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A Karlin

Publications and source records attributed to A Karlin.

At least 55 records · Page 3Linked to original sources
PubMed1989

The intactness and orientation of acetylcholine receptor-rich membrane from Torpedo californica electric tissue.

By a mild and highly reproducible fractionation of Torpedo californica electric tissue, we prepared membrane which was 30 times enriched in nicotinic acetylcholine receptor (AChR). This preparation was neither alkali-stripped nor reconstituted and consequently contained nu (43-kDa protein), which is associated with the cytoplasmic aspect of the receptor. We tested this membrane for the presence of sealed vesicles and determined the orientation of these vesicles by combining three methods. Two of these methods were based on the accessibilities, in the presence and absence of detergent, of the extracellular acetylcholine binding site to alpha-bungarotoxin and of the intracellular nu to trypsin. These two methods are specific for AChR-containing membrane. The third method was morphometry of electron micrographs, by which we estimated the proportion of sequestered membrane. These methods taken together indicated that approximately 45% of the AChR-containing membrane was in the form of leaky vesicles or sheets, 33% was sealed right-side-out vesicles, 11% was sealed inside-out vesicles, and 11% was sequestered within multilamellar or multivesicular vesicles. The complexity of this membrane needs to be taken into account in sidedness studies of the AChR.

Animals↗
PubMed1988

Rate of induction of hypotension with trimetaphan modifies the intracranial pressure response in cats.

An infusion of 0.1% trimetaphan was administered to eight cats with artificially increased intracranial pressure (ICP) in order to decrease their mean arterial pressure (MAP) from 121 +/- 9.5 (SEM) to 58 +/- 4.6 mm Hg in less than 1 min. All cats developed an increase in intracranial pressure (ICP) (from 16 +/- 1.4 to 23 +/- 3.2 mm Hg) accompanied by a partial rebound in MAP. Eight additional cats received 0.1% trimetaphan to decrease their MAP from 128 +/- 13.4 to 52 +/- 8.1 mm Hg over more than 2 min. Four of these cats followed the same pattern, with ICP increases from 19 +/- 1.1 to 31 +/- 3.9 mm Hg, while in the other four ICP did not change. In nine of the 12 cats with an ICP increase, that increase was initiated before the partial MAP rebound. We conclude that trimetaphan causes clinically significant ICP increases in cats with increased ICP, that partial rebound in MAP frequently exacerbates these increases in ICP, and that rapid induction of hypotension tends to increase the frequency with which trimetaphan increases ICP.

Animals↗
PubMed1986

Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues.

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.

Acetylcholine↗
PubMed1986

Photoaffinity labeling of the epithelial sodium channel.

Sodium enters tight epithelia across the apical plasma membrane through a sodium channel, a process inhibited by submicromolar concentrations of amiloride and benzamil. Using membrane vesicles from bovine kidney cortex, we found that sodium transport through the sodium channel was inhibited by benzamil with an IC50 of 4 nM. Amiloride (IC50 = 400 nM) was a weaker inhibitor of sodium transport. [3H]Benzamil bound to the vesicles at a single class of high affinity binding sites with a Kd of 5 nM, the similarity of which to the IC50 suggests that these binding sites are associated with the sodium channel. Amiloride displaced bound [3H]benzamil with a Ki of 2,500 nM. Bromobenzamil is a photoactive amiloride analog with potency similar to benzamil in inhibiting sodium transport (IC50 = 5 nM) and binding to the sodium channel (Kd = 6 nM). [3H]Bromobenzamil was specifically photoincorporated into three molecular weight classes of polypeptides with apparent Mr values of 176,000, 77,000, and 47,000. The photoincorporation of [3H]bromobenzamil into these three classes of polypeptides was blocked by addition of excess benzamil and by amiloride in a dose-dependent manner. These data suggest that these polypeptides are components of the epithelial sodium channel.

Affinity Labels↗
PubMed1986

Functional domains of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor is a multisubunit, membrane-spanning protein that contains a gated, cation-conducting channel. Our approach to the understanding of the function of this receptor in molecular terms has been to locate its functionally significant sites in the sequences of its subunits and in its three-dimensional structure. In addition, we have tried to correlate transitions in the properties of these sites with functional transitions of the receptor. On binding acetylcholine, the nicotinic acetylcholine receptor enters at least two transient states, the open state and the rapid-onset desensitized state, and, in the continued presence of agonist, finally subsides into the slow-onset desensitized state. The transitions of the receptor between these various states are susceptible to regulation by acetylcholine and its congeners acting at one type of site and by a broad class of noncompetitive inhibitors (NCIs), including local anesthetics, acting at other sites. The chain composition of the receptor is alpha 2 beta gamma delta. The two acetylcholine binding sites are on the alpha chains, and two residues contributing to these sites, Cys-192 and Cys-193, have been identified. Furthermore, these adjacent Cys residues are cross-linked by a disulfide bond. In the quaternary structure of the receptor, the chains appear to be arranged in the order alpha gamma alpha beta delta around a central channel. Both the alpha and beta chains contribute to functionally significant NCI binding sites. The addition to receptor-rich membrane from Torpedo electric tissue of agonists (but not competitive antagonists) renders these NCI sites susceptible to photolabeling by the NCI quinacrine azide (QA). Furthermore, this susceptibility is transient, arising in milliseconds and subsiding in hundreds of milliseconds. These transiently susceptible sites are protected by other NCIs against photolabeling by QA. The time-course of the susceptibility and its dependence on agonist-concentration suggest that it might be the transient, rapid-onset desensitized state of the receptor that is most susceptible to photolabeling by QA.

Acetylcholine↗
PubMed1985

Binding of a curarimimetic toxin from cobra venom to the nicotinic acetylcholine receptor. Interactions of six biotinyltoxin derivatives with receptor and avidin.

We have reacted N-hydroxysuccinimidyl biotin with the principal curarimimetic toxin in Naja naja siamensis venom, biotinylating each of the five lysine residues and the N-terminal isoleucine. The six monobiotinyl-toxins were isolated by ion-exchange chromatography, and the residue modified in each was identified by peptide mapping and amino acid analysis. We evaluated the role of each lysine in the binding of toxin to the acetylcholine receptor by measuring the affinity of each biotinyltoxin for receptor and by determining which biotinyltoxins could bind receptor and avidin simultaneously. The effect of biotinylation of each residue decreased the affinity of toxin for receptor in the order Lys 23 greater than Lys 49 greater than Lys 35 greater than Lys 69 congruent to Lys 12 greater than Ile 1. Biotinyltoxin modified either at Lys 12 or at Lys 69 is effective in cross-linking avidin to receptor, while biotinyltoxin modified at Lys 49 can form a low-affinity avidin-biotinyltoxin-receptor complex. Taken together, these results help define the surface of toxin that binds to receptor.

Amino Acid Sequence↗
PubMed1985

Time-resolved photolabeling by quinacrine azide of a noncompetitive inhibitor site of the nicotinic acetylcholine receptor in a transient, agonist-induced state.

Local anesthetics and other noncompetitive inhibitors (NCIs) of the nicotinic acetylcholine receptor, acting at sites other than the acetylcholine-binding sites, block channel opening and/or cation translation through the open channel. In order to characterize the NCI sites and to decide among possible mechanisms of NCI action, we have photolabeled the receptor in membrane from Torpedo electric tissue with the photolyzable NCI [3H]quinacrine azide ([3H]QA), using a continuous-flow, rapid-mixing device and millisecond-duration irradiation. Membrane, [3H]QA, and effectors were mixed, and, after delay times of 20 ms or greater, the mixture was irradiated for 2 ms, quenched, and collected. Brief exposure of the receptor to acetylcholine, but not to hexamethonium or d-tubocurarine, induced a state particularly susceptible to photoincorporation of [3H]QA. This acetylcholine-induced photoincorporation was exclusively into the alpha and beta chains of the receptor, peaked at 100-ms delay time, declined to 15% of maximum after delay times of minutes, and was blocked by the NCIs proadifen and histrionicotoxin. At 20-ms delay, the dependence of labeling by 2 microM [3H]QA on acetylcholine concentration was characterized by an apparent dissociation constant of about 15 microM and a Hill coefficient of 1. The kinetics of the development of susceptibility to photolabeling and the apparent lack of positive cooperativity in the effect of acetylcholine on this development suggest that the preferentially photolabeled state is a transient, rapidly developing, desensitized state, rather than an open-channel state.

Affinity Labels↗
PubMed1984

Identification of the alpha subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine receptor binding site.

Nicotinic acetylcholine receptors contain a readily reducible disulfide bond at the periphery of the acetylcholine binding site. Following reduction of this disulfide, the binding site is susceptible to affinity labeling by electrophilic reagents with quaternary ammonium moieties. We reduced purified receptor from Torpedo californica electric tissue and affinity alkylated it with 4-(N-maleimido)benzyltri[3H]methylammonium iodide. The label was incorporated solely into the alpha subunit of the receptor. Isolated, labeled alpha subunit was cleaved with CNBr, and the fragments were separated by reverse-phase high-performance liquid chromatography. A uniquely labeled CNBr fragment was isolated, and its partial sequence was determined by automated Edman degradation. This CNBr fragment was cleaved at tryptophan residues, the subfragments were separated, and the labeled subfragments were partially sequenced. From our protein sequence information, we identify the labeled CNBr fragment as residues 179 to 207 of the sequence of alpha predicted from the cDNA sequence (Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Furutani, Y., Hirose, T., Asai, M., Inayama, S., Miyata, T., and Numa, S. (1982) Nature (Lond.) 299, 793-797). From the cycle of the Edman degradation in which radioactive residues are released, we conclude that Cys 192 and, possibly in addition, Cys 193 are the residues specifically labeled by 4-(N-maleimido)benzyltri[3H]methylammonium iodide. They are, therefore, close to the acetylcholine binding site.

Affinity Labels↗
PubMed1984

A continuous-flow, rapid-mixing, photolabeling technique applied to the acetylcholine receptor.

A continuous-flow technique is described in which a photoaffinity label, membrane rich in acetylcholine receptor, and various effectors are rapidly mixed, passed through a delay tube, through a tube in which they are irradiated, and are collected in a tube containing quencher. Delay times as short as 20 ms between mixing and photolysis are achievable. Because the flow is continuous, milliliter volumes of membrane can be labeled in a single run, which is convenient for the analysis of both the functional effects and sites of photolabeling. Using this technique, we have found that receptor in its transitory, active state, in which the channel is open, is more susceptible to photolabeling by the noncompetitive inhibitor analog [3H] quinacrine azide than is receptor in either its resting or desensitized states, in which the channel is closed. This technique should prove generally useful for the photolabeling of transient conformational states of macromolecules.

Acetylcholine↗
PubMed1983

The arrangement of the subunits of the acetylcholine receptor of Torpedo californica.

The monomeric form of the acetylcholine receptor from torpedo is composed of five, membrane-spanning chains with the stoichiometry alpha 2 beta gamma delta. The native receptor is predominantly a dimer cross-linked by a disulfide bridge between delta chains. We reduced native dimer to monomer and generated a different dimer by diamide-induced disulfide formation specifically between beta chains. Purified beta-beta cross-linked dimer, when adsorbed to a carbon film and negatively stained, appears in the electron microscope as two contiguous disks, frequently with central, stain-filled pits; i.e. it looks like native receptor in situ viewed normal to the plane of the membrane. We take the region of closest approach of the disks to mark the portions of the beta chains involved in the cross-link. In addition, we tagged the acetylcholine binding sites (one on each alpha chain) for electron microscopic identification, using a complex of monobiotinylated cobratoxin and avidin. Based on the locations of avidins bound to the beta-beta cross-linked dimer, the two toxin binding sites/monomer appear to be separated on the average by 110 degrees, as measured between lines from the center of the monomer to the centers of the avidins. One toxin binding site appears close to the beta-beta cross-link and the other close to the end of the monomer opposite to the cross-link; these locations are similar to the locations of the toxin binding sites relative to the delta-delta cross-link in native dimer. On the assumptions that the chains are compact units and are arranged in a unique order around the central pit, we interpret these results as indicating that the alpha chains are not contiguous and that neither the beta chain nor the delta chain lies between them. Therefore, the arrangement of the chains most easily reconciled with our assumptions and observations is alpha gamma alpha beta delta.

Animals↗
PubMed1983

Reaction of quinacrine mustard with the acetylcholine receptor from Torpedo californica.

Amines with local anesthetic activity are typically also noncompetitive inhibitors of the agonist-induced increase in cation permeability mediated by the nicotinic acetylcholine receptor. Quinacrine is such an agent, and we have synthesized tritiated quinacrine mustard, a derivative capable of reacting with nucleophiles. Quinacrine mustard was reacted with receptor-rich membrane from torpedo electric tissue, excess reagent was removed by partition into liposomes, and the modified receptor was extracted and reconstituted with exogenous phospholipid. After reaction of the native membrane with 10 microM quinacrine mustard for 5 min, binding of cobratoxin to the acetylcholine binding sites is inhibited 15%; in contrast, receptor-mediated 86Rb uptake in the reconstituted vesicles is inhibited 70%. When the reaction with quinacrine mustard is carried out in the presence of 10 microM carbamylcholine or 10 microM d-tubocurarine, there is no block of the acetylcholine binding sites; nevertheless, the inhibition of Rb uptake is greater than that resulting from reaction in the absence of acetylcholine binding site ligands. Conversely, when the reaction is carried out in the presence of either 100 microM quinacrine or 100 microM proadifen (also a potent noncompetitive inhibitor), either with or without carbamylcholine or d-tubocurarine, the inhibition of 86Rb uptake is about 70% smaller. Under the same conditions that we used in the functional studies, quinacrine mustard reacts with the four types of chains that constitute the receptor complex, alpha 2 beta gamma delta. The presence of the acetylcholine binding site ligands, however, results in increased reaction with the alpha and beta chains, while the presence of the noncompetitive inhibitors, with or without the acetylcholine binding site ligands, results in decreased reaction with the alpha and beta chains. We conclude that the alpha and beta chains contribute to one or more functionally significant binding sites for noncompetitively inhibiting amines.

Animals↗
PubMed1982

Electron microscopy of complexes of isolated acetylcholine receptor, biotinyl-toxin, and avidin.

The principal curarimimetic toxin of Naja naja siamensis derivatized with biotinyl groups binds specifically both to acetylcholine receptor, isolated from Torpedo californica electric tissue, and to avidin. Isolated complexes of receptor monomer or dimer, biotinyl-toxin, and avidin were negatively stained and examined in the scanning transmission electron microscope. We measured the angle made by the radius of each avidin bound at the periphery of a monomeric unit in dimer to the axis connecting the centers of the monomers, starting at the crosslink between the monomers. We infer from the distribution of these angles that one toxin binding site is located in the range of 45 degrees to 85 degrees and another at about 100 degrees further from the crosslink between the monomers. Because it is known that there are two toxin binding sites per monomer, associated with the two alpha chains, the bound avidins presumably point to portions of the alpha chains, indicating their positions relative to that portion of the delta chain located at the crosslink between monomers in dimer.

Animals↗
PubMed1981

Relative locations of the beta and delta chains of the acetylcholine receptor determined by electron microscopy of isolated receptor trimer.

The monomeric unit of the acetylcholine receptor of electric tissue of Torpedo californica has previously been shown to have a subunit composition of alpha 2 beta gamma delta. Receptor in membrane isolated from Torpedo electric tissue occurs as both monomer and dimer. In the dimer which is the predominant form, the monomeric units are cross-linked via a disulfide bond between delta chains. The addition of diamide to receptor-rich membrane causes the formation of trimer and higher oligomers in which the monomeric units are linked by disulfide bonds alternately between pairs of delta chains and between pairs of beta chains. We have isolated receptor trimer and determined the relative locations of the monomeric units by scanning transmission electron microscopy of negatively stained preparations. In face view, the trimer appears as three approximately 90 A disks, each with a central, densely staining pit. From the angles of the triangle formed by the lines connecting the centers of the monomers in the trimer, we infer that the beta-beta disulfide bond is separated from the delta-delta disulfide bond by an angle in the range of 50-80 degrees.

Animals↗
PubMed1981

Structure of acetylcholine receptor dimer determined by neutron scattering and electron microscopy.

Previous work has shown that the predominant native form of the acetylcholine receptor from the electric tissue of Torpedo californica is a dimer of Mr = 500,000, cross-linked by a disulfide bond between the largest (delta) of the five chains (alpha 2 beta gamma delta) that comprise the monomer. Small-angle neutron scattering of purified receptor dimer in Triton X-100 solution containing 18% D2O, in which the Triton X-100 is contrast-matched, yields a radius of gyration of the dimer of 66 A. Based on the assumptions that the dimer is symmetrical and that the radius of gyration of the monomer does not change in forming dimer, this value, together with the radius of gyration of the receptor monomer (46 A), determined previously, allows the calculation of the distance separating the centers of neutron scattering density of monomers in a dimer; the result is 96 A. Electron microscopy of negatively stained dimers permits an independent measurement of the distance between the apparent centers of mass of the monomers; the average is 96 A, in agreement with the result of the neutron scattering analysis. The electron micrographs of dimer also permit the location of the delta chains at the region of contact of the monomers. A model for the receptor dimer consistent with all available structural information is presented.

Acetylcholine↗