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M Montal

Publications and source records attributed to M Montal.

At least 55 records · Page 3Linked to original sources
PubMed1995

Formation of ion channels in lipid bilayers by a peptide with the predicted transmembrane sequence of botulinum neurotoxin A.

Synthetic peptides patterned after the predicted transmembrane sequence of botulinum toxin A were used as tools to identify an ion channel-forming motif. A peptide denoted BoTxATM, with the sequence GAVILLEFIPEIAI PVLGTFALV, forms cation-selective channels when reconstituted in planar lipid bilayers. As predicted, the self-assembled conductive oligomers express heterogeneous single-channel conductances. The most frequent openings exhibit single-channel conductance of 12 and 7 pS in 0.5 M NaCl, and 29 and 9 pS in 0.5 M KCl. In contrast, ion channels are not formed by a peptide of the same amino acid composition as BoTxATM with a scrambled sequence. Conformational energy calculations show that a bundle of four amphipathic alpha-helices is a plausible structural motif underlying the measured pore properties. These studies suggest that the identified module may play a functional role in the ion channel-forming activity of intact botulinum toxin A.

Amino Acid Sequence↗
PubMed1995

Molecular mimicry in channel-protein structure.

The approach to probing the sequence-structure relationship of ion-channel proteins using small peptides stems from the abundance of sequence information and the virtual absence of structures at atomic resolution. It is anticipated that model peptides may fold predictably into stable structures and reproduce functional properties of specific proteins. Model peptides are well suited to the application of NMR methods to determine protein structure in a membrane environment or to high-resolution X-ray diffraction analysis. It is timely to ask what we have learned through this strategy and where it may lead in our quest to understand the sequence-structure determinism.

Amino Acid Sequence↗
PubMed1995

Design of molecular function: channels of communication.

The ultimate goal in protein design is to elucidate the fundamental principles that determine structure. With increased understanding of the molecular basis underlying the sequence-structure relationship may come the ability to control it and, thereby, to generate proteins with desired specifications. Channel proteins, which mediate cell signaling, are ideally suitable for protein design. Plausible molecular blueprints for the pore-forming structure are bundles of amphipathic alpha-helices or beta-barrels that cluster together to generate a hydrophilic channel. This review focuses on the progress achieved to produce such designs and on the approximation of the synthetic channels to the targeted biological function.

Amino Acid Sequence↗
PubMed1994

Identification of an ion channel-forming motif in the primary structure of CFTR, the cystic fibrosis chloride channel.

Synthetic peptides with sequences representing putative transmembrane (M) segments of CFTR (the cystic fibrosis transmembrane conductance regulator) were used as tools to identify the involvement of such segments in forming the ionic pore of the CFTR Cl- channel. Peptides with sequences corresponding to M2 and M6 form anion-selective channels after reconstitution in lipid bilayers. In contrast, peptides with the sequences of M1, M3, M4, and M5, or peptides of the same amino acid composition as M2 and M6 but with scrambled sequences, do not form channels. Conductive heterooligomers of M2 and M6 exhibit a single channel conductance of 8 pS (in 0.15 M KCl) and a 95% selectivity for anions over cations, properties that emulate both the conductance and the selectivity of the authentic CFTR channel. The identification of sequence-specific motifs that account for key functional attributes of the CFTR channel suggests that such modules may represent fundamental units of function and are plausible constituents of the pore-forming structure of the CFTR Cl- channel.

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↗
PubMed1994

Chemical synthesis and characterization of peptides and oligomeric proteins designed to form transmembrane ion channels.

A strategy for the synthesis of peptides and oligomeric proteins designed to form transmembrane ion channels is described. A folding motif that exhibits a functional ionic pore encompasses amphipathic alpha-helices organized as a four-helix bundle around a central hydrophilic pore. The channel-forming activity of monomeric amphipathic peptides may be examined after reconstitution in lipid bilayers in which peptides self-assemble into conductive oligomers. The covalent attachment of channel-forming peptides to the lysine epsilon-amino groups of a template molecule (KKKPGKEKG) specifies oligomeric number and facilitates the study of ionic permeation and channel blockade. Here we describe detailed protocols for the total synthesis of peptides and template-assembled four-helix bundle proteins, exemplified with the sequence of M2 delta (EKM-STAISVLLAQAVFLLLTSQR), considered involved in lining the pore of the nicotinic acetylcholine receptor channel. For comparison, the synthesis of a second four-helix bundle, T4CaIVS3 with the sequence of predicted transmembrane segment S3 (DPWNVFDFLIVIGSIIDVILSE) of the fourth repeat of the L-type voltage-gated calcium channel, is included. Peptides and proteins are synthesized step-wise by solid-phase methods, purified by reversed-phase HPLC, and homogeneity ascertained by analytical HPLC, capillary zone electrophoresis, SDS/PAGE, amino acid analysis and sequencing. Optimization of synthetic procedures for hydrophobic molecules include reducing resin substitution to avoid steric hindrance and aggregation of the final product. Protocols for the preparation of the samples prior to HPLC purification as well as the conditions and columns required for successful purification are presented. The methods developed are generally applicable for the chemical synthesis, purification and characterization of amphipathic peptides and template directed helical bundle proteins.

Amino Acid Sequence↗
PubMed1993

Two distinct modalities of NMDA-receptor inactivation induced by calcium influx in cultured rat hippocampal neurons.

Repetitive stimulation of glutamate (glu) receptors elicits increasingly smaller ionic currents in hippocampal neurons. To investigate mechanisms underlying this phenomenon, voltage clamp whole-cell currents evoked by glu (100 microM) were recorded from hippocampal neurons in culture. These currents were primarily carried by N-methyl-D-aspartate-receptor (NMDA-R) channels, as shown by the voltage-dependent sensitivity to extracellular Mg2+ blockade, and inhibition by the specific antagonist MK-801. In the presence of 2.2 mM extracellular Ca2+ ([Ca2+]e), repetitive glu applications (15 episodes of 4 s/min) elicited progressively smaller currents that stabilized at 45% of their initial peak value. Replacement of [Ca2+]e by Ba2+ produced similar effects. This phenomenon, defined as interepisode inactivation, was exacerbated by elevating [Ca2+]e to 11 mM, attenuated by reducing [Ca2+]e to 0.22 mM, and further diminished by shortening the length of the glu pulse to 2 s. Current decay exhibited during individual stimuli, or intraepisode inactivation, was dependent on [Ca2+]e yet remained stable during repetitive stimulation. We conclude that interepisode and intraepisode inactivations of NMDA-R currents are the expression of two distinct processes triggered by Ca2+. These modalities of inactivation may arise from Ca2+ binding either to the receptor or to closely associated regulatory proteins.

Adolescent↗
PubMed1993

Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. I. Transmembrane segment M2 of the nicotinic cholinergic receptor channel is a key pore-lining structure.

Monomeric peptides and four-helix bundle proteins with amino acid sequences of the predicted transmembrane segment M2 of nicotinic acetylcholine receptors (AChR) were designed, synthesized, and used as probes to elucidate the pore-forming structure of the authentic AChR channel. Peptides M2 delta and M2 alpha 4 with sequences of M2 from muscle-like Torpedo californica AChR delta subunit and from rat neuronal AChR alpha 4 subunit form cation-selective channels in lipid bilayers with predominant single-channel conductances in 0.5 M KCl of 20 pS and 27 pS, respectively. Corresponding analogs with presumed pore-lining residues serine 8 or phenylalanine 16 specifically substituted by alanine retain the ability to self-assemble into conductive oligomers and form channels with primary conductances of 16 pS and 22 pS for M2 delta analogs and of 14 pS and 26 pS for M2 alpha 4 analogs. In contrast, peptides with randomized sequences and the same amino acid composition as M2 delta do not form channels. Four-helix bundle proteins T4M2 delta and T4M2 alpha 4 exhibit conductances in 0.5 M KCl of 20 pS and 26 pS. Analogs of T4M2 delta with selective replacement of serine 8 for alanine exhibit lower conductances, whereas substitution of phenylalanine 16 for alanine increases the single-channel conductance. T4M2 delta channels are blocked by open channel blockers such as the quaternary derivative of lidocaine QX-222 and by chlorpromazine. Channel open probability is reduced, and open time is abbreviated. Conduction properties of T4M2 delta and analogs are in accord with several properties of authentic AChRs. These comprehensive studies provide insights into the components of the pore-forming structure of nicotinic AChRs.

Amino Acid Sequence↗
PubMed1993

Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. II. Transmembrane segment M2 of the brain glycine receptor is a plausible candidate for the pore-lining structure.

A synthetic 23-mer peptide (M2GlyR) with the amino acid sequence of the putative transmembrane segment M2 of the strychnine-binding alpha subunit of the inhibitory glycine receptor forms anion-selective channels in phospholipid bilayers. The most frequent events show single-channel conductances, gamma, of 25 pS and 49 pS in symmetric 0.5 M KCl with channel open lifetimes, tau o, in the millisecond time range. These properties match those of authentic glycine receptors studied in inside-out patches of cultured rat spinal cord neurons, namely gamma = 27 pS and gamma = 45 pS, and tau o in the millisecond time range. The channel activity of M2GlyR is sequence-specific: 1) a synthetic peptide with the sequence of putative transmembrane segment M1 (M1GlyR), not considered to contribute to the channel lining, does not form channels; 2) an analog of M2GlyR with site-specific substitutions displays distinct channel properties: 2 arginine residues at the N and C termini of M2, postulated to contribute to the anion selectivity of the channel, are substituted by glutamic acids, and the analog peptide ([Glu3,22]M2GlyR) forms cation-selective channels. Further, a four-helix bundle protein (T4M2GlyR) formed by tethering four identical M2GlyR modules to a carrier template forms homogeneous anion-selective channels with gamma = 25 pS in 0.5 M KCl. These channels are blocked by picrotoxin and by the anion channel blockers 9-anthracene carboxylic acid and niflumic acid, but not by an analog of the local anesthetic lidocaine (QX-222), a cation channel blocker. Observed single-channel properties suggest that a pentameric assembly of alpha and beta subunits with a central pore lined by M2 segments would account for conductance properties of the authentic glycine receptor and the 2 arginines at either end of M2 could confer anion specificity to the receptor channel.

Amino Acid Sequence↗
PubMed1993

A negative charge in the M2 transmembrane segment of the neuronal alpha 7 acetylcholine receptor increases permeability to divalent cations.

Threonine-244 (T244) in the putative channel-forming M2 segment of the neuronal alpha 7 acetylcholine receptor (AChR), a residue proposed to form part of the selectivity filter, was mutated to aspartic acid to examine the influence of a negative charge on AChR ion permeation properties. Wild type (AChR alpha 7wt) and mutant (AChR alpha 7D244) acetylcholine receptors expressed in Xenopus oocytes give rise to acetylcholine (ACh)-activated, alpha-bungarotoxin-sensitive, cation-selective ionic currents. AChR alpha 7D244 exhibited larger currents than AChR alpha 7wt that, in addition, activated at lower ACh concentrations. The relative ionic permeability (Px/PNa) of AChR alpha 7wt to K+ was PK/PNa = 1.2, and to Ba2+, P'Ba/PNa = 1.4. In contrast, AChR alpha 7D244 was less selective in discriminating between K+ and Na+, PK/PNa = 0.95, but exhibited a remarkable increase in permeability to Ba2+, P'Ba/PNa = 3.7. Furthermore, only mutant receptors were permeable to Mg2+. Hence, a ring of negatively charged residues in the putative pore-forming segment of the receptor increases the permeability to divalent cations. Our results substantiate the notion that T244, or its equivalent, in the M2 transmembrane segment of cholinergic receptor channels is a key structural determinant of the selectivity filter.

Acetylcholine↗
PubMed1993

Molecular cloning, functional expression, and pharmacological characterization of an N-methyl-D-aspartate receptor subunit from human brain.

A cDNA encoding a full-length N-methyl-D-aspartate (NMDA) receptor subunit 1, hNR1, was isolated from a human brain cDNA library. The hNR1 cDNA encodes an open reading frame of approximately 2.7 kb that shares high homology with the rat brain NMDA receptor subunit 1 and the mouse zeta 1 subunit. The hNR1 sequence, however, diverges from the rodent and murine homologs near the C terminus, suggesting that they represent alternatively spliced messages of the same gene. Oocytes injected with cRNA synthesized from the hNR1 cDNA express glutamate and NMDA-activated currents in the presence of glycine. Currents are blocked by the NMDA-receptor-specific antagonists 2-amino-5-phosphovaleric acid and 7-chlorokynurenate, and the open channel blockers MK-801 and phencyclidine, by Mg2+ ions in a voltage-dependent manner, and by Zn2+. Expressed hNR1 homomeric receptor channels exhibit the high Ca2+ permeability characteristic of neuronal NMDA receptors. Therefore, the cDNA clone hNR1 codes for a human brain NMDA receptor subunit cognate to the rodent and murine brain NR1 subunits.

2-Amino-5-phosphonovalerate↗
PubMed1993

Design, synthesis and functional characterization of a pentameric channel protein that mimics the presumed pore structure of the nicotinic cholinergic receptor.

Nicotinic cholinergic receptors are membrane proteins composed of five subunits organized around a central aqueous pore. A pentameric channel protein, T5M2 delta, that emulates the presumed pore-forming structure of this receptor was generated by assembling five helix-forming peptide modules at the lysine epsilon-amino groups of the 11-residue template [K*AK*KK*PGK*EK*G], where * indicates attachment sites. Helical modules represent the sequence of the M2 segment of the Torpedo californica acetylcholine receptor (AChR) delta subunit; M2 segments are considered involved in pore-lining. Purified T5M2 delta migrates in SDS-PAGE with an apparent M(r) approximately 14,000, concordant with a protein of 126 residues. T5M2 delta forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in symmetric 0.5 M KCl is 40 pS. This value approximates the 45 pS single channel conductance characteristic of authentic purified Torpedo AChR, recorded under otherwise identical conditions. These results, together with conformational energy calculations, support the notion that a bundle of five amphipathic alpha-helices is a plausible structural motif underlying the inner bundle that forms the pore of the pentameric AChR channel.

Amino Acid Sequence↗
PubMed1993

Design of a functional calcium channel protein: inferences about an ion channel-forming motif derived from the primary structure of voltage-gated calcium channels.

To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (S1-S6) predicted to form membrane-spanning, alpha-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation-selective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by microM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1993

Mapping of the human NMDA receptor subunit (NMDAR1) and the proposed NMDA receptor glutamate-binding subunit (NMDARA1) to chromosomes 9q34.3 and chromosome 8, respectively.

A role for the N-methyl-D-aspartate (NMDA) receptor in the molecular pathology underlying Huntington disease (HD) has been proposed on the basis of neurochemical studies in HD and the ability of the NMDA receptor to mediate neuronal cell death. The molecular cloning of the human NMDA receptor subunit (NMDAR1) and a proposed glutamate-binding subunit of the NMDA receptor (NMDARA1) have provided an opportunity to test the hypothesis that either of these genes might be directly involved in the causation of HD. We have mapped NMDAR1 to 9q34.3 using in situ hybridization studies and NMDARA1 to human chromosome 8 using a somatic cell hybrid panel. Because the gene causing HD has been localized to chromosome 4p16.3, the chromosome assignments reported here are inconsistent with either of these genes playing a causative role in the molecular pathology of HD. However, it is noteworthy that the gene for torsion dystonia has also been localized by genetic studies to 9q34.3, the same regional map location as NMDAR1.

Animals↗
PubMed1992

Identification of an ion channel-forming motif in the primary structure of tetanus and botulinum neurotoxins.

Synthetic peptides with amino acid sequences corresponding to predicted transmembrane segments of tetanus toxin were used as probes to identify a channel-forming motif. A peptide denoted TeTx II, with sequence GVVLLLEYIPEITLPVIAALSIA, forms cation-selective channels when reconstituted in planar lipid bilayers. The single channel conductance in 0.5 M NaCl or KCl is 28 +/- 3 and 24 +/- 2 pS, respectively. In contrast, a peptide with sequence NFIGALETTGVVLLLEYIPEIT, denoted as TeTx I, or a peptide with the same amino acid composition as TeTx II but with a randomized sequence, do not form channels. Conformational energy calculations show that a bundle of four amphipathic alpha-helices is a plausible structural motif underlying observable pore properties. The identified functional module may account for the channel-forming activity of both tetanus toxin and the homologous botulinum toxin A.

Amino Acid Sequence↗
PubMed1992

Protein folding funnels: a kinetic approach to the sequence-structure relationship.

A lattice model of protein folding is developed to distinguish between amino acid sequences that do and do not fold into unique conformations. Although Monte Carlo simulations provide insights into the long-time processes involved in protein folding, these simulations cannot systematically chart the conformational energy surface that enables folding. By assuming that protein folding occurs after chain collapse, a kinetic map of important pathways on this surface is constructed through the use of an analytical theory of probability flow. Convergent kinetic pathways, or "folding funnels," guide folding to a unique, stable, native conformation. Solution of the probability flow equations is facilitated by limiting treatment to diffusion between geometrically similar collapsed conformers. Similarity is measured in terms of a reconfigurational distance. Two specific amino acid sequences are deemed foldable and nonfoldable because one gives rise to a single, large folding funnel leading to a native conformation and the other has multiple pathways leading to several stable conformers. Monte Carlo simulations demonstrate that folding funnel calculations accurately predict the fact of and the pathways involved in folding-specific sequences. The existence of folding funnels for specific sequences suggests that geometrically related families of stable, collapsed conformers fulfill kinetic and thermodynamic requirements of protein folding.

Algorithms↗
PubMed1992

Molecular cloning, chromosomal mapping, and functional expression of human brain glutamate receptors.

A full-length cDNA clone encoding a glutamate receptor was isolated from a human brain cDNA library, and the gene product was characterized after expression in Xenopus oocytes. Degenerate PCR primers to conserved regions of published rat brain glutamate receptor sequences amplified a 1-kilobase fragment from a human brain cDNA library. This fragment was used as a probe for subsequent hybridization screening. Two clones were isolated that, based on sequence information, code for different receptors: a 3-kilobase clone, HBGR1, contains a full-length glutamate receptor cDNA highly homologous to the rat brain clone GluR1, and a second clone, HBGR2, contains approximately two-thirds of the coding region of a receptor homologous to rat brain clone GluR2. Southern and PCR analysis of a somatic cell-hybrid panel mapped HBGR1 to human chromosome 5q31.3-33.3 and mapped HBGR2 to chromosome 4q25-34.3. Xenopus oocytes injected with in vitro-synthesized HBGR1 cRNA expressed currents activated by glutamate receptor agonists with the following specificity sequence: domoate greater than kainate much greater than quisqualate greater than or equal to alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid greater than or equal to L-glutamate much greater than N-methyl-D-aspartate. The kainate-elicited currents were specifically blocked by 6-cyano-7-nitroquinoxaline-2,3-dione but were insensitive to 2-amino-5-phosphonovalerate and kynurenic acid. These results indicate that clone HBGR1 codes for a glutamate receptor of the kainate subtype cognate to members of the glutamate receptor family from rodent brain.

Amino Acid Sequence↗