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M Montal

Publications and source records attributed to M Montal.

At least 109 records · Page 6Linked to original sources
PubMed1984

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers.

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.

Animals↗
PubMed1984

The acetylcholine receptor channel from Torpedo californica has two open states.

We recorded single channel currents activated by agonist binding to purified acetylcholine receptors from the electric organ of Torpedo californica reconstituted in planar lipid bilayers. Analysis of single channel records indicate that the acetylcholine receptor channel displays two kinetically distinct open states which differ in their mean open times but have similar channel conductances.

Animals↗
PubMed1983

Single-channel recordings from purified acetylcholine receptors reconstituted in bilayers formed at the tip of patch pipets.

The channel of the purified acetylcholine receptor from Torpedo californica electric organ reconstituted in lipid vesicles was assayed by direct electrical recording using patch-clamp pipets. High-resistance seals were obtained by gentle suction of vesicles into the pipet or after the formation of lipid bilayers from monolayers at the tip of the pipet. Single-channel currents were activated by three cholinergic ligands: acetylcholine, carbamylcholine, and suberyldicholine. The single-channel conductance, gamma, was 40 +/- 5 pS in 0.5 M NaCl, irrespective of the agonist used. The distributions of channel open times were fitted by a sum of two exponentials. The lifetimes of the two exponential components were a factor of 2 longer for suberyldicholine than for acetylcholine or carbamylcholine. At desensitizing concentrations of agonists the single events appeared in paroxysms of channel activity followed by quiescent periods. These results suggest that the full cycle of solubilization, purification, and reconstitution of this membrane receptor can be achieved without impairment of channel function.

Acetylcholine↗
PubMed1983

A small angle x-ray scattering study of a rhodopsin-lipid complex in hexane.

A small angle x-ray scattering study of a rhodopsin-lipid complex in hexane demonstrates the presence of two distinct particle populations with corresponding radii of gyration of approximately 22 and 160 A. A model based on the existence of inverted lipid micelles surrounding the protein polar moieties while the protein hydrophobic domains act as cross-links between the inverted micelles is presented. It accounts for the results herewith reported and explains most of the properties hitherto characterized for the rhodopsin-lipid complex in hexane.

Animals↗
PubMed1983

Rhodopsin in reconstituted phospholipid vesicles. 1. Structural parameters and light-induced conformational changes detected by resonance energy transfer and fluorescence quenching.

The structure of purified rhodopsin was investigated by steady-state resonance energy transfer and fluorescence quenching techniques: (1) Fluorescence parameters and relative distances between rhodopsin sites labeled with fluorescent probes and the endogenous chromophore 11-cis-retinal were measured in micellar detergent solution and in reconstituted phospholipid vesicles. (2) The accessibility of the labeled rhodopsin sites in reconstituted vesicles to N-methyl- and N-benzylpicolinium was studied in the dark and subsequent to rhodopsin bleaching. Fluorescent-labeled rhodopsin was affinity purified in octyl glucoside from rod outer segments which were previously reacted with either the sulfhydryl-specific reagents, pyrenylmaleimide or monobromobimane, or reagents specific to amino groups, dansyl chloride or fluorescein isothiocyanate. The purified protein was recombined with phospholipids, and vesicles were formed by detergent dialysis. All four fluorophores appear to react greater than or equal to 30 A away from the endogenous chromophore as estimated from the efficiency of energy transfer and presumably probe rhodopsin domains exposed at the membrane surface. The maximal fraction of quenchable fluorescence and the mean quenching constant were determined in dark and bleached vesicles: bleaching did not affect the quenching of the fluorophores attached to sulfhydryl groups but markedly decreased the quenching constants of the fluorophores coupled to amino groups. The apparent collisional rate constant decreased by 20- and 4-fold for dansyl and fluorescein, respectively. The results suggest that bleaching reduced the accessibility of these sites which, in turn, may reflect light-induced displacements of rhodopsin domains at the membrane surface. Such structural changes may regulate rhodopsin-rhodopsin as well as rhodopsin-enzyme interactions.

Chemical Phenomena↗
PubMed1983

Rhodopsin in reconstituted phospholipid vesicles. 2. Rhodopsin-rhodopsin interactions detected by resonance energy transfer.

The interactions between rhodopsin molecules in a micellar detergent solution (octyl glucoside) and in reconstituted phospholipid vesicles were studied in the dark and after bleaching. Resonance energy transfer measurements were used to monitor the proximity between rhodopsin monomers conjugated with a fluorescent donor or a fluorescent acceptor. Reactive sulfhydryl groups of rhodopsin were labeled with pyrenylmaleimide (donor) or monobromobimane (acceptor), whereas amino groups were labeled with dansyl chloride (donor) or fluorescein isothiocyanate (acceptor). The results suggest that in the micellar solution rhodopsin was monomeric in the dark and aggregated after bleaching. If the aggregate were to be a dimer, the labeled sulfhydryl groups of the monomers would be approximately 40 A apart, while the labeled amino groups would be at least 68 A distant from each other. Rhodopsin reconstituted in phospholipid vesicles appeared aggregated both in the dark and after bleaching. The proximity between the sulfhydryl groups of the monomers was not influenced by illumination. In contrast, the labeled amino groups seemed to be largely separated in the dark and closer to each other once the vesicles were bleached. If the aggregate were to be a dimer, the labeled sulfhydryl groups would be approximately 40 A apart both in the dark and after bleaching, whereas the labeled amino groups would be greater than 60 A apart in the dark and approximately 44 A from each other after bleaching. These findings are discussed in the context of rhodopsin structure, its ability to regenerate after bleaching, and the light-induced events initiated by rhodopsin photoexcitation.

Chemical Phenomena↗
PubMed1982

Incorporation of acetylcholine receptors into liposomes. Vesicle structure and acetylcholine receptor function.

Functionally intact acetylcholine receptors can be solubilized from electric organ membranes of Torpedo californica and incorporated into liposomes by the cholate dialysis technique. Freezing and thawing of the reconstituted preparation appears to seal a population of initially leaky vesicles and leads to vesicle fusion. Inclusion of supplementary cholesterol at an optimal concentration of 20% (w/w) greatly enhances vesicle fusion during the freeze-thaw cycle. Size analysis by electron microscopy of negatively stained preparations indicates that fusion is accompanied by shifts in size and volume distributions of the vesicle population. Liposomes formed in the absence of acetylcholine receptors are distributed over a substantially smaller size range than liposomes containing receptors. Acetylcholine receptors appear in those liposomes as dimers of 80 A doughnut-shaped particles. Freeze-fracture replicas of reconstituted preparations reveal the presence of large vesicles containing particles which correspond in size to acetylcholine receptors and smaller liposomes devoid of particles. The distribution of particles in the reconstituted membranes is sparse compared to their dense packing in native electric organ membranes. The activation and desensitization of reconstituted acetylcholine receptors mediated by acetylcholine or carbamylcholine is dose dependent. The reconstituted receptors distinguish between these agonists in terms of binding affinity in a way similar to receptors in the native membrane. Correlation of the fractional occupancy of ligand binding sites by cobratoxin with inhibition of receptor function is used to demonstrate that in the reconstituted system the doubly liganded acetylcholine receptor prevails in controlling channel gating. The potential experimental advantages as well as limitations of this reconstituted system are discussed.

Animals↗
PubMed1981

Stabilization of acetylcholine receptor channels by lipids in cholate solution and during reconstitution in vesicles.

Acetylcholine receptors were solubilized from electric organ membranes of Torpedo californica in mixed micelles of sodium cholate and soybean lipids. Sodium cholate, when supplemented with relatively low amounts of soybean lipids (cholate:lipid, 20:1, molar ratio), was effective in solubilizing receptors without denaturing their agonist-regulated cation channels. Another dialyzable detergent, octylglucoside, denatured the ion channel even in the presence of excess lipids. Reassembly of receptors and lipids into vesicles was achieved by cholate dialysis. About 70% of the receptors were oriented with their toxin binding sites on the external surface of the vesicles. Evidence suggests that all of the receptors in a single vesicle were oriented either right side out or inside out. During the reassembly process about 10-fold greater lipid concentrations were required for the preservation of channel function. At lipid/protein ratios greater than 16:1 (w/w), receptors reassembled into vesicles at a constant protein/lipid ratio. These vesicles contained approximately 7% receptors by weight, 5-fold less than the native membrane. The remainder of the lipid assembled into small vesicles which did not contain receptors. At lipid/protein ratios less than 16:1 (w/w), receptors reassociated with lipids in higher weight ratios. Irreversible inactivation of a fraction of the acetylcholine receptor channels occurred in proportion to the greater packing density. This channel denaturation was accompanied by a lowered susceptibility of the disulfide bond between the delta subunits of the acetylcholine receptor dimer to reducing agents. Toxin binding and the orientation of the receptors in the reconstituted vesicles was not affected by reduced lipid/protein ratios. The unexpected constant acetylcholine receptor/lipid ratio in the reconstituted vesicles and the unexpected uniform orientation of the acetylcholine receptors within a vesicle are discussed in terms of the interactions occurring during the initial nucleation events of the reassembly process.

Acetylcholine↗
PubMed1981

Diphtheria toxin forms transmembrane channels in planar lipid bilayers.

When exposed to a lipid bilayer, diphtheria toxin binds to it and forms transmembrane, voltage-dependent, anion-selective channels. The mean (+/- SD) conductance of these channels in asolectin membranes is 6.2 +/- 0.7 pmho (pS) in 0.2 M NaCl and 20 +/- 2 pmho in 1.0 M NaCl. The rate of channel formation depends on the pH in the toxin-containing compartment; it is very low at pH greater than 5.0 and increases abruptly as the pH decreases from 4.9 to 4.0. Binding of toxin to the membrane is also pH dependent, being unmeasurable at pH 7 and increasing monotonically with decreasing pH. The rate of channel formation depends upon membrane potential as well; channels form only at negative potentials. These channels are permanent in the time scale of the experiments (about 1 hr). The membrane conductance caused by the channels is also voltage dependent, being constant at positive potentials and decreasing at negative potentials. Hence, the current-voltage curve is linear at positive potentials and sublinear at negative potentials. The conditions necessary for insertion of toxin into the bilayer and formation of channels are similar to those that prevail inside the lysosome. Thus, these results lend credence to the idea that toxin enters the cytoplasm from the lysosomal compartment.

Anions↗
PubMed1980

Purification of acetylcholine receptors, reconstitution into lipid vesicles, and study of agonist-induced cation channel regulation.

We report the purification of acetylcholine receptors with active agonist-regulated cation channels from Torpedo californica electric organ tissue by five methods. In one method, previously used by others, contaminating proteins were removed from partially purified membranes by alkaline extraction, preserving membrane integrity throughout the procedure. In the other four methods, acetylcholine receptors were purified after solubalization with sodium cholate. The continual presence of soybean lipid in mixed micelles with cholate was required to prevent irreversible inactivation of the cation channel. Solubilized receptors were purified by affinity chromatography using either Naga naja siamensis toxin III or concanavalin A coupled to agarose. Sucrose gradient centrifugation was also used to purify solubilized receptors. The best method combined affinity chromatography on toxin-agarose and concanavalin A agarose. Receptors purified by all five methods were incorporated into soybean lipid vesicles by the cholate dialysis technique. The agonist-regulated cation channels of the receptors were equally active after reconstitution, independent of the method used for purification. All reconstituted vesicle preparations were similar in preferential orientation of acetylcholine receptor toward the external surface, dose-response to carbamylcholine, carbamylcholine-induced desensitization, and carbamycholine-induced influx of 22Na+ per mol of receptor. Carbamylcholine-induced 22Na+ influx/receptor was greater after reconstitution than in native vesicles. This was because, in native vesicles, carbamylcholine-induced 22Na+ influx was limited by equilibration of the internal volume of the vesicles with the external 22Na+ concentration, whereas in reconstituted vesicles 22Na+ influx was limited by desensitization of the receptor molecule. We demonstrate that only one of the two toxin binding sites on the receptor monomer, the one which can be affinity alkylated with 4-(N-maleimido)benzyltrimethylammonium, controls the carbamylcholine-induced opening of the cation channel.

Acetylcholine↗
PubMed1980

Reassembly of protein-lipid complexes into large bilayer vesicles: perspectives for membrane reconstitution.

Protein-lipid complexes in apolar solvents reassemble into large bilayer protein-lipid vesicles (PLVs) with diameters of several micrometers. PLVs form spontaneously upon hydration of the protein-lipid complex residue after solvent removal. This procedure has been applied to the following membrane proteins: bovine and squid rhodopsin, reaction centers from Rhodopseudomonas sphaeroides, beef heart cytochrome c oxidase, and acetylcholine receptors from Torpedo californica. PLVs have a large internal aqueous space (e.g., 790 mul/mg of lipid for cattle rhodopsin vesicles). Freeze-fracture replicas of PLVs revealed that both internal and external leaflets contained numerous intramembranous particles with diameters between 80 and 120 A, depending on the specific protein incorporated in the membrane. The optical spectral properties of rhodopsin and reaction centers in PLVs were similar to those recorded in the respective natural membrane. Furthermore, bovine rhodopsin in PLVs was chemically regenerable with 9-cis-retinal. Actinic illumination induced proton efflux from reaction center vesicles that was abolished by proton ionophores. Therefore, this method is suitable for the incorporation of some membrane proteins in their functional state. PLVs were penetrated with microelectrodes and visualized by the injection of a fluorescent dye. Preliminary electrical recordings were obtained by sealing PLVs to a hole in a septum separating two aqueous compartments. These studies suggest that PLVs assembled by this procedure permit the simultaneous analysis of reconstituted membranes by chemical, optical, and electrical techniques.

Animals↗
PubMed1980

Reconstitution of purified acetylcholine receptors with functional ion channels in planar lipid bilayers.

Acetylcholine receptor, solubilized and purified from Torpedo californica electric organ under conditions that preserve the activity of its ion channel, was reconstituted into vesicles of soybean lipid by the cholate-dialysis technique. The reconstituted vesicles were then spread into monolayers at an air-water interface and planar bilayers were subsequently formed by apposition of two monolayers. Addition of carbamoylcholine caused an increase in membrane conductance that was transient and relaxed spontaneously to the base level (i.e., became desensitized). The response to carbamoylcholine was dose dependent and competitively inhibited by curare. Fluctuations of membrane conductance corresponding to the opening and closing of receptor channels were observed. Fluctuation analysis indicated a single-channel conductance of 16 +/- 3 pS (in 0.1 M NaCl) with a mean channel open time estimated to be 35 +/- 5 ms. Thus, purified acetylcholine receptor reconstituted into lipid bilayers exhibited the pharmacological specificity, activation, and desensitization properties expected of this receptor in native membranes.

Animals↗
PubMed1980

Functional equivalence of monomeric and dimeric forms of purified acetylcholine receptors from Torpedo californica in reconstituted lipid vesicles.

Acetylcholine receptors from Torpedo californica electric organ were solubilized and purified under conditions which prevent inactivation of the agonist-regulated cation channels. The dimer form of the receptors was preserved during purification. Treatment with reducing agents converted dimers into monomers. Receptor monomers and dimers were separately reconstituted into soybean lipid vesicles by the cholate dialysis technique. Reconstituted monomers and dimers were functionally equivalent with respect to their carbamylcholine-induced dose-dependent uptake of 22Na+, the total flux of 22Na+ per receptor during the permeability response, and the occurrence of desensitization. Evidence against non-covalent association of monomers to produce dimeric functional units was obtained using glutaraldehyde as a crosslinking agent. These results show that both the acetylcholine-binding sites and the agonist-regulated cation-specific channel are contained within the alpha 2 beta gamma delta subunit structure of the acetylcholine receptor monomer.

Acetylcholine↗