA receptor channel with IC50 approximately 100 nM, a potency similar to clinically tolerated blockers such as memantine, and only marginally blocked on non-NMDA glutamate receptors. These peptides prevent neuronal cell death elicited by an excitotoxic insult on hippocampal cultures.

Animals↗
PubMed1997

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.

Adrenal Medulla↗
PubMed1996

Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV-1-infected cells.

HIV-1 Vpu catalyzes two independent functions, degradation of the virus receptor CD4 in the endoplasmic reticulum and enhancement of virus release from the cell surface. These activities are confined to distinct structural domains of Vpu, the cytoplasmic tail and the transmembrane (TM) anchor, respectively. It was recently reported that Vpu forms cation-selective ion channels in lipid bilayers. Here we report that this property of Vpu is a characteristic of its TM anchor. Expression of full-length Vpu in Xenopus oocytes increases membrane conductance. The Vpu-induced conductance is selective to monovalent cations over anions, does not discriminate Na+ over K+ and shows marginal permeability to divalent cations. Notably, introduction of the scrambled TM sequence into full-length Vpu abrogates its capacity to increase membrane conductance in oocytes and to promote virus release from infected cells. Reconstitution of synthetic Vpu fragments in lipid bilayers identified an ion channel activity for a sequence corresponding to the TM domain of Vpu. In contrast, a peptide with the same amino acid composition but with a scrambled sequence does not form ion channels. Our findings therefore suggest that the ability of Vpu to increase virus release from infected cells may be correlated with an ion channel activity of the TM domain, thereby providing a potential target for drug intervention based on the development of Vpu-specific channel blockers.

Amino Acid Sequence↗
PubMed1996

Tyrosine phosphorylation modulates the activity of clostridial neurotoxins.

Clostridial neurotoxins' metalloprotease domain selectively cleaves proteins implicated in the process of synaptic vesicle fusion with the plasma membrane and, accordingly, blocks neurotransmitter release into the synaptic cleft. Here we investigate the potential modulation of these neurotoxins by intracellular cascades triggered by environmental signals, which in turn may alter its activity on target substrates. We report that the nonreceptor tyrosine kinase Src phosphorylates botulinum neurotoxins A, B, and E and tetanus neurotoxin. Protein tyrosine phosphorylation of serotypes A and E dramatically increases both their catalytic activity and thermal stability, while dephosphorylation reverses the effect. This suggests that the biologically significant form of the neurotoxins inside neurons is phosphorylated. Indeed, in PC12 cells in which tyrosine kinases such as Src and PYK2 are highly abundant, stimulation by membrane depolarization in presence of extracellular calcium induces rapid and selective tyrosine phosphorylation of internalized light chain, the metalloprotease domain, of botulinum toxin A. These findings provide a conceptual framework to connect intracellular signaling pathways involving tyrosine kinases, G-proteins, phosphoinositides, and calcium with the action of botulinum neurotoxins in abrogating vesicle fusion and neurosecretion.

Animals↗
PubMed1996

Pentameric subunit stoichiometry of a neuronal glutamate receptor.

Ionotropic glutamate receptors, neurotransmitter-activated ion channels that mediate excitatory synaptic transmission in the central nervous system, are oligomeric membrane proteins of unknown subunit stoichiometry. To determine the subunit stoichiometry we have used a functional assay based on the blockade of two alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor subunit 1 (GluR1) mutant subunits selectively engineered to exhibit differential sensitivity to the open channel blockers phencyclidine and dizolcipine (MK-801). Coinjection into amphibian oocytes of weakly sensitive with highly sensitive subunit complementary RNAs produces functional heteromeric channels with mixed blocker sensitivities. Increasing the fraction of the highly sensitive subunit augmented the proportion of drug-sensitive receptors. Analysis of the data using a model based on random aggregation of receptor subunits allowed us to determine a pentameric stoichiometry for GluR1. This finding supports the view that a pentameric subunit organization underlies the structure of the neuronal ionotropic glutamate receptor gene family.

Animals↗
PubMed1996

A single tryptophan on M2 of glutamate receptor channels confers high permeability to divalent cations.

Ionotropic glutamate receptors (iGluRs) of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate subtype display lower permeability to Ca2+ than the N-methyl-D-aspartate (NMDA) subtype. The well-documented N/Q/R site on the M2 transmembrane segment (M2) is an important determinant of the distinct Ca2+ permeability exhibited by members of the non-NMDA receptor subfamily. This site, however, does not completely account for the different permeation properties displayed by non-NMDA and NMDA receptors, suggesting the involvement of other molecular determinants. We have identified additional molecular elements on M2 of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor GluR1 that specify its permeation properties. Higher permeability to divalent over monovalent cations is conferred on GluR1 by a tryptophan at position 577, whereas blockade by external divalent cations is imparted by an asparagine at position 582. Hence, the permeation properties of ionotropic glutamate receptors appear to be primarily specified by two distinct determinants on M2, the well-known N/Q/R site and the newly identified L/W site. These findings substantiate the notion that M2 is a structural component of the pore lining.

Amino Acid Sequence↗
PubMed1996

The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein is an integral membrane phosphoprotein that induces CD4 degradation in the endoplasmic reticulum and enhances virus release from the cell surface. CD4 degradation is specific, requires phosphorylation of Vpu, and involves the interaction between Vpu and the CD4 cytoplasmic domain. In contrast, regulation of virus release is less specific and not restricted to HIV-1 and may be mechanistically-distinct from CD4 degradation. We show here that a mutant of Vpu, Vpu35, lacking most of its cytoplasmic domain has residual biological activity for virus release but is unable to induce CD4 degradation. This finding suggests that the N terminus of Vpu encoding the transmembrane (TM) anchor represents an active domain important for the regulation of virus release but not CD4 degradation. To better define the functions of Vpu's TM anchor and cytoplasmic domain, we designed a mutant, VpuRD, containing a scrambled TM sequence with a conserved amino acid composition and alpha-helical structure. The resulting protein was integrated normally into membranes, was able to form homo-oligomers, and exhibited expression levels, protein stability, and subcellular localization similar to those of wild-type Vpu. Moreover, VpuRD was capable of binding to CD4 and to induce CD4 degradation with wild-type efficiency, confirming proper membrane topology and indicating that the alteration of the Vpu TM domain did not interfere with this function of Vpu. However, VpuRD was unable to enhance the release of virus particles from infected or transfected cells, and virus encoding VpuRD had replication characteristics in T cells indistinguishable from those of a Vpu-deficient HIV-1 isolate. Mutation of the phosphorylation sites in VpuRD resulted in a protein which was unable to perform either function of Vpu. The results of our experiments suggest that the two biological activities of Vpu operate via two distinct molecular mechanisms and involve two different structural domains of the Vpu protein.

Amino Acid Sequence↗
PubMed1995

Mutation of conserved negatively charged residues in the S2 and S3 transmembrane segments of a mammalian K+ channel selectively modulates channel gating.

Voltage-gated channel proteins sense a change in the transmembrane electric field and respond with a conformational change that allows ions to diffuse across the pore-forming structure. Site-specific mutagenesis combined with electrophysiological analysis of expressed mutants in amphibian oocytes has previously established the S4 transmembrane segment as an element of the voltage sensor. Here, we show that mutations of conserved negatively charged residues in S2 and S3 of a brain K+ channel, thought of as countercharges for the positively charged residues in S4, selectively modulate channel gating without modifying the permeation properties. Mutations of Glu235 in S2 that neutralize or reverse charge increase the probability of channel opening and the apparent gating valence. In contrast, replacements of Glu272 by Arg or Thr268 by Asp in S3 decrease the open probability and the apparent gating valence. Residue Glu225 in S2 tolerated replacement only by acidic residues, whereas Asp258 in S3 was intolerant to any attempted change. These results imply that S2 and S3 are unlikely to be involved in channel lining, yet, together with S4, may be additional components of the voltage-sensing structure.

Amino Acid Sequence↗
PubMed1995

A peptide that mimics the carboxy-terminal domain of SNAP-25 blocks Ca(2+)-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 Ca(2+)-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 Ca(2+)-dependent catecholamine release with an IC50 = 20 microM. 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 Ca(2+)-dependent exocytosis, presumably mediated via interactions with other protein components of the fusion complex.

Adrenal Medulla↗
PubMed1995

Molecular design of the N-methyl-D-aspartate receptor binding site for phencyclidine and dizolcipine.

The N-methyl-D-aspartate receptor (NMDAR), a pivotal entity for synaptic plasticity and excitotoxicity in the brain, is a target of psychotomimetic drugs such as phencyclidine (PCP) and dizolcipine (MK-801). In contrast, a related glutamate receptor, the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate/kainate receptor GluR1, is weakly sensitive to these drugs. Three point mutations on GluR1, mimicking homologous residues on the NMDAR, confer the PCP and MK-801 blockade properties that are characteristic of the NMDAR--namely, high potency, voltage dependence, and use dependence. The molecular determinants that specify the PCP block appear confined to the putative M2 transmembrane segment, whereas the sensitivity to MK-801 requires an interplay between residues from M2 and M3. Given the plausible involvement of the NMDAR in the etiology of several neurodegenerative diseases and in excitotoxic neuronal cell death, tailored glutamate receptors with specific properties may be models for designing and screening new drugs targeted to prevent glutamate-mediated neural damage.

Amino Acid Sequence↗
PubMed1994

Primary structure and functional expression of the AMPA/kainate receptor subunit 2 from human brain.

A full-length cDNA clone encoding the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate (KA) receptor subunit 2 (HBGR2) was isolated from a human brain cDNA library. The HBGR2 cDNA has an open reading frame of approximately 2.7 kb that codes for an 883-residue protein. At the amino acid level, HBGR2 is 98% identical to its rat counterpart GluR2, and 69% to the AMPA/KA receptor subunit 1 from human brain (HBGR1). Injection of cRNA transcripts from the HBGR2 into oocytes produces barely detectable kainate-activated ionic currents, indicating that the HBGR2 subunit alone weakly expresses homomeric receptor channels. Coexpression of HBGR2 and HBGR1 transcripts, however, evokes kainate-dependent currents which activate at higher agonist concentration than those required by homomeric HBGR1 receptor channels. Coexpressed receptors display a linear current-to-voltage relationship at variance with the inwardly rectifying profile exhibited by HBGR1 homomers. Hence, the HBGR2 subunit coassembles with the HBGR1 subunit to form heteromeric receptor channels akin to the glutamate receptors from rodent brain.

Amino Acid Sequence↗