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A Karlin

Publications and source records attributed to A Karlin.

At least 37 records · Page 2Linked to original sources
PubMed1996

Structure of the NMDA receptor channel M2 segment inferred from the accessibility of substituted cysteines.

The structure of the NMDA receptor channel M2 segment was investigated by probing the extracellular and cytoplasmic faces of cysteine-substituted NR1-NR2C channels with charged sulfhydryl-specific reagents. The pattern of accessible positions suggests that the M2 segment forms a channel-lining loop originating and ending on the cytoplasmic side of the channel, with the ascending limb in an alpha-helical structure and the descending limb in an extended structure. A functionally critical asparagine (N-site) is positioned at the tip of the loop, and a cluster of hydrophilic residues of the descending limb, adjacent to the tip, forms the narrow constriction of the channel. An apparent asymmetric positioning of the NR1- and NR2-subunit N-site asparagines may account for their unequal role in Ca2+ permeability and Mg2+ block.

Amino Acid Sequence↗
PubMed1995

Identification of acetylcholine receptor channel-lining residues in the M1 segment of the alpha-subunit.

The muscle-type acetylcholine (ACh) receptor has the composition alpha 2 beta gamma delta. The subunits are arranged quasisymmetrically around a central, ion-conducting, water-filled channel. Each subunit has four membrane-spanning segments, M1-M4, and the channel through the membrane is formed among these segments. Substituting cysteine for each of the residues in and flanking the alpha M2 segment, we previously found that, at 10 of the 21 mutated positions, the cysteine was accessible to a small, positively charged, sulfhydryl-specific reagent, methanethiosulfonate ethylammonium (MTSEA), and inferred that the residues at these positions are exposed in the channel lumen. We have now applied the substituted-cysteine-accessibility method to alpha M1. We analyzed 15 consecutive residues, starting at alpha Pro211 at the extracellular end of M1. Wild-type alpha contains Cys222, which is inaccessible to MTSEA. We mutated each of the other 14 residues to cysteine and expressed the mutant alpha subunits, together with wild-type beta, gamma, and delta subunits, in Xenopus oocytes. Thirteen of the fourteen mutants gave robust ACh-induced currents. MTSEA irreversibly altered the ACh-induced response of seven cysteine-substitution mutants: alpha Y213C was susceptible to MSTEA added in the presence or the absence of ACh, alpha P211C, alpha I215C, alpha V216C, alpha N217C, and alpha I220C were susceptible in the absence of ACh, and alpha V218C was susceptible in the presence of ACh. These results imply that M1 is exposed in the channel, and its exposure changes during gating or desensitization.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Structure of the nicotinic receptor acetylcholine-binding site. Identification of acidic residues in the delta subunit within 0.9 nm of the 5 alpha subunit-binding.

In the nicotinic receptor, the quaternary ammonium group of acetylcholine (ACh) binds to a negative subsite at most 1 nm from a readily reducible disulfide formed between alpha-subunit residues Cys192 and Cys193. The cross-linker S-(2-[3H]glycylamidoethyl)dithio-2-pyridine formed a disulfide bond with reduced alpha Cys192/Cys193 and an amide bond with an acidic residue in the delta subunit (Czajkoswski, C., and Karlin, A. (1991) J. Biol. Chem. 266, 22603-22612). The fully extended cross-linking moiety -NHCH2CONHCH2CH2S- is is 0.9 nm long. After the disulfide bond linking -NHCH2CONHCH2CH2S- to the alpha subunit was reduced, -NHCH2CONHCH2CH2SH remained linked to the delta subunit by an amide bond. The delta subunit was cleaved at Met residues, and the radioactive fragments were isolated and sequenced by automated Edman degradation. Additionally, the isolated radioactive fragments were further cleaved at Trp residues and sequenced. Peaks of release of radioactivity were obtained in the sequencing cycles corresponding to delta Asp165, delta Asp180, and delta Glu182. The mutation of delta Asp180 to Asn decreased the affinity of the receptor for ACh 100-fold, whereas the mutation of either delta Asp165, delta Glu182, or 8 other acidic residues in the same region of delta decreased the affinity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin, A. (1993) Proc. Natl. Acad. Sci. U.S.A 90, 6285-6289). Because delta Asp180 both contributes to ACh binding and is suitably close to the binding site disulfide, it is likely to be part of the ACh-binding site formed in the interface between the alpha and the delta subunits.

Amino Acid Sequence↗
PubMed1995

Mapping the binding-site crevice of the dopamine D2 receptor by the substituted-cysteine accessibility method.

The binding site of the dopamine D2 receptor, like that of other homologous G protein-coupled receptors, is contained within a water-accessible crevice formed among its seven membrane-spanning segments. We have developed a method to map systematically all the residues forming the surface of this binding-site crevice, and we have applied this method to the third membrane-spanning segment (M3). We mutated, one at a time, 23 residues in and flanking M3 to cysteine and expressed the mutant receptors heterologously. Ten of these mutants reacted with charged, hydrophilic, lipophobic, sulfhydryl-specific reagents, added extracellularly, and were protected from reaction by a reversible dopamine antagonist. Thus, the side chains of these residues are exposed in the binding-site crevice, which like M3 extends from the extracellular to the intracellular side of the membrane. The pattern of exposure is consistent with a short loop followed by six turns of an alpha helix.

Binding Sites↗
PubMed1994

A cysteine residue in the third membrane-spanning segment of the human D2 dopamine receptor is exposed in the binding-site crevice.

The binding site in G-protein-linked neurotransmitter receptors is formed among their membrane-spanning segments. Because the binding site is in the plane of the bilayer and is accessible to charged, water-soluble agonists, it must lie in a crevice open to the extracellular, aqueous medium. Information about the structure of these receptors can be obtained by identifying the residues in the membrane-spanning segments which face this water-filled crevice. Human D2 dopamine receptor was expressed in human embryonic kidney 293 cells. Small, charged, sulfhydryl-specific methanethiosulfonate (MTS) derivatives irreversibly inhibited the binding of the D2-specific antagonist [3H]YM-09151-2 to these cells. The highly polar MTS derivatives should react with cysteine sulfhydryl groups only at the water-accessible surface of the receptor, which includes the surface of the binding-site crevice. In contrast, these reagents will have little access to sulfhydryls facing the lipid bilayer or buried in the protein interior. Positively charged MTS reagents irreversibly inhibited binding several hundredfold faster than a negatively charged MTS reagent, consistent with the affinity of the binding site for positively charged dopamine agonists and antagonists. Furthermore, both agonists and antagonists of the D2 receptor protected against irreversible inhibition by the MTS reagents. To identify the susceptible cysteine, we mutated, one at a time, five transmembrane and two extracellular cysteine residues to serine. Only the mutation of Cys118 to serine decreased the susceptibility of antagonist binding to irreversible inhibition by the MTS reagents. Thus, Cys118, a residue in the middle of the third membrane-spanning segment, is exposed in the D2 receptor binding-site crevice.

Amino Acid Sequence↗
PubMed1994

Electrostatic potential of the acetylcholine binding sites in the nicotinic receptor probed by reactions of binding-site cysteines with charged methanethiosulfonates.

All of the potent agonists and competitive antagonists of the acetylcholine receptors are positively charged, onium compounds. Among the interactions involved in the binding of these compounds, electrostatic forces undoubtedly make an important contribution. There is evidence that the acetylcholine binding site contains both acidic and aromatic amino acids. The acidic side chains could provide long-range charge-charge interactions with acetylcholine, while the aromatic side chains could provide short-range cation-pi-electron and hydrophobic interactions. To probe the long-range electrostatic interactions in the binding site, the rate constants for the reactions of sulfhydryl-specific reagents with cysteines in the binding site have been determined as a function of ionic strength. The reagents are the positively charged methanethiosulfonate ethylammonium and methanethiosulfonate ethyltrimethylammonium, the negatively charged methanethiosulfonate ethylsulfonate, and the neutral methyl methanethiosulfonate. In addition, the rate constants of the reactions of these methanethiosulfonates with positively charged, negatively charged, and uncharged simple thiol compounds have been similarly determined. An analysis of these rate constants in terms of absolute rate theory and Debye-Hückel theory is consistent with the acetylcholine binding site containing two to three negative charges and an electrostatic potential at zero ionic strength of about -80 mV relative to bulk solution.

Acetylcholine↗
PubMed1994

Identification of acetylcholine receptor channel-lining residues in the entire M2 segment of the alpha subunit.

Each residue in and flanking the M2 membrane-spanning segment of the alpha subunit, from Glu-241 to Glu-262, was mutated to cysteine, and the mutant subunits were expressed together with wild-type beta, gamma, and delta subunits in Xenopus oocytes. Cysteines substituted for Glu-262, Leu-258, Val-255, Ser-252, Leu-251, Leu-250, Ser-248, Leu-245, Thr-244, and Glu-241 reacted with the positively charged, hydrophilic, sulfhydryl-specific reagent methanethiosulfonate ethylammonium (MTSEA), added extracellularly. These 10 residues, therefore, are exposed in the channel lumen. The pattern of exposure is compatible with an alpha helix, interrupted by an extended structure from Leu-250 to Ser-252. Acetylcholine caused subtle changes in the accessibilities of some of the engineered cysteines. Since all 10 residues are accessible to MTSEA in the closed state of the channel, the channel gate is at least as cytoplasmic as Glu-241, the most cytoplasmic of the residues tested.

Acetylcholine↗
PubMed1994

Expression and characterization of human dopamine D2 receptor in baculovirus-infected insect cells.

Multiple types of dopamine D2-like receptors (D2, D3, D4) have been identified. Differences in pharmacology among these receptors may have profound clinical ramifications for the treatment of psychosis. Analysis of the structure and function of their binding sites requires a source of large amounts of receptor, uncontaminated by the other types of D2-like receptor. We engineered a recombinant baculovirus containing the human D2 receptor cDNA (DRD2) to express this receptor in insect cells. Spodoptera frugiperda cells (Sf9 and Sf21) and Trichoplusia ni cells (TN-5) were infected with the recombinant baculovirus. Binding of the D2 antagonist [3H]YM-09151-2 to membranes fractions of these cells peaked at a specific activity of 5-8 pmol/mg protein, approximately 40 times that of membranes from bovine striatum. The receptor expressed in Sf9 cells was similar to that of striatum in its affinities for D2 agonists and antagonists. Sodium ion stimulated [3H]YM-09151-2 binding to D2 receptor in infected Sf9 cell membranes. This effect was fit by an allosteric model which predicted the apparent affinity of [3H]YM-09151-2. The D2 receptor expressed in Sf9 and TN-5 cells was photolabeled with N-(p-azido-m-[125I]iodophenylethyl)spiperone. The specifically labeled component(s) ran as a broad band of apparent molecular weight between 54,000 and 60,000. Deglycosylation of the labeled component(s) reduced its molecular weight to 46,000.

Animals↗
PubMed1993

Negatively charged amino acid residues in the nicotinic receptor delta subunit that contribute to the binding of acetylcholine.

In nicotinic receptors, the binding sites for acetylcholine are likely to contain negatively charged amino acid side chains that interact with the positively charged quaternary ammonium group of acetylcholine and of other potent agonists. We previously found that a 61-residue segment of the delta subunit contains aspartate or glutamate residues within 1 nm of cysteines in the acetylcholine binding site on the alpha subunit. We have now mutated, one at a time, the 12 aspartates and glutamates in this segment of the mouse muscle delta subunit and have expressed the mutant receptors in Xenopus oocytes. Both the concentration of acetylcholine eliciting half-maximal current (Kapp) and the Ki for the inhibition by acetylcholine of alpha-bungarotoxin binding were increased 100-fold by the mutation of delta Asp180 to Asn and 10-fold by the mutation of delta Glu189 to Gln. These two residues, and their homologs in the gamma and epsilon subunits, are likely to contribute to the acetylcholine binding sites.

Acetylcholine↗
PubMed1993

Structure of nicotinic acetylcholine receptors.

Nicotinic acetylcholine (ACh) receptors convert the binding of ACh into the opening of a cation-conducting channel. New information about the regions of the receptor most immediately involved in its function, namely the ACh-binding sites, the gate and the channel, has come from two approaches. One is the identification by labelling and by mutagenesis of residues contributing to these regions. Another is the determination of the three-dimensional structure of the receptor by electron microscopy. Although the identification of functionally relevant residues is incomplete and residues cannot yet be resolved in the three-dimensional structure, the two approaches are converging. There is still room in the gap for speculation.

Acetylcholine↗
PubMed1992

Acetylcholine receptor channel structure probed in cysteine-substitution mutants.

In order to understand the structural bases of ion conduction, ion selectivity, and gating in the nicotinic acetylcholine receptor, mutagenesis and covalent modification were combined to identify the amino acid residues that line the channel. The side chains of alternate residues--Ser248, Leu250, Ser252, and Thr254--in M2, a membrane-spanning segment of the alpha subunit, are exposed in the closed channel. Thus alpha 248-254 probably forms a beta strand, and the gate is closer to the cytoplasmic end of the channel than any of these residues. On channel opening, Leu251 is also exposed. These results lead to a revised view of the closed and open channel structures.

Acetylcholine↗
PubMed1991

Agonist binding site of Torpedo electric tissue nicotinic acetylcholine receptor. A negatively charged region of the delta subunit within 0.9 nm of the alpha subunit binding site disulfide.

The positively charged quaternary ammonium group of agonists of the nicotinic acetylcholine (ACh) receptor binds to a negative subsite at most about 1 nm from a readily reducible disulfide. This disulfide is formed by alpha Cys192 and Cys193 (Kao and Karlin, 1986). In order to identify Asp or Glu residues that may contribute to the negative subsite, we synthesized S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. Purified ACh receptor from Torpedo californica was mildly reduced and reacted with S-(2-[3H]glycylamidoethyl)dithio-2-pyridine. The predominant product was a mixed disulfide between the 3H-N-glycylcysteamine moiety and alpha Cys192 or Cys193. In the extended conformation of [3H] N-glycylcysteamine, the distance from the glycyl amino group to the cysteamine thio group is 0.9 nm. Thus, the amino group of disulfide-linked [3H]N-glycylcysteamine could react with carboxyls within 0.9 nm of Cys192/Cys193. To promote amide bond formation between the tethered amino group and receptor carboxyls, we added 1-ethyl-3-(3'-dimethylaminopropyl)-carbodiimide. The predominant sites of amide coupling were on the delta subunit, in CNBr fragment 4 (delta 164-257). This reaction was inhibited by ACh. Only the first 61 residues of delta CNBr 4 are predicted to be extracellular, and there are 11 Asp or Gly residues in this region. One or more of these residues is likely to contribute to the binding of ACh.

Amino Acid Sequence↗
PubMed1991

Paraben preservatives do not increase intracranial pressure in cats.

It has been hypothesized recently that succinylcholine-associated increases in intracranial pressure (ICP) are caused by the paraben preservatives contained in multidose vials. We tested that hypothesis in a standard feline model to determine the effects on ICP of equal-volume injections of preservative-free succinylcholine, succinylcholine with preservatives from multi-dose vials that contain both propylparaben and methylparaben, these preservatives alone at five times the dose contained in the succinylcholine, and normal saline. The preservatives alone increased ICP by 0.08 +/- 0.08 mmHg (+/- standard error; not significant). Normal saline had no effect on ICP. Preservative-free succinylcholine and succinylcholine with preservatives increased ICP by 4.2 +/- 0.10 and 3.8 +/- 0.07 mmHg respectively (P less than 0.01 compared to the preservatives alone and normal saline). The 99% upper confidence limit for the increase in ICP induced by the preservatives alone was 0.42 mmHg. This result suggests that parabens do not cause or substantially augment the ICP increase associated with succinylcholine administration.

Animals↗
PubMed1990

Mapping the alpha-subunit site photolabeled by the noncompetitive inhibitor [3H]quinacrine azide in the active state of the nicotinic acetylcholine receptor.

We have characterized the time-resolved labeling of a site on the Torpedo californica electrocyte acetylcholine receptor (ACHR) by the photoreactive noncompetitive inhibitor derivative quinacrine azide (QA). The dependence of [3H]QA labeling on acetylcholine (ACH) concentration and on time is consistent with the preferential labeling by [3H]QA of ACHR in the open state. The ACH-dependent [3H]QA labeling, which was associated predominantly with the alpha-subunit, was blocked by other noncompetitive inhibitors including quinacrine, chlorpromazine, proadifen, histrionicotoxin, and bupivacaine. alpha-Subunit from ACHR labeled with [3H]QA 20 ms after the addition of ACH was cleaved with CNBr, and the fragments were separated by high pressure liquid chromatography. A peptide containing a major site of specific labeling was purified on two different reverse-phase columns. By N-terminal sequencing, amino acid composition, binding to mercurial-agarose, and apparent molecular weight, this [3H]QA-labeled peptide was identified as alpha-208-243, a CNBr fragment containing the putative membrane-spanning helix M1.

Acetylcholine↗
PubMed1989

The sidedness of the COOH terminus of the acetylcholine receptor delta subunit.

The nicotinic acetylcholine receptor from Torpedo sp. occurs as a dimer, disulfide-cross-linked between delta subunits. We determined the sidedness of the COOH terminus of the acetylcholine receptor delta subunit by locating the delta-delta disulfide relative to the membrane and by identifying the Cys residue forming the disulfide. We used receptor-rich native membrane vesicles isolated from Torpedo californica electric tissue and characterized as to orientation and intactness. These vesicles had not been extracted and retained v ("43-kDa protein") as a marker of the cytoplasmic surface. Using the reduction of v as an assay of permeability, we showed that two reductants, 2-mercaptoethanesulfonate and reduced glutathione, were relatively impermeant. Both of these reductants reduced the delta-delta disulfide in sealed right-side-out vesicles equally in the presence and absence of saponin, and 2-mercaptoethanesulfonate reduced this disulfide equally in the presence and absence of Triton X-100. By contrast, surfactants enhanced the reduction of dimer in inside-out and sequestered vesicles. We conclude that the disulfide is extracellular. To identify the Cys residue forming the disulfide, we labeled the sulfhydryls both in receptor dimer and in monomer generated by mild reduction of dimer. By high performance liquid chromatography and NH2-terminal sequencing of cyanogen bromide fragments of labeled delta-delta dimer and delta monomer, we found that the penultimate residue, delta-Cys-500, uniquely formed an intersubunit disulfide and that this disulfide was uniquely reduced when receptor dimer was reduced to monomer. Therefore, the delta COOH terminus is extracellular.

Amino Acid Sequence↗