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M Criado

Publications and source records attributed to M Criado.

At least 55 records · Page 3Linked to original sources
PubMed1988

Single channel recordings of reconstituted ion channel proteins: an improved technique.

Single channel recording of reconstituted ion channels is possible by patch clamp measurements of giant liposomes formed by dehydration-rehydration of lipid films. This "hydration technique" consists of carefully controlled dehydration of a suspension of small vesicles followed by rehydration of the residue resulting in formation of large liposomes. Patch pipettes can be attached to the liposome surface, yielding stable, high resistance seals between membranes and glass pipettes. This method allows the study of the properties of reconstituted ion channels from different tissues. The hydration technique was used to characterize the reconstituted K+-channel of sarcoplasmic reticulum from rabbit skeletal muscle. In a solution of 100 mM KCl, the sarcoplasmic reticulum K+-channel studied displays a conductance gamma K+ of 145 pS. The single channel conductance in 100 mM Rb+ and Na+ is gamma Rb+ = 98 pS and gamma Na+ = 65 pS respectively. A concentration of 0.5 mM decamethonium causes a flickering channel block. These properties are in good agreement with the ones found in sarcoplasmic reticulum K+-channels characterized by other methods. Other ion channels have also been reconstituted and studied by this technique. This improved method is compared with previous approaches and its applicability for the characterization of reconstituted ion channel proteins is discussed.

Animals↗
PubMed1987

A membrane fusion strategy for single-channel recordings of membranes usually non-accessible to patch-clamp pipette electrodes.

Membranes of cellular organelles and plasma membranes of some type of cells are not accessible to the high-resolution recordings that the conventional patch-clamp technique allows. However, when these purified membranes are dehydrated together with small lipid vesicles and hydrated again, cell-size vesicles (5-100 micron diameter) are obtained, on which single-channel recordings are possible. This approach, which has been proven successful with about ten different membrane preparations of varied origin, is further illustrated with two examples. First, a known conductivity of the sarcoplasmic reticulum membrane is compared with data obtained by using other techniques. Second, a new sodium current, present at purified postsynaptic membranes from the Torpedo electric organ, is described.

Animals↗
PubMed1986

Structural heterogeneity of the alpha subunits of the nicotinic acetylcholine receptor in relation to agonist affinity alkylation and antagonist binding.

The structural basis for the heterogeneity of the two agonist binding sites of the Torpedo californica acetylcholine receptor with respect to antagonist binding and reactivity toward affinity alkylating reagents was investigated. There is one agonist binding site on each of the two alpha subunits in a receptor monomer. One of these sites is easily affinity labeled with bromoacetylcholine, while more extreme conditions are required to label the other. Evidence is presented that the site which is easily labeled with bromoacetylcholine is the site with higher affinity for the antagonist d-tubocurarine. Digestion of purified alpha subunits with staphylococcal V8 protease gave two limit fragments with apparent molecular weights of 17K and 19K. Both of these fragments began at residue 46 of the alpha sequence, and both reacted with monoclonal antibodies specific for the sequence alpha 152-159 but not with antibodies specific for alpha 235-242. Their tryptic peptide maps and reactivity with a number of monoclonal antibodies were virtually identical. Only the 17-kilodalton (17-kDa) fragments stained heavily for sugars with Schiff's reagent. However, both fragments bound 125I-labeled concanavalin A. Complete removal of carbohydrate detectable with concanavalin A from V8 protease digests of alpha subunits resulted in two fragments of lower apparent molecular weights, indicating that these fragments differed not only in carbohydrate content but also in their C-termini or by another covalent modification. Covalent labeling of one of the two agonist sites of the intact receptor with bromo[3H]acetylcholine followed by digestion with V8 protease resulted in labeling of only the 19-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1986

Evidence that the acetylcholine binding site is not formed by the sequence alpha 127-143 of the acetylcholine receptor.

The sequence alpha 127-143 of the alpha subunit of the acetylcholine receptor has been proposed to contain several important features: (1) the acetylcholine binding site, (2) the only N-glycosylation site of the alpha subunit, at asparagine-alpha 141, and (3) two cysteine residues, at alpha 128 and alpha 142, that may participate in a disulfide bond known to be near the binding site. We tested these hypotheses by using antisera to receptor and its subunits and monoclonal antibodies to the synthetic peptide alpha 127-143 cyclized by a disulfide bond between alpha 128 and alpha 142. Antisera to receptor and its alpha subunit were able to immunoprecipitate the iodinated peptide, and this reaction was inhibited by soluble receptor, but not by membrane-bound receptor. alpha-Bungarotoxin did not inhibit antiserum binding to solubilized receptor. Similarly, cholinergic ligands had little or no effect on binding to immobilized receptors of anti-peptide monoclonal antibodies. In addition, these monoclonal antibodies, when bound to the receptor, did not affect toxin binding kinetics. By contrast, preincubation with concanavalin A did inhibit monoclonal antibody binding. Reduction of the receptor significantly decreased the binding of three of the monoclonal antibodies, but subsequent alkylation with N-ethylmaleimide or the affinity labeling reagent bromoacetylcholine had no additional effect on binding. A dithiothreitol concentration about 100-fold higher that the one needed to reduce the disulfide near the acetylcholine binding site was necessary to inhibit monoclonal antibody binding. We conclude that the sequence alpha 127-143 is not fully exposed on the surface when the receptor is in the membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1986

Location of antigenic determinants on primary sequences of subunits of nicotinic acetylcholine receptor by peptide mapping.

The binding domains of 28 monoclonal antibodies (mAbs) against the alpha, beta, and delta subunits of the Torpedo acetylcholine receptor were mapped on the primary sequences of these subunits. Small peptide fragments (2000-20,000 daltons) of the purified subunits were obtained by digestion with staphylococcal V8 protease and papain, separated on a discontinuous polyacrylamide gel electrophoretic system, and electroblotted onto diaminophenyl thioether paper. The blots were probed with the various monoclonal antibodies and also with antibodies against carboxy-terminal decapeptides of the alpha, beta, and delta subunits to identify the carboxy-terminal fragments. From inspection of the binding patterns of the various antibodies to the subunits fragments and the molecular weights of these fragments, and by using the carboxy termini of the subunits as reference points, it was possible to deduce the regions on the primary sequence of each subunit in which the antibodies bound and in some cases to order the binding sites within these sequences. mAb 148, which inhibits receptor function by cross-linking receptor molecules on the cytoplasmic side, was mapped to the sequence beta 368-406. The main immunogenic region of the native receptor, which is of pathological importance in the autoimmune disease myasthenia gravis, was mapped by using mAb 210 to within 80 amino acid residues (alpha 46-127). The overall antigenic structure of alpha subunits was examined. Synthetic peptides have been used to locate determinants responsible for 83% of the antibodies in antisera to denatured alpha subunits and 46% of the antibodies to denatured alpha subunits in antisera to intact receptor. Theoretical models of the transmembrane orientation of the subunit polypeptide chains were tested by determining whether mapped monoclonal antibodies bound to the extracellular or intracellular surface of receptor-rich membranes. Our results confirm previous reports that the carboxy termini of the subunits are exposed on the intracellular surface, as is part of the region between a putative channel-forming domain (M5) and a putative membrane-spanning region (M3). However, contrary to current theoretical models, the region between M5 and the putative membrane-spanning sequence M4 also appears to be on the intracellular surface, implying that M4 and M5 are not membrane-spanning domains.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1985

Structural localization of the sequence alpha 235-242 of the nicotinic acetylcholine receptor.

Two monoclonal antibodies (mAb 254 and 255) were obtained against a synthetic peptide corresponding to the sequence 235-242 of the alpha-subunit of Torpedo acetylcholine receptor. These mAbs could bind to receptor in native membrane vesicles only when these vesicles were permeabilized, suggesting that the sequence alpha 235-242 is exposed on the cytoplasmic surface of the receptor. Further evidence for the cytoplasmic localization of this sequence was partial competition for binding between these mAbs and mAbs previously demonstrated to bind to the cytoplasmic part of the receptor. A model is proposed which accounts for all the experimental data obtained thus far on the transmembrane orientation of the subunit polypeptide chains.

Amino Acid Sequence↗
PubMed1985

Evidence for unpredicted transmembrane domains in acetylcholine receptor subunits.

Two monoclonal antibodies (mAbs 236 and 237) against a synthetic peptide composed of the same amino acid residues as the sequence 152-167 of the alpha subunit of the acetylcholine receptor were obtained, and their crossreaction with the synthetic peptide, alpha subunit, and solubilized receptor was demonstrated. Crossreaction with the synthetic peptide alpha 159-169 was less by a factor of 10(4), suggesting that the mAbs bind primarily to the sequence alpha 152-159. Cholinergic ligands did not inhibit mAb binding. No crossreaction was observed with the receptor in native membranes, but the mAbs could bind to receptor reconstituted into liposomes in which 50% of the receptors have their cytoplasmic surface oriented outside. When native membranes were permeabilized with saponin, mAbs directed against cytoplasmic determinants of the receptor could bind to them, but mAbs 236 and 237 could not. However, after treatments that removed peripheral proteins from the cytoplasmic surface, binding of both mAbs was observed. Further evidence for the cytoplasmic localization of this sequence was provided by observation of partial competition for binding between mAbs 236 and 237 and mAbs previously demonstrated to bind to the cytoplasmic surface of the receptor. To account for these findings, a model for the organization of the polypeptide chains in receptor subunits is proposed that has a total of seven transmembrane domains in each subunit, two of which are amphipathic and one of which is not alpha-helical.

Amino Acid Sequence↗
PubMed1985

Cytotoxic granules from killer cells: specificity of granules and insertion of channels of defined size into target membranes.

The channel-forming polyperforins P1 and P2 are thought to be formed from the contents of dense core vesicles of cytolytic effector cells. To test this hypothesis, granules from various cytotoxic effector cells were assayed for cytolytic activity on nucleated or unnucleated targets. The results show that in general, granules from cytolytic effector cells are cytolytic, whereas granules from noncytotoxic cells are not. Cytotoxicity of granules is not specific, but there appears to be a preference in that nucleated targets are lysed better than are erythrocytes by granules from T killer or natural killer cells. Granules from CTLL-2, however, preferentially lyse erythrocyte targets. This cell line has been in culture for a long period of time and has lost its cytotoxicity. We tested whether granules from CTLL-2 caused formation of transmembrane pores in erythrocyte target membranes. We found that granule- and complement-induced lesions have similar pore sizes. They are big enough to allow the total release of alpha-bungarotoxin, an 8000 Mr polypeptide with dimensions of 4 X 2.5 nm. Larger molecules are released partially or not at all. Under acidic conditions (pH 5.4) granules do not permeabilize target membranes. This may suggest a pH-dependent control mechanism in the formation, insertion, or function of polyperforin channels, in addition to a previously recognized Ca2+-dependent mechanism. Permeabilization of lipid vesicles by granules was studied to explore what the molecular requirements for channel insertion into membranes may be. Release of alpha-bungarotoxin induced by granules was observed in liposomes made of soybean lipid with or without cholesterol, suggesting that no membrane component other than lipid is required for the insertion of polyperforins, and that the action of polyperforins does not require other mechanisms in the target cell. When pure lecithin from soybean and egg, or synthetic phosphatidylcholines were used, slower release or no release of macromolecules was observed. We suggest that some kind of lipid specificity is required for perforin action. This may be related to the hydrophobic region of the lipid bilayer rather than to the polar portion, because different lecithins with varying fatty acid composition gave similar results.

Animals↗
PubMed1984

Functional properties of the acetylcholine receptor incorporated in model lipid membranes. Differential effects of chain length and head group of phospholipids on receptor affinity states and receptor-mediated ion translocation.

Torpedo acetylcholine receptor was reconstituted into liposomes of pure synthetic lipids in order to study the influence of the lipid environment on affinity state transitions and the ion translocation function of the receptor. A critical concentration of 30 to 40% of cholesteryl hemisuccinate was necessary in liposomes made of cholesteryl hemisuccinate and dimyristoyl phosphatidylcholine to mimic the kinetics of agonist-induced state transitions observed in native membranes. With increasing chain length of the saturated lecithins, a marked increase in carbamylcholine dissociation constants was observed. Substitution by other dimyristoyl phospholipids for dimyristoyl phosphatidylcholine had the same, though quantitatively less pronounced effects. Introduction of unsaturation in the acyl chains reverted the effect of increasing chain length. Unsaturated phosphatidylethanolamines in combination with 28-35 mol% of cholesteryl hemisuccinate was the best lipid mixture for reconstitution of the receptor-gating function. When phosphatidylethanolamine was replaced totally or partially by other phospholipids with the same or different acyl chain composition, a marked decrease of ion transport was apparent, even when similar vesicle size, receptor incorporation, and agonist-induced affinity transitions were obtained. Therefore, the maintenance of the affinity state transitions of the reconstituted receptor is a necessary but not sufficient condition for the manifestation of the ion-gating receptor activity. On the other hand, the more unsaturated the acyl chains of phosphatidylethanolamine are, the higher the response that was observed, suggesting that a critical lipid packing is essential for the ion translocation function of the receptor.

Animals↗
PubMed1984

Conversion of acetylcholine receptor dimers to monomers upon depletion of non-receptor peripheral proteins.

The influence of treatments for extracting non-receptor peripheral proteins on the oligomeric states of the acetylcholine receptor has been studied in receptor-rich membranes from Torpedo marmorata. Conventional alkaline treatment of non-alkylated membranes resulted in the extraction of peripheral proteins (30% of total membrane proteins). Concomitantly, partial conversion of the dimer into the monomer was observed in the absence of exogenous reduction. Alkaline extraction at high ionic strength resulted in a marked decrease in protein solubilization, and no conversion of the dimer to the monomer occurred. Alkaline treatment extracted only one half of the peripheral proteins (15% of total protein) from membranes previously alkylated with N-ethylmaleimide or iodoacetamide, or oxidized by sodium periodate. Conversion of dimer to monomer was totally prevented by these treatments. Similar results were obtained by treatment of the membranes with lithium 3,5-diiodosalicylate. The above effects of alkaline extraction on the acetylcholine receptor can be interpreted in the context of two mutually non-exclusive mechanisms: (a) some of the peripheral proteins may directly participate in the thiol-dependent receptor aggregational states. Their extraction destroys this dynamic control. (b) Extraction of peripheral proteins destabilizes the receptor and makes it more susceptible to inter or intramolecular sulfhydryl-disulfide exchange, leading to the endogenous reduction of a proportion of the dimers.

Alkylation↗
PubMed1983

Sulphydryl groups and iodo-[3H]acetic acid labeling in proteolipids from Torpedo electroplax.

Several fractions of proteolipids from Torpedo electroplax were separated by DEAE-cellulose chromatography in organic solvents, and the sulphydryl groups were determined by a spectrophotometric method. On the same fractions the covalent labeling with iodo-[3H]acetic acid to sulphydryl groups was studied. In total proteolipids there were 30.3 nmol/mg protein of sulphydryl groups of which 20.6 nmoles were in the form of disulfide bonds and 10.9 nmol as free--SH groups. The highest content of sulphydryl groups (36.7 nmol/mg protein) was found in fraction II; while fraction I, that binds the cholinergic ligands, has a lower content (23.7 nmol/mg protein). The 42 Kdaltons polypeptide, which is the major band in Fraction II, has the strongest labeling with iodo-[3H]acetic acid, while the 39 Kdaltons cholinergic polypeptide shows a lower labeling. The importance of proteolipids as channel-forming macromolecules is discussed in connection with the possible significance of the 42 Kdaltons polypeptide.

Animals↗
PubMed1983

The delipidation of brain proteolipid protein by ultrafiltration.

It has been very difficult to prepare the apoprotein moiety of brain white matter proteolipid so that it is completely devoid of complex lipids, without suffering aggregation and protein denaturation. The reason is that complex lipids are tightly bound to the proteolipid apoprotein. Using a new ultrafiltration method, we obtained, in a gradual way and in a relatively short time, more than 99% delipidation in water-saturated n-butanol, with and without 0.1 M acetic acid, and recovered up to 86% of the protein with no detectable reducing sugars remaining. The delipidated protein remained in solution and in a relatively nondenatured state for several days. In 2% sodium dodecyl sulfate (SDS)-aqueous media, 90% of the lipids were removed and the yield of recovered protein in solution was near 90%; nearly 6% of the reducing sugars remained in the apoprotein. A higher delipidation was obtained by washing with 0.1 M NaOH. The content of reducing sugars was greater but the protein was less stable. When 10% SDS was employed to dissociate lipid-protein interaction, an almost complete delipidation was obtained and reducing sugars disappeared.

Animals↗
PubMed1982

Size dependence of the translational diffusion of large integral membrane proteins in liquid-crystalline phase lipid bilayers. A study using fluorescence recovery after photobleaching.

The translational diffusion of bovine rhodopsin, the Ca2+-activated adenosinetriphosphatase of rabbit muscle sarcoplasmic reticulum, and the acetylcholine receptor monomer of Torpedo marmorata has been examined at a high dilution (molar ratios of lipid/protein greater than or equal to 3000/1) in liquid-crystalline phase phospholipid bilayer membranes by using the fluorescence recovery after photobleaching technique. These integral membrane proteins having molecular weights of about 37 000 for rhodopsin, about 100 000 for the adenosinetriphosphatase, and about 250 000 for the acetylcholine receptor were reconstituted into membranes of dimyristoylphosphatidylcholine (rhodopsin and acetylcholine receptor), soybean lipids (acetylcholine receptor), and a total lipid extract of rabbit muscle sarcoplasmic reticulum (adenosinetriphosphatase). The translational diffusion coefficients of all the proteins at 310 K were found to be in the range (1-3) X 10(-8) cm2/s. In consideration of the sizes of the membrane-bound portions of these proteins, this result is in agreement with the weak dependence of the translational diffusion coefficient upon diffusing particle size predicted by continuum fluid hydrodynamic models for the diffusion in membranes [Saffman, P. G., & Delbrück, M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3111-3113]. Lipid diffusion was also examined in th same lipid bilayers with the fluorescent lipid derivative N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dimyristoylphosphatidylethanolamine. The translational diffusion coefficient for this lipid derivative was found to be in the range (9-14) X 10(-8) cm2/s at 310 K. In consideration of the dimensions of the lipid molecule, this value for the lipid diffusion coefficient is in agreement with the continuum fluid hydrodynamic model only if a near-complete slip boundary condition is assumed at the bilayer midplane. Alternatively, kinetic diffusion models [Träuble, H., & Sackmann. E. (1972) J. Am. Chem. Soc. 94, 4499-4510] may have to be invoked to explain the lipid diffusion behavior.

Acetylcholine↗