Unconventional oil and gas extraction and animal health.

The extraction of hydrocarbons from shale formations using horizontal drilling with high volume hydraulic fracturing (unconventional shale gas and tight oil extraction), while derived from methods that have been used for decades, is a relatively new innovation that was introduced first in the United States and has more recently spread worldwide. Although this has led to the availability of new sources of fossil fuels for domestic consumption and export, important issues have been raised concerning the safety of the process relative to public health, animal health, and our food supply. Because of the multiple toxicants used and generated, and because of the complexity of the drilling, hydraulic fracturing, and completion processes including associated infrastructure such as pipelines, compressor stations and processing plants, impacts on the health of humans and animals are difficult to assess definitively. We discuss here findings concerning the safety of unconventional oil and gas extraction from the perspectives of public health, veterinary medicine, and food safety.

Identification of C-terminal domain residues involved in protein kinase A-mediated potentiation of kainate receptor subtype 6.

Glutamate receptors are the major excitatory receptors in the vertebrate CNS and have been implicated in a number of physiological and pathological processes. Previous work has shown that glutamate receptor function may be modulated by protein kinase A (PKA)-mediated phosphorylation, although the molecular mechanism of this potentiation has remained unclear. We have investigated the phosphorylation of specific amino acid residues in the C-terminal cytoplasmic domain of the rat kainate receptor subtype 6 (GluR6) as a possible mechanism for regulation of receptor function. The C-terminal tail of rat GluR6 can be phosphorylated by PKA on serine residues as demonstrated using [gamma-32P]ATP kinase assays. Whole cell recordings of transiently transfected human embryonic kidney (HEK) 293 cells showed that phosphorylation by PKA potentiates whole cell currents in wildtype GluR6 and that removal of the cytoplasmic C-terminal domain abolishes this potentiation. This suggested that the C-terminal domain may contain residue(s) involved in the PKA-mediated potentiation. Single mutations of each serine residue in the C-terminal domain (S815A, S825A, S828A, and S837A) and a truncation after position 855, which removes all threonines (T856, T864, and T875) from the domain, do not abolish PKA potentiation. However, the S825A/S837A mutation, but no other double mutation, abolishes potentiation. These results demonstrate that phosphorylation of the C-terminal tail of GluR6 by PKA leads to potentiation of whole cell response, and the combination of S825 and S837 in the C-terminal domain is a vital component of the mechanism of GluR6 potentiation by PKA.

Specific activation of the alpha 7 nicotinic acetylcholine receptor by a quaternary analog of cocaine.

Effects of cocaine and cocaine methiodide were evaluated on the homomeric alpha 7 neuronal nicotinic receptor (nAChR). Whereas cocaine itself is a general nAChR noncompetitive antagonist, we report here the characterization of cocaine methiodide, a novel selective agonist for the alpha 7 subtype of nAChR. Data from (125)I-alpha-bungarotoxin binding assays indicate that cocaine methiodide binds to alpha 7 nAChR with a K(i) value of approximately 200 nM while electrophysiology studies indicate that the addition of a methyl group at the amine moiety of cocaine changes the drug's activity profile from inhibitor to agonist. Cocaine methiodide activates alpha 7 nAChR with an EC(50) value of approximately 50 microM and shows comparable efficacy to ACh in oocyte experiments. While agonist effects are specific for the alpha 7 neuronal nAChR and are not observed with heteromeric neuronal or skeletal muscle nAChR, antagonist effects are present for heteromeric nAChR combinations. Studies of PC12 cells transiently transfected with human alpha 7 cDNA and expressing a variety of functional nicotinic receptor subtypes confirm the specificity of cocaine methiodide agonist effects. Our results indicate that a quaternary structural derivative of cocaine can be used as a specific agonist for the alpha 7 subtype of neuronal nicotinic receptor.

Cysteine mutagenesis and homology modeling of the ligand-binding site of a kainate-binding protein.

Glutamate receptors comprise the most abundant group of neurotransmitter receptors in the vertebrate central nervous system. Cysteine mutagenesis in combination with homology modeling has been used to study the determinants of kainate binding in a glutamate receptor subtype, a low molecular weight goldfish kainate-binding protein, GFKARbeta. A construct of GFKARbeta with no cysteines in the extracellular domain was produced, and single cysteine residues were introduced at selected positions. N-Ethylmaleimide or derivatized methanethiosulfonate reagents (neutral or charged) were used to modify the introduced cysteines covalently, and the effect on [(3)H]kainate binding was determined. In addition, cysteine mutants of GFKARbeta transiently expressed in HEK293 cells were labeled with a membrane-impermeable biotinylating reagent followed by precipitation with streptavidin beads and specific detection of GFKARbeta by Western blot analysis. The results are consistent with the proposal that the energy driving kainate binding is contributed both from residues within the binding site and from interactions between two regions (i.e. two lobes) of the protein that are brought into contact upon ligand binding in a manner analogous to that seen in bacterial amino acid-binding proteins.

Identification of the binding surface on Cdc42Hs for p21-activated kinase.

The Ras superfamily of GTP-binding proteins is involved in a number of cellular signaling events including, but not limited to, tumorigenesis, intracellular trafficking, and cytoskeletal organization. The Rho subfamily, of which Cdc42Hs is a member, is involved in cell morphogenesis through a GTPase cascade which regulates cytoskeletal changes. Cdc42Hs has been shown to stimulate DNA synthesis as well as to initiate a protein kinase cascade that begins with the activation of the p21-activated serine/threonine kinases (PAKs). We have determined previously the solution structure of Cdc42Hs [Feltham et al. (1997) Biochemistry 36, 8755-8766] using NMR spectroscopy. A minimal-binding domain of 46 amino acids of PAK was identified (PBD46), which binds Cdc42Hs with a KD of approximately 20 nM and inhibits GTP hydrolysis. The binding interface was mapped by producing a fully deuterated sample of 15N-Cdc42Hs bound to PBD46. A 1H,15N-NOESY-HSQC spectrum demonstrated that the binding surface on Cdc42Hs consists of the second beta-strand (beta2) and a portion of the loop between the first alpha-helix (alpha1) and beta2 (switch I). A complex of PBD46 bound to 15N-Cdc42Hs.GMPPCP exhibited extensive chemical shift changes in the 1H,15N-HSQC spectrum. Thus, PBD46 likely produces structural changes in Cdc42Hs which are not limited to the binding interface, consistent with its effects on GTP hydrolysis. These results suggest that the kinase-binding domain on Cdc42Hs is similar to, but more extensive than, the c-Raf-binding domain on the Ras antagonist, Rap1 [Nassar et al. (1995) Nature 375, 554-560)].

Ligand-binding characteristics and related structural features of the expressed goldfish kainate receptors: identification of a conserved disulfide bond and three residues important for ligand binding.

Low-molecular-weight kainate receptors from nonmammalian vertebrate brain belong structurally to the ionotropic glutamate receptor superfamily. In this study, two previously cloned goldfish kainate receptor subunits (GFKAR alpha and GFKAR beta) were transiently expressed in human embryonic kidney 293 cells, and their ligand-binding properties and some associated structural features were characterized, resulting in the following findings. 1) Both subunits form homomeric receptors with high affinity for [3H]kainate (KD = 16 and 31 nM, respectively) and L-glutamate (KD = 2 and 40 microM, respectively). 2) A deletion mutant lacking the originally proposed second-transmembrane domain was efficiently expressed and retains the overall ligand-binding properties of wild-type GFKAR alpha, strongly indicating that this region is not a transmembrane domain. 3) Mutations of Q12, A53, and Y54 of GFKAR beta indicate that these three residues are important for ligand binding (particularly L-glutamate), which is consistent with the sequence homology to bacterial periplasmic binding proteins. 4) Mutation of the three extracellular cysteine residues of GFKAR beta indicated that the two conserved cysteine residues (C305 and C385), located between two transmembrane segments, form a solvent-accessible disulfide bond. Analysis of [3H]kainate binding to wild-type and cysteine mutations of GFKAR beta indicate that in the absence of the disulfide bond, the affinity for kainate is increased 3-fold. These data lend further evidence in support of a model of glutamate receptor topology with three transmembrane segments and reveal several general structural features of the extracellular ligand-binding domain of the kainate receptors. These results are consistent with the notion that the ligand-binding domain has close structural similarities to bacterial periplasmic binding proteins.

A topological analysis of goldfish kainate receptors predicts three transmembrane segments.

Glutamate receptors are the most abundant excitatory neurotransmitter receptors in vertebrate brain. We have previously cloned cDNAs encoding two homologous kainate receptors (GFKAR alpha, 45 kDa, and GFKAR beta, 41 kDa) from goldfish brain and proposed a topology with three transmembrane domains (Wo, Z. G., and Oswald, R. E. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 7154-7158). These studies have been extended using an in vitro translation/translocation system in conjunction with site-specific antibodies and point and deletion mutations. We report here that the entire region between the previously proposed third and fourth transmembrane segments is translocated and likely to be extracellular in mature receptors. This was based on the following results. 1) The entire segment was protected from Proteinase K and trypsin digestion and could be immunoprecipitated by a site-specific antibody. 2) Functional sites for N-glycosylation are present in the C-terminal half of the segment, and 3) a mutation, constructed with an additional consensus site for N-glycosylation in the N-terminal half of the segment, was found to be glycosylated at that site. Given the fact that the N terminus of the protein is likely to be extracellular, this would place an even number of transmembrane segments between the extracellular N terminus and the glycosylated segment. In addition, results of N-glycosylation and proteolysis protection assays of GFKAR alpha mutations indicated that the previously proposed second transmembrane segment is not a true transmembrane domain. These results provide further evidence in support of a topology with three transmembrane domains that has important implications for the relationship of structure to function in ionotropic glutamate receptors.

Interaction of the frog brain kainate receptor expressed in Chinese hamster ovary cells with a GTP-binding protein.

A protein that binds kainate with high affinity has been purified and cloned from frog brain (Rana pipiens) and has approximately 35% sequence homology with mammalian non-N-methyl-D-aspartate glutamate receptors, some of which have been shown to be ligand-gated ion channels. Frog brain membranes and membranes from Chinese hamster ovary (CHO) cells transfected with the cDNA coding for the frog kainate-binding protein (CHO-4 cells) bound kainate with essentially identical affinity (KD values of 1.9 and 2.1 nM, respectively). In both tissues, the affinity for kainate decreased 9-fold in the presence of 100 microM GTP gamma S (guanosine 5'-O-(3-thio)triphosphate). No specific kainate binding to nontransfected CHO cell membranes was observed. GTP gamma S and GDP were effective inhibitors of kainate binding, while cGMP and adenosine 5'-O-(3-thio)triphosphate had no effect in either frog brain membranes or CHO-4 membranes. Pretreatment of CHO-4 cell membranes with pertussis toxin led to a 34% decrease in kainate binding. Kainate increased the binding of [3H]5'-guanylyl imidodiphosphate by 61%, and the rate of GTP hydrolysis by up to 5-fold. These results indicate that the kainate receptor cloned from frog brain can interact functionally with a G protein present in CHO-4 cell membranes.

Biochemical characterization of kainate receptors from goldfish brain.

Kainate receptors from goldfish brain were purified by affinity chromatography. Unlike previously published purifications, which have yielded single proteins of 48-50 kDa from frog, chick, and pigeon brain, our preparations contained two polypeptides, of 41 kDa and 45 kDa. In addition, a broad band centered at 120 kDa was present. Some of the 41-kDa and 45-kDa polypeptides were derived from the higher molecular mass protein. All of these proteins were recognized by a monoclonal antibody produced against a purified frog kainate receptor. The distribution of the 41-kDa and 45-kDa proteins varied independently in different major brain regions, suggesting that they can exist as separate independent proteins. A partial amino acid sequence of the 41-kDa polypeptide is very similar (40-60% identity) to specific segments of the frog and chick kainate-binding proteins and the alpha-amino-3-hydroxy-5-methylisoxazolepropionate/kainate ion channels. The characteristics of the 41-kDa and 45-kDa polypeptides suggest that these two proteins are distinct. Photo-affinity labeling with [3H]kainate showed that a [3H]kainate binding site is associated with the 41-kDa polypeptide, and peptide mapping suggests that the two proteins are not identical. In addition, the two peptides do not appear to be related by differential glycosylation or phosphorylation. The 41-kDa and 45-kDa polypeptides, therefore, appear to be distinct and may represent kainate receptor subtypes or, in some cases, possibly two different subunits of a kainate receptor complex.

The interaction of a kainate receptor from goldfish brain with a pertussis toxin-sensitive GTP-binding protein.

Kainate receptors are present in high concentrations in goldfish brain (Henley and Oswald, 1988a and b; Ziegra et al., 1990), possibly in neuronal and glial cells. In a number of systems, the kainate receptor has been assumed to be an integral ion channel (Watkins and Evans, 1981); but, for some kainate receptors, ion channel activity has not been demonstrated (Wada et al., 1989). This study presents evidence that a portion of the [3H]kainate-binding sites in goldfish brain is sensitive to guanine nucleotides, with a loss of high affinity binding in the presence of nonhydrolyzable GTP analogs. Pertussis toxin pretreatment of membranes causes a loss of high affinity [3H]kainate binding and of the guanine nucleotide-sensitive binding. Pertussis toxin catalyzes the specific [32P]ADP-ribosylation of a 40-kDa substrate in a kainate-sensitive manner. In addition, incorporation of [alpha-32P]GTP-gamma-azidoanilide by photoaffinity labeling was enhanced in the presence of kainate. These results indicate that a subpopulation of [3H]kainate-binding sites in goldfish brain may be coupled to G proteins.

Diffusion model in ion channel gating. Extension to agonist-activated ion channels.

Previously, we described a model which treats ion channel gating as a discrete diffusion problem. In the case of agonist-activated channels at high agonist concentration, the model predicts that the closed lifetime probability density function from single channel recording approximates a power law with an exponent of -3/2 (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988a. Proc. Natl. Acad. Sci. USA. 85: 1503-1507). This prediction is consistent with distributions derived from a number of ligand-gated channels at high agonist concentration (Millhauser, G. L., E. E. Salpeter, and R. E. Oswald. 1988b. Biophys. J. 54: 1165-1168.) but does not describe the behavior of ion channels at low activator concentrations. We examine here an extension of this model to include an agonist binding step. This extended model is consistent with the closed time distributions generated from the BC3H-1 nicotinic acetylcholine receptor for agonist concentrations varying over three orders of magnitude.

Characterization of nicotinic acetylcholine receptor channels of the TE671 human medulloblastoma clonal line.

Acetylcholine (ACh)-gated single channel events were studied on the TE671 human medulloblastoma clonal cell line by the use of the cell-attached patch clamp technique. Channel activity was detected (86% probability) in the presence of 0.1-2 microM ACh but not (0% probability) in the absence of agonist or in the presence of 1 microM alpha-bungarotoxin (Bgt). This effect of Bgt was reversible within 1 h. The most prominent channel type had a conductance of 50 pS. The kinetics of opening and closing of the channel were similar to that for skeletal muscle nicotinic acetylcholine receptors.

Interaction of monoclonal antibodies with alpha-bungarotoxin and (-)-nicotine binding sites in goldfish brain. Identification of putative nicotinic acetylcholine receptor subtypes.

Monoclonal antibodies raised against the nicotinic acetylcholine receptor of Electrophorus electricus electroplaque have been used as probes to characterize putative nicotinic acetylcholine receptors in goldfish brain. One monoclonal antibody (mAb), mAb 47, recognized a protein which binds both (-)-[3H]nicotine and 125I-alpha-bungarotoxin with high affinity. Another monoclonal antibody (mAb 172) recognized a protein which binds (-)-[3H]nicotine but not 125I-alpha-bungarotoxin. Both antibodies precipitated a protein(s) (biosynthetically labeled with [35S]methionine) in the absence, but not in the presence, of excess purified nicotinic acetylcholine receptor from Torpedo nobiliana. The dilution of mAb 47 that precipitated half of the maximum amount of 125I-alpha-bungarotoxin binding protein was the same as that which precipitated half of the maximum amount of (-)-[3H]nicotine binding activity. When used in combination, the two antibodies precipitated more (-)-[3H]nicotine radioactivity than either antibody alone. The (-)-[3H]nicotine and 125I-alpha-bungarotoxin binding component-mAb complexes were characterized by sucrose density centrifugation. In the presence of either mAb 172 or 47, the (-)-[3H] nicotine binding component migrated further into the gradient, but only mAb 47 shifted the 125I-alpha-bungarotoxin peak. Incubation of solubilized brain extract with alpha-bungarotoxin-coupled Sepharose reduced the amount of (-)-[3H]nicotine radioactivity precipitated by mAb 47 but not by mAb 172. These data suggest that the antibodies may recognize distinct subtypes of (-)-nicotine binding sites in goldfish brain, one subtype which binds both 125I-alpha-bungarotoxin and (-)-[3H]nicotine and a second subtype which binds only (-)-[3H] nicotine.

Functional reconstitution of skeletal muscle Ca2+ channels: separation of regulatory and channel components.

Regulatory properties of a partially purified Ca2+ -channel preparation from isolated rabbit skeletal muscle triads were examined in proteoliposomes. These properties included (i) inhibition by phenylalkylamine antagonists, such as verapamil, (ii) inhibition by the GTP-binding protein Go in the presence of guanosine 5'-[gamma-thio]triphosphate, and (iii) regulation of phenylalkylamine inhibition as a result of phosphorylation by a polypeptide-dependent protein kinase (PK-P). By selective reconstitution of protein fractions obtained by wheat germ lectin and ion-exchange chromatography, a separation of Ca2+-channel activity (fraction C) from regulatory component(s) (fraction R) responsible for verapamil sensitivity was achieved. Reconstitution of fraction C alone yielded vesicles that exhibited channel-mediated 45Ca2+ uptake that could be directly inhibited by coreconstitution of Go in the presence of guanosine 5'-[gamma-thio]triphosphate. However, the 45Ca2+ uptake obtained with fraction C was not inhibited by verapamil. Coreconstitution of fractions C and R yielded vesicles in which the sensitivity of 45Ca2+ uptake to verapamil was restored. The verapamil sensitivity of this preparation could be inhibited by PK-P. Fraction C, obtained by wheat germ agglutinin-Sepharose chromatography followed by DEAE-Sephacel chromatography, included a 180-kDa protein that was phosphorylated by cAMP-dependent protein kinase (PK-A) but not by PK-P and a 145-kDa protein (180 kDa under nonreducing conditions) that was not phosphorylated by either kinase. Fraction R contained proteins that did not adsorb to wheat germ lectin and included 165-kDa and 55-kDa proteins that were phosphorylated by PK-P but not by PK-A. These results suggest a complex model for Ca2+-channel regulation in skeletal muscle involving a number of distinct, separable protein components.

Two distinct (-)nicotine binding sites in goldfish brain. Identification and characterization of putative neuronal nicotinic acetylcholine receptor subtypes.

The binding of (-)-[3H]nicotine to membrane fragments and a detergent solubilized fraction of goldfish brain was characterized. (-)-[3H]nicotine binding was not displaced by alpha-bungarotoxin, but was displaced by (-)nicotine and carbamoylcholine with Ki of approximately 8.6 and 86 nM, respectively. Preincubation of solubilized membrane extract with alpha-bungarotoxin-coupled Sepharose resulted in the loss of approximately 50% of the (-)-[3H]nicotine binding protein from the eluent and an increase in (-)-[3H]nicotine binding to the gel compared to control, non-alpha-bungarotoxin Sepharose. 125I-alpha-bungarotoxin binding protein in the eluent from the same preincubation experiments was totally removed. In addition, incubation of the solubilized tissue extracts with alpha-bungarotoxin-coupled Sepharose resulted in an increase in the affinity for (-)-[3H]nicotine in the eluent (mean KD = 3.1) compared to control solubilized tissue extracts (KD = 6.4 nM). Specific (-)-[3H]nicotine binding sites could be eluted from the alpha-bungarotoxin-coupled Sepharose with carbamoylcholine and D-tubocurarine. Similar to previously reported 125I-alpha-bungarotoxin binding data, eye removal resulted in an approximately 40% decrease in (-)-[3H]nicotine binding in the contralateral tectum compared to that in the ipsilateral tectum. These data indicate that at least two distinct subtypes of (-)nicotine binding sites may be present in goldfish brain, one which binds alpha-bungarotoxin and (-)nicotine and another which binds only (-)nicotine.