Solid state NMR studies of ligands bound to the nicotinic acetylcholine receptor.
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Acetylcholine↗
Biomedical subjects
K W Miller
Publications and source records attributed to K W Miller.
Production of active chimeric pediocin AcH in Escherichia coli in the absence of processing and secretion genes from the Pediococcus pap operon.
Minimum requirements have been determined for synthesis and secretion of the Pediococcus antimicrobial peptide, pediocin AcH, in Escherichia coli. The functional mature domain of pediocin AcH (Lys+1 to Cys+44) is targeted into the E. coli sec machinery and secreted to the periplasm in active form when fused in frame to the COOH terminus of the secretory protein maltose-binding protein (MBP). The PapC-PapD specialized secretion machinery is not required for secretion of the MBP-pediocin AcH chimeric protein, indicating that in Pediococcus, PapC and PapD probably are required for recognition and processing of the leader peptide rather than for translocation of the mature pediocin AcH domain across the cytoplasmic membrane. The chimeric protein displays bactericidal activity, suggesting that the NH2 terminus of pediocin AcH does not span the phospholipid bilayer in the membrane-interactive form of the molecule. However, the conserved Lys(+1)-Tyr-Tyr-Gly-Asn-Gly-Val(+7)-sequence at the NH2 terminus is important because deletion of this sequence abolishes activity. The secreted chimeric protein is released into the culture medium when expressed in a periplasmic leaky E. coli host. The MBP fusion-periplasmic leaky expression system should be generally advantageous for production and screening of the activity of bioactive peptides.
ATP-Binding Cassette Transporters↗
Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity.
A collection of pediocin AcH amino acid substitution mutants was generated by PCR random mutagenesis of DNA encoding the bacteriocin. Mutants were isolated by cloning mutagenized DNA into an Escherichia coli malE plasmid that directs the secretion of maltose binding protein-pediocin AcH chimeric proteins and by screening transformant colonies for bactericidal activity against Lactobacillus plantarum NCDO955 (K. W. Miller, R. Schamber, Y. Chen, and B. Ray, 1998. Appl. Environ. Microbiol. 64:14-20, 1998). In all, 17 substitution mutants were isolated at 14 of the 44 amino acids of pediocin AcH. Seven mutants (N5K, C9R, C14S, C14Y, G37E, G37R, and C44W) were completely inactive against the pediocin AcH-sensitive strains L. plantarum NCDO955, Listeria innocua Lin11, Enterococcus faecalis M1, Pediococcus acidilactici LB42, and Leuconostoc mesenteroides Ly. A C24S substitution mutant constructed by other means also was inactive against these bacteria. Nine other mutants (K1N, W18R, I26T, M31T, A34D, N41K, H42L, K43N, and K43E) retained from <1% to approximately 60% of wild-type activity when assayed against L. innocua Lin11. One mutant, K11E, displayed approximately 2. 8-fold-higher activity against this indicator. About one half of the mutations mapped to amino acids that are conserved in the pediocin-like family of bacteriocins. All four cysteines were found to be required for activity, although only C9 and C14 are conserved among pediocin-like bacteriocins. Several basic amino acids as well as nonpolar amino acids located within the hydrophobic C-terminal region also were found to be important. The mutations are discussed in the context of structural models that have been proposed for the bacteriocin.
ATP-Binding Cassette Transporters↗
Age and sex-related variation in hyoid bone morphology.
Although the relationship between hyoid bone shape and fracture pattern figures prominently in forensic investigations of strangulation, few quantitative data exist on age and sex differences in hyoid morphology. An image analysis system was used to take a series of 30 measurements on digitized radiographs of 315 hyoid bones from people of known age and sex. The degree of fusion of the greater cornua to the hyoid body was also recorded. Statistical analysis of these data shows that there is a continuous distribution of hyoid bone shapes and the most bones are highly symmetrical. Based on smaller samples, previous researchers have suggested that non-fusion is more common in women than in men. In contrast, our data suggest that men and women have similar non-fusion rates. Analysis of sexual dimorphism shows that the greatest length differences are in the greater cornua. There are also significant sex differences in hyoid shape. For example, the distal ends of the greater cornua of women are significantly longer than those of men.
Adolescent↗
A thermodynamic analysis of the partitioning of cholesterol and related compounds between trioleoylglycerol and egg phosphatidylcholine bilayers.
The free energy of transfer of a number of alcohols, including cholesterol, from a bulk isotropic lipid phase, trioleoylglycerol (TG), to an anisotropic lipid phase, egg phosphatidylcholine (PC), was determined. n-Alkane-1-ols partitioned preferentially into the bilayer phase; for example, the free energy of transfer of octanol-1 from TG to PC was about -1.0 kcal/mol. This preference declined with increasing number of carbons at a rate of 40 cal/mol of CH2. Cholesterol had a much stronger preference for the bilayer with a free energy of -1.3 kcal/mol, compared to an extrapolated value of -0.2 kcal/mol for a normal alkane-1-ol with the same number of carbon atoms. Thus, the excess free energy of -1.1 kcal/mol represents the favourable interaction of the cholesterol skeleton with the bilayer phase. This conclusion was confirmed by comparing cholesterol 3-hemisuccinate to oleic acid. Substituting TG for water as the standard state has eliminated the large hydrophobic effect and has permitted us to identify for the first time the subtle binding increment of the steroid ring system.
Alcohols↗
p-(4-Hydroxybenzoyl)phenylalanine: a photoreactive amino acid analog amenable to radioiodination for elucidation of peptide-protein interaction. Application to substance P receptor.
Benzoylphenylalanine, a photoreactive phenylalanine analog that can be incorporated into a peptide during solid-phase synthesis, is a useful probe for investigating the interactions of bioactive peptides with their receptors. This probe, however, lacks versatility because it is not detectable by Edman sequencing and because it cannot be labeled with radioiodine, requiring radiolabeling of the peptide ligand at a site distal to the photoreactive amino acid. The separation of the radioisotope and photoaffinity labels along the primary sequence limits identification of the photoinsertion site to a peptide fragment rather than a specific amino acid of the receptor protein. We have now synthesized p-(4-hydroxybenzoyl)phenylalanine by a synthetic route involving reaction of 4-(chloromethyl)benzoic anhydride with phenol in polyphosphoric acid to give the 4-(chloromethyl)benzoyl ester of 4-(chloromethyl)-4'-hydroxybenzophenone followed by reaction of the benzophenone derivative with ethyl acetamidocyanoacetate and subsequent hydrolysis of the product to give p-(4-hydroxybenzoyl)phenylalanine. The novel photolabile amino acid was incorporated into substance P (replacing Phe8 or Lys3) to give 11-mer peptides that bind with high (nM) affinity and specificity to the substance P receptor. Radioiodination of the substance P analogs resulted in the incorporation of 125I at the photoreactive amino acid residue, yielding probes of high (approximately 2000 Ci/mmol) specific activity. Subsequent photolysis of the radiolabeled peptides in the presence of substance P receptor caused covalent attachment of the peptide to the receptor with high photoinsertion yield (approximately 30%); photolabeling was abolished in the presence of excess unlabeled SP. p-(4-Hydroxybenzoyl)phenylalanine retains p-benzoylphenylalanine's high insertion yield and low reactivity with water, but in contrast allows placement of radioiodine and the photoactive moieties within the same residue, providing the ability to identify the specific site(s) of interaction, and identification of the residue by Edman sequencing. This novel amino acid may be useful in the elucidation of the interaction of a variety of peptides with their receptors.
Affinity Labels↗
Comparison of olestra absorption in guinea pigs with normal and compromised gastrointestinal tracts.
Female guinea pigs (12/group) were given a single dose of [14C]olestra by gavage after consuming either 3% poligeenan in tap water (Compromised group) or just tap water (Normal group) for 5 weeks. A Sentinel group (N = 2) was given 3% poligeenan for 5 weeks. Ten sentinel animals were killed 1 day before and 10 1 day after the other animals were dosed with [14C]olestra and their gastrointestinal tracts were examined by histology. The Compromised and Normal animals were endoscoped just before dosing with [14C]olestra. Urine and feces were collected continuously and CO2 was collected for 7 days after dosing. The samples were analyzed for 14C and urine was also analyzed for [14C]sucrose. Animals (3/group) were killed 1, 3, 7, and 21 days after dosing, and tissues were collected and assayed for 14C. Tissue lipids were extracted, fractionated by high-pressure liquid chromatography, and analyzed for [14C]olestra by liquid scintillation. Animals fed poligeenan showed mucosal edema, congestion, ulceration, and fibrin deposition within the distal colon and rectum. Histology revealed inflammation, epithelial degeneration, and multifocal ulceration of the cecum, distal colon, and rectum. The gastrointestinal mucosae of nonpoligeenan fed animals were normal. No [14C]olestra was detected in liver lipids and no [14C]sucrose was found in the urine for any animal in the Normal or Compromised groups, indicating that intact olestra was not absorbed. The amount, distribution, and elimination of absorbed 14C did not differ between guinea pigs with normal and compromised gastrointestinal tracts. The poligeenan-treated animals displayed mucosal damage similar to that seen in human inflammatory bowel diseases; therefore, these results suggest that patients with inflammatory bowel conditions will not absorb olestra to any greater extent than normal healthy people.
Administration, Oral↗
The cholesterol dependence of activation and fast desensitization of the nicotinic acetylcholine receptor.
When nicotinic acetylcholine receptors are reconstituted into lipid bilayers lacking cholesterol, agonists no longer stimulate cation flux. The kinetics of this process are difficult to study because variations in vesicle morphology cause errors in flux measurements. We developed a new stopped-flow fluorescence assay to study activation independently of vesicle morphology. When receptors were rapidly mixed with agonist plus ethidium, the earliest fluorescence increase reported the fraction of channels that opened and their apparent rate of fast desensitization. These processes were absent when the receptor was reconstituted into dioleoylphosphatidylcholine or into a mixture of that lipid with dioleoylphosphatidic acid (12 mol%), even though a fluorescent agonist reported that resting-state receptors were still present. The agonist-induced channel opening probability increased with bilayer cholesterol, with a midpoint value of 9 +/- 1.7 mol% and a Hill coefficient of 1.9 +/- 0.69, reaching a plateau above 20-30 mol% cholesterol that was equal to the native value. On the other hand, the observed fast desensitization rate was comparable to that for native membranes from the lowest cholesterol concentration examined (5 mol%). Thus the ability to reach the open state after activation varies with the cholesterol concentration in the bilayer, whereas the rate of the open state to fast desensitized state transition is unaffected. The structural basis for this is unknown, but an interesting corollary is that the channels of newly synthesized receptors are not fully primed by cholesterol until they are inserted into the plasma membrane--a novel form of posttranslational processing.
Animals↗
The feasibility of a street-intercept survey method in an African-American community.
OBJECTIVES: This study evaluates the feasibility of a nonquota, street-intercept survey method that utilized random selection of interview sites. METHODS: The street-intercept survey was compared with a random digit-dial telephone survey conducted in the same catchment area among African-American adults aged 18 or older. RESULTS: The street-intercept survey's response rate was 80.2%; residence rate, 85.3%; interview completion rate, 97.9%; interference rate, 4.0%; and yield rate, 2.5 interviews per interviewer per hour. The street-intercept method produced more representative distributions of age and sex than the random-digit-dial survey. CONCLUSIONS: The street-intercept method is a feasible alternative to traditional population survey methods and may provide better access to harder-to-reach segments of the urban population in a safe manner.
Adolescent↗
Efficient insertion of odd-numbered transmembrane segments of the tetracycline resistance protein requires even-numbered segments.
Functional membrane insertion elements in the pBR322 tetracycline resistance protein were identified by comparing the ability of odd-numbered transmembrane segments and their attached periplasmic loops to insert into the membrane individually or when combined with the next even-numbered segment in the tetracycline resistance protein sequence. The efficiency with which individual odd-numbered segments and periplasmic loops inserted was probed by treating proteins truncated at the distal ends of periplasmic loops P2-P6 with carboxypeptidases and endoproteases in inside-out membrane vesicles. Insertion of odd-numbered segments and attached loops is inefficient when they occupy a C-terminal position in the protein. The C-terminal odd-numbered segment and loop sequences of 34-54% of the molecules of periplasmic loop truncation mutants could be removed by carboxypeptidase Y. In contrast, odd-numbered segments and loops insert efficiently if the next even-numbered segment in the sequence is present. In such cytoplasmic loop truncation mutants, only the cytoplasmic tail sequences of the proteins could be removed by carboxypeptidases. Remarkably, insertion of individual odd-numbered segments and loops is inefficient even though free energies for insertion of these sequences are highly favorable. The results indicate that pairs of adjacent segments, possibly "helical hairpins," are necessary for efficient membrane insertion of the tetracycline resistance protein.
Amino Acid Sequence↗
A concordance of nucleotide substitutions in the first and second hypervariable segments of the human mtDNA control region.
A new and easily accessible concordance of nucleotide substitutions in the hypervariable segments of the human mitochondrial DNA (mtDNA) control region has been constructed. The concordance indexes all population-specific mtDNA sequences in a standardized format. The first edition of the concordance includes 1,440 sequences representing 762 mtDNA types from over 65 populations for hypervariable region 1, and 520 sequences representing 260 mtDNA types from over 26 populations for hypervariable region 2. Investigators are invited to submit new sequences to the database, and details for doing so are given in the text.
Anthropology↗
Disposition of ingested olestra in weanling mini-pigs.
The disposition of ingested olestra in Hanford mini-pigs was examined by following a single oral gavage dose of radiolabelled (U-14C-sucrose) olestra Eight dosed animal (four/sex) and one undosed animal were killed 1, 3 and 7 days after dosing, and tissues were collected and counted. Urine and faeces were collected continuously and counted. Tissue lipids were extracted and analysed for intact radiolabelled olestra by size exclusion chromatography. Sucrose will be excreted in urine if olestra is absorbed and metabolized. Mean recovery of radiolabel was 96.6% of the administered dose. Of the recovered radiolabel, more than 99.4%, on average, was not absorbed and found in faeces, or cage and animal wash solutions. The absorbed radiolabel (0.6%), was distributed across the carcass, all tissues and blood, or excreted in urine. This radiolabel primarily came from the metabolism of glucose and fructose resulting from the hydrolysis of the trace levels of penta- and lower sucrose esters present in the test material. No radiolabel was found in the olestra-containing fraction of liver lipids, the primary measure of absorbed and non-metabolized olestra, at a detection limit of 0.0002% of dose. A conservative estimate of the amount of 14C-sucrose excreted in the urine was 0.0012%. The total absorption of intact olestra was thus less than 0.0014% of the dose, the sum of the two measures. These results indicate that intact olestra is essentially not absorbed by the weanling mini-pig, an animal with a young developing gastrointestinal tract similar to that of young children (2-5 yr).
Administration, Oral↗
Identification of a topology control domain in the tetracycline resistance protein.
Two N-terminal fusion proteins combining Escherichia coli maltose-binding protein (MBP) and the 12-transmembrane-segment pBR322 tetracycline resistance protein (Tet) have been constructed to determine the strength and location of topology control signals within the N-terminal portion of the Tet protein. The fusions contain either a secretable (wild-type) or a nonsecretable (MBP delta 2-26) MBP domain joined to the normally cytoplasmic N-terminus of the Tet protein. The effects of MBP targeting on Tet topology were investigated by analyzing the susceptibility of fusion strains to tetracycline and by proteolysis of the fusion proteins in inverted membrane vesicles and spheroplasts. The fusion protein containing MBP delta 2-26 conferred tetracycline resistance to the host strain and gave a normal pattern of Tet digestion fragments, indicating that its Tet domain is oriented and folded properly in the membrane. In contrast, the fusion containing secretable MBP was catalytically inactive apparently due to transfer of the Tet N-terminus to the periplasm with MBP. However, protease treatment of this fusion revealed that MBP secretion seems to affect only the topology of segments 1 and/or 2 of the Tet domain. Therefore, a strong topology control sequence appears to be located in the first cytoplasmic loop of the protein.
ATP-Binding Cassette Transporters↗
"Prohormone thiol protease" (PTP) processing of recombinant proenkephalin.
The "prohormone thiol protease" (PTP) from adrenal medullary chromaffin granules has been demonstrated as a novel cysteine protease that converts the model enkephalin precursor, ([35S]Met)-preproenkephalin, to appropriate enkephalin related peptide products [Krieger, T. J., & Hook, V. Y. H. (1991) J. Biol. Chem. 266, 8376-8383; Kreiger, T. J., Mende-Mueller, L., & Hook, V. Y. H. (1992) J. Neurochem. 59, 26-31; Azaryan, A. V., & Hook, V. Y. H. (1994) FEBS Lett. 341, 197-202]. In this report, PTP processing of authentic proenkephalin (PE) was examined with respect to production of appropriate intermediate products, and kinetics of PE processing were assessed. Recombinant PE was obtained by high level expression in Escherichia coli, with the pET3c expression vector; PE was then purified from E. coli by DEAE-Sepharose chromatography, preparative gel electrophoresis, and reverse-phase HPLC. Authentic purified PE was confirmed by amino acid composition analyses and peptide microsequencing. In time course studies, PTP converted PE (12 microM) to intermediates of 22.5, 21.7, 12.5, and 11.0 kDa that represented NH2-terminal fragments of PE, as assessed by peptide microsequencing. Differences in molecular masses of the 22.5, 21.7, 12.5, and 11.0 kDa products reflect PTP processing of PE within the COOH-terminal region of PE, which resembles PE processing in vivo [Liston, D. L., Patey, G., Rossier, J., Verbanck, P., & Vanderhaeghen, J. (1983) Science 225, 734-737; Udenfriend, S., & Kilpatrick, D. L. (1983) Arch. Biochem. Biophys. 221, 309-314]. Products of 12.5, 11.0, and 8.5 kDa were generated by PTP cleavage between Lys-Arg at the COOH-terminus of (Met)enkephalin-Arg6-Gly7-Leu8.(ABSTRACT TRUNCATED AT 250 WORDS)
Amino Acid Sequence↗
Disposition of ingested olestra in the Fischer 344 rat.
Four studies were conducted in the Fischer 344 rat to determine the disposition of orally gavaged olestra. Twenty-four rats were used for each study. Three olestra samples, differing in the degree of saturation of the fatty acid chains, were tested; one sample was heated to simulate olestra's intended use in preparing fried foods. In addition, a sucrose polyester (SPE) sample containing 28% short-chain penta- and lower polyesters was tested. All test samples were uniformly labeled with 14C in the sucrose moiety. Urine, feces, and CO2 were continuously collected and counted. Urine also was analyzed for [14C]sucrose by HPLC. If olestra were absorbed and systemically metabolized, [14C]sucrose would be excreted in the urine. Rats were killed 1, 3, 7, and 21 days after dosing, and tissues were collected and counted. Tissue lipids were extracted and analyzed for intact olestra or SPE by HPLC. Less than 0.15% of the dose of 14C was absorbed from the olestra samples, and about 1.3% from the SPE sample. The disposition of the absorbed radiolabel suggested that its source was glucose and fructose resulting from the intestinal hydrolysis of short-chain penta- and lower sucrose polyesters. For rats dosed with olestra, < 8 x 10(-4)% of the dose of radiolabel was recovered in the olestra-containing fraction of lipids extracted from liver, the target organ for absorbed olestra, and no [14C]sucrose was found in urine, indications that olestra essentially was not absorbed. Heating olestra did not change this result.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Electron spin resonance studies of acyl chain motion in reconstituted nicotinic acetylcholine receptor membranes.
The electron spin resonance spectra of spin-label positional isomers of stearic acid (n-SASL) incorporated into nicotinic acetylcholine receptors (nAcChoR) reconstituted into dioleoylphosphatidylcholine (DOPC) were deconvoluted into bilayer- and protein-associated components by subtraction under conditions of slow exchange. The selectivity of n-SASL (n = 6, 9, 12, and 14) for the lipid-protein interface of the nAcChoR was threefold greater than that of DOPC and independent of the spin label position. The temperature at which exchange became apparent as judged from lineshape broadening of the mobile lipid component spectrum was dependent upon the position of the spin-label moiety; near the bilayer center, exchange broadening occurred at lower temperatures than it did closer to the lipid headgroup. This suggests that the lipid headgroup region of boundary lipids is relatively fixed, whereas its acyl chain whips on and off the protein with increasing frequency near the bilayer center. Motions on the microsecond time scale were examined by microwave power saturation. Each n-SASL saturated more readily when incorporated into vesicles containing the nAcChoR than when in pure DOPC liposomes. Therefore, lipid mobility is perturbed by the nAcChoR on the microsecond time scale with an apparent magnitude that is relatively modest, probably due to exchange on this time scale.
Animals↗
General anesthetics modify the kinetics of nicotinic acetylcholine receptor desensitization at clinically relevant concentrations.
BACKGROUND: General anesthetics are thought to induce anesthesia through their actions on ligand-gated ion channels. One such channel, the nicotinic acetylcholine receptor (nAcChoR), can be found in different subtypes in the central nervous system and at the periphery in the neuromuscular junction. The latter subtype of the nAcChoR is a useful model for examining interactions between general anesthetics and ligand-gated ion channels, because it can be isolated and purified in sufficient quantities to allow for biophysical and biochemical studies. This study examines the actions of general anesthetics on agonist-induced conversion of the nAcChoR to inactive desensitized conformational states. METHODS: Nicotinic acetylcholine receptor membranes were purified from the electric organ of Torpedo nobiliana. Agonist-induced desensitization was characterized from the time-dependent increase in fluorescence intensity that results from the binding of the fluorescent acetylcholine analog, Dns-C6-Cho, to the nAcChoR. RESULTS: Mixing Dns-C6-Cho with nAcChoR-rich membranes results in an increase in fluorescence that is characterized by four rate processes. Concentrations of isoflurane and butanol, which range from subclinical to toxic increase the rates of the third and fourth components of fluorescence, corresponding to fast and slow desensitization, respectively. At concentrations that are twice their EC50s for anesthesia, isoflurane, butanol, chloroform, methanol, and cyclopentanemethanol increase the apparent rates of fast and slow desensitization by an average of 92 +/- 22% and 108 +/- 22%, respectively. CONCLUSIONS: The concentration range over which general anesthetics modify the kinetics of nAcChoR desensitization is similar to those reported for anesthetic actions on the GABAA receptor. Thus, the nAcChoR, like other members of this superfamily, is a sensitive target of general anesthetics.
1-Butanol↗
A discrete site for general anesthetics on a postsynaptic receptor.
General anesthetics depress central nervous system excitability via a mechanism that probably involves effects on synaptic ion channels, but the fundamental molecular nature of the site where they act is unknown. Although the importance of hydrophobicity for general anesthetic drug potency has long been established, it remains uncertain whether these "nonspecific" drugs act on membrane proteins directly or by modification of the physical properties of the lipid membrane or the lipid-protein interface. We find that specific mutations in the acetylcholine receptor pore-forming M2 domains enhance the sensitivity of the receptor to the general anesthetics isoflurane, hexanol, and octanol, suggesting that these agents act by binding directly to a discrete protein site at or near these residues. The sensitivity of the receptor to block by general anesthetics increases with increased hydrophobicity of these residues, demonstrating that hydrophobic forces dominate the interaction of drugs with their protein site. Furthermore, octanol inhibits both wild-type and mutant nicotinic acetylcholine receptors preferentially after channel opening, which is consistent with a mechanism where drugs bind within the receptor's pore. Similar sites on postsynaptic ion channels in brain may represent general anesthetic targets for modulating consciousness.
Amino Acid Sequence↗