Acetylcholine receptor synthesis in retina and transport to optic tectum in goldfish.

Previous studies have suggested that the retinotectal system of the goldfish contains a nicotinic acetylcholine receptor (nAChR) that is sensitive to alpha-bungarotoxin. Extracellularly recorded field potentials elicited in response to visual stimulation can be blocked by alpha-bungarotoxin, and alpha-bungarotoxin can interfere with the maintenance of retinotectal synaptic connections. Whether the transmission between the retinal ganglion cells and the tectal cells is mediated by acetylcholine and whether nAChR's exist on the dendrites of tectal cells are questions that remain. The experiments described in this report were designed to determine the site of synthesis of the nAChR's associated with the goldfish retinotectal projection. Radioactive (35S-labeled) methionine was injected into either the eye or the tectal ventricle, and the incorporation of radioactivity into the nAChR was measured by immunoprecipitation. The use of this technique provides evidence that an nAChR associated with the goldfish retinotectal projection is synthesized in the retina and transported to the optic tectum, which suggests a presynaptic site of acetylcholine action on retinal terminals.

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.

Unraveling the modular design of glutamate-gated ion channels.

Glutamate receptors that function as ligand-gated ion channels are essential components of cell-cell communication in the nervous system. Despite a wealth of information concerning these receptors, details of their structure are just beginning to emerge. We propose that glutamate receptors comprise four modules: two modules that are related to bacterial periplasmic-binding proteins, one module that is related to the pore-forming region of K+ channels, and one regulatory module of unknown origin. A K(+)-channel-like domain inserted into a crucial region of a periplasmic-binding protein-like domain suggests a mechanism for transduction of binding energy to channel opening. This modular design also suggests an evolutionary link between a ligand-gated ion-channel family and voltage-gated ion channels.

Solubilization and characterization of kainate receptors from goldfish brain.

The binding of [3H]kainate to goldfish brain membrane fragments was investigated. Scatchard analysis revealed a single class of binding sites in Tris-HCl buffer with a Kd of 352 nM and a Bmax of 3.1 pmol/mg wet weight. In Ringer's saline, [3H]kainate bound with a Bmax of 1.8 pmol/mg wet weight and a Kd of 214 nM. Binding in Ringer's saline, but not Tris-HCl buffer, displayed positive cooperativity with a Hill coefficient of 1.15. The [3H]kainate binding sites were solubilized in Ringer's saline using the nonionic detergent n-octyl-beta-D-glucopyranoside. Approximately 30-50% of the total number of membrane-bound binding sites were recovered on solubilization. The Kd of [3H]kainate for solubilized binding sites was approximately 200 nM. The rank order of potency for glutamatergic ligands at inhibiting [3H]kainate binding was identical and the competitive ligands had similar Ki values in both membranes and solubilized extracts. In membrane preparations, [3H]kainate displayed a two component off-rate with koff values of 0.97 min-1 and 0.07 min-1; in solubilized extracts, however, only a single off-rate (koff = 0.52 min-1) was observed. The hydrodynamic properties of n-octyl-beta-D-glucopyranoside solubilized [3H]kainate binding sites was investigated by sucrose density centrifugation. A single well defined peak was detected which yielded a sedimentation coefficient of 8.3 S. The results presented in this report suggest that goldfish brain may provide an ideal system in which to study kainate receptor biochemistry.

Rapid incorporation of the solubilized dihydropyridine receptor into phospholipid vesicles.

We describe the rapid incorporation of the CHAPS solubilized dihydropyridine receptor into phospholipid vesicles. A series of sucrose gradient sedimentation experiments demonstrate that the (+)-[3H]PN200-110-labeled dihydropyridine receptor is associated with lipid vesicles following detergent removal by Extracti-gel chromatography. Solubilization of the receptor results in a loss of (+)-[3H]PN200-110 binding affinity relative to that observed in native membranes; the high affinity binding of (+)-[3H]PN200-110 can be restored upon reincorporation of the receptor into phospholipid vesicles. Similarly, the incorporation of the receptor restores its stability to incubation at 37 degrees C relative to that of the detergent solubilized receptor, thereby mimicking the properties of the membrane bound form of the receptor. The dissociation rate of (+)-[3H]PN200-110 from the reconstituted receptor is shown to be allosterically regulated by verapamil and diltiazem, indicating that the binding sites for these calcium antagonists have been inserted along with the dihydropyridine receptor into phospholipid vesicles. The results presented in this report, thus demonstrate the successful reconstitution of the dihydropyridine receptor into phospholipid vesicles by a variety of criteria. The reconstitution method described here is rapid and efficient, and should now facilitate structure-function studies of this receptor and its interrelationships with other regulatory components of the voltage-sensitive calcium channel system.

Mechanisms of noncompetitive inhibition of acetylcholine-induced single-channel currents.

The functional mechanisms of noncompetitive blockade of the nicotinic acetylcholine receptor from the BC3H-1 cell line were examined using single-channel currents recorded from cell-attached patches. Channel open times were distributed as sums of two exponentials and the closed times as sums of at least four exponentials. The single-channel currents of the receptor were analyzed in terms of activation schemes in which the receptor exists in two open states and a number of closed or blocked states. The existence of two distinct open states for the acetylcholine receptor allows for predictions to be made that will distinguish between different mechanisms of blockade. Notably, predictions could be made based on the model for the sequential block of open channels, that would allow us to discriminate such a mechanism, even for ligands that appear to dissociate so slowly that sequential openings of the same channel do not appear as distinct bursts. Four noncompetitive blockers of the acetylcholine receptor were studied: tetracaine, phencyclidine, and the (+) and (-) isomers of N-allylnormetazocine (SKF-10047). All four of these ligands decreased the duration of single-channel currents without increasing the number of fast closures per burst. The data suggest that the ligands block the channel in at least two distinct ways, one of which involves a specific interaction with open channels and the other is most consistent with the blockade of channels that may be either open or closed. In addition, the duration of the open state may be allosterically lengthened by the interaction of certain blockers with another class of sites.

Asn-265 of frog kainate binding protein is a functional glycosylation site: implications for the transmembrane topology of glutamate receptors.

Kainate binding proteins (KBPs) from frog and goldfish brain are glycosylated, integral membrane proteins. These KBPs are homologous (35-40%) to the C-terminal half of AMPA and kainate receptors which have been shown to form glutamate-gated ion channels. We report here that the frog KBP has three functional N-glycosylation sites. Of particular interest, Asn-265, a residue located between two putative membrane spanning regions of the frog KBP, is a functional N-glycosylation site. A mutation of Ser-267 to Gly renders this site non-functional as shown using an in vitro translation system and by transient expression in human embryonic kidney (HEK 293) cells. The mutant receptor protein (S267G), when expressed in HEK cells, binds kainate with high affinity (Kd = 16 nM). These results further support a topology with three transmembrane segments for KBPs and, by sequence homology, for glutamate-gated ion channels.

Goldfish brain GluR2: multiple forms, RNA editing, and alternative splicing.

cDNA coding for a full-length goldfish alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR2, was cloned by screening unidirectional and bidirectional goldfish brain cDNA libraries. The clone has an open reading frame of 2679 bp, encoding a protein of 893 amino acids. Partial cDNA clones for three other GluR2 subunits were identified. GluR2 from goldfish brain exhibits RNA editing and alternative splicing. RNA editing occurred at the two sites demonstrated for mammalian GluR2 (Q/R and R/G). Unlike rat GluR2, GFGluR2a has a long (68 amino acids) C-terminal tail. Analysis of genomic DNA suggests that an alternatively spliced shorter C-terminal tail can be produced, similar to the rat protein. Thus, in goldfish brain, GluR2 exhibits diversity arising from multiple subtypes, RNA editing, and alternative splicing.

Linear prediction and single-channel recording.

The measurement of individual single-channel events arising from the gating of ion channels provides a detailed data set from which the kinetic mechanism of a channel can be deduced. In many cases, the pattern of dwells in the open and closed states is very complex, and the kinetic mechanism and parameters are not easily determined. Assuming a Markov model for channel kinetics, the probability density function for open and closed time dwells should consist of a sum of decaying exponentials. One method of approaching the kinetic analysis of such a system is to determine the number of exponentials and the corresponding parameters which comprise the open and closed dwell time distributions. These can then be compared to the relaxations predicted from the kinetic model to determine, where possible, the kinetic constants. We report here the use of a linear technique, linear prediction/singular value decomposition, to determine the number of exponentials and the exponential parameters. Using simulated distributions and comparing with standard maximum-likelihood analysis, the singular value decomposition techniques provide advantages in some situations and are a useful adjunct to other single-channel analysis techniques.

Interaction of monoclonal antibodies to electroplaque acetylcholine receptors with the alpha-bungarotoxin binding site of goldfish brain.

Polyclonal and monoclonal antibodies raised against acetylcholine receptors from Torpedo californica and Electrophorus electricus electroplaque were tested for interaction with the [125I]alpha-bungarotoxin binding protein of goldfish brain. A subset of monoclonal antibodies which recognize the main immunogenic region of the alpha subunit of the Electrophorus acetylcholine receptor interacted at high affinity with the [125I]alpha-bungarotoxin binding protein. Using immunofluorescence, these antibodies were shown to label the same layers of the optic tectum as [125I]alpha-bungarotoxin.

[3H]kainate localization in goldfish brain: receptor autoradiography and membrane binding.

The anatomical distribution of specific [3H]kainate binding in goldfish brain was investigated by membrane binding and autoradiographical techniques. Saturation binding of the radioligand was determined in 8 anatomically defined regions and demonstrated a single class of high affinity sites with Kd values ranging from 290 to 650 nM. Kainate receptor densities, however, varied significantly. The cerebellum contained the highest concentration of binding sites (964 pmol/mg prot.), while the optic tectum had the lowest (96 pmol/mg prot.). Binding site distributions determined by autoradiographic studies demonstrated the same regional variation and allowed more specific localization of the binding sites. Within the cerebellum, the molecular layers of the corpus, valvula and lobus caudalis displayed a uniform and highly intense image while the granule cell layers (except for the medial granule cell mass of the lobus caudalis) did not. Other areas of intense binding were the posterior tubercle of the diencephalon, inferior lobes of the hypothalamus and layers 1 and 2 of the optic tectum (deep to the periventricular granule cells).

Localization of alpha-bungarotoxin binding sites to the goldfish retinotectal projection.

The optic tectum of the goldfish Carassius auratus is a rich source of alpha-bungarotoxin (alpha-Btx) binding protein. In order to determine whether some fraction of these receptors is present at retinotectal synapses, we have compared the histological distribution of receptors revealed by the use of [125I]alpha-Btx radioautography to the distribution of optic nerve terminals revealed by the use of cobalt and horseradish peroxidase (HRP) techniques. The majority of alpha-Btx binding is concentrated in those tectal layers containing primary retinotectal synapses. The same layers contain high concentrations of acetylcholinesterase (AChE), revealed histochemically. Following enucleation of one eye, there is a loss of alpha-Btx binding in the contralateral tectum, observed both by radioautography and by a quantitative binding assay of alpha-Btx binding. Approximately 40% of the alpha-Btx binding sites are lost within two weeks following enucleation. By contrast, no significant change in AChE activity could be demonstrated up to 6 months following enucleation. These results are discussed in light of recent studies which show that the alpha-Btx binding protein and the nicotinic acetylcholine receptor are probably identical in goldfish tectum. We conclude that the 3 main classes of retinal ganglion cells projecting to the goldfish tectum are nicotinic cholinergic and that little or no postdenervation hypersensitivity due to receptor proliferation occurs in tectal neurons following denervation of the retinal input.

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.

Binding of phencyclidine to the detergent solubilized acetylcholine receptor from Torpedo marmorata.

The binding of phencyclidine to the acetylcholine receptor from Torpedo marmorata electroplaque was measured following solubilization of the receptor in sodium cholate followed by the exchange of cholate for Tween 80. In both the membrane-bound and solubilized AChR, the addition of cholinergic agonists simultaneously with the addition of PCP results in a 100 to 1000 fold increase in the PCP association rate and a 5 to 10 fold increase in the dissociation rate as compared to the unliganded AChR or AChR equilibrated with agonist prior to PCP addition. In addition, the number of binding sites and the pharmacological properties of the binding are not markedly changed in the soluble receptor. These results suggest that the acetylcholine receptor can undergo similar conformational transitions in the membrane-bound and the Tween 80 solubilized form and that phencyclidine can monitor these transitions in both cases.

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.