Identification and localization of an immunoreactive AMPA-type glutamate receptor subunit (GluR4) with respect to identified photoreceptor synapses in the outer plexiform layer of goldfish retina.

L-glutamate, the main excitatory synaptic transmitter in the retina, is released from photoreceptors and evokes responses in second-order retinal neurons (horizontal, bipolar cells) which utilize both ionotropic and metabotropic types of glutamate receptors. In the present study, to elucidate the functional roles of glutamate receptors in synaptic transmission, we have identified a specific ionotropic receptor subunit (GluR4) and determined its localization with respect to photoreceptor cells in the outer plexiform layer of the goldfish retina by light and pre-embedding electron-microscopical immunocytochemistry. We screened antisera to mammalian AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate)-preferring ionotropic glutamate receptors (GluR 1-4) of goldfish retina by light- and electron-microscopical immunocytochemistry. Only immunoreactive (IR) GluR4 was found in discrete clusters in the outer plexiform layer. The cones contacted in this manner were identified as long-wavelength ("red") and intermediate-wavelength ("green") cones, which were strongly immunoreactive to monoclonal antibody FRet 43 and antisera to goldfish red and green-cone opsins; and short-wavelength ("blue") cones, which were weakly immunoreactive to FRet 43 but strongly immunoreactive with antiserum to blue-cone opsin. Immunoblots of goldfish retinal homogenate with anti-GluR4 revealed a single protein at M(r) = 110 kDa. Preadsorption of GluR4 antiserum with either the immunizing rat peptide, or its goldfish homolog, reduced or abolished staining in retinal sections and blots. Therefore, we have detected and localized genuine goldfish GluR4 in the outer plexiform layer of the goldfish retina. We characterized contacts between photoreceptor cells and GluR4-IR second-order neurons in the electron microscope. IR-GluR4 was localized to invaginating central dendrites of triads in ribbon synapses of red cones, semi-invaginating dendrites in other cones and rods, and dendrites making wide-cleft basal junctions in rods and cones; the GluR4-IR structures are best identified as dendrites of OFF-bipolar cells. The results of our studies indicate that in goldfish retina GluR4-expressing neurons are postsynaptic to all types of photoreceptors and that transmission from photoreceptors to OFF-bipolars is mediated at least in part by AMPA-sensitive receptors containing GluR4 subunits.

Molecular cloning of a mineralocorticoid (type I) receptor complementary DNA from rat hippocampus.

Rat brain expresses two types of corticosteroid-binding proteins. The type I receptor binds corticosterone with high affinity and is structurally related to the kidney mineralocorticoid receptor (MR), while the type II or classical glucocorticoid receptor binds corticosterone with lower affinity and displays an in vivo preference for dexamethasone. Here we describe the isolation and characterization of a cDNA coding for the MR, from a rat hippocampus cDNA library, by low stringency hybridization to radiolabeled human glucocorticoid receptor cDNA. The nucleotide and deduced amino acid sequence for rat hippocampal MR displays extensive homology to a MR cDNA isolated from human kidney, suggesting that they are orthologous genes. Southern analysis suggests that there is only one gene for the MR, and in vitro expression of the receptor generates a high affinity corticosterone-binding protein. These data provide evidence to support the contention that a single gene gives rise to the MR in renal tissues and type I receptors in the brain.

In vitro methylation and demethylation of methyl-accepting chemotaxis proteins in Bacillus subtilis.

Bacillus subtilis responds to attractants by demethylating a group of integral membrane proteins referred to as methyl-accepting chemotaxis proteins (MCPs). We have studied the methylation and demethylation of these proteins in an in vitro system, consisting of membrane vesicles, and purified methyltransferase and methylesterase. The chemoattractant aspartate was found to inhibit methylation and stimulate demethylation of MCPs. Escherichia coli radiolabeled membranes in the presence of B. subtilis enzyme do not respond to aspartate by an increase demethylation rate. We also report that B. subtilis MCPs are multiply methylated, demethylation resulting in slower migrating proteins on sodium dodecyl sulfate-polyacrylamide gels.

Purification and characterization of chemotactic methylesterase from Bacillus subtilis.

By utilization of methanol evolution as an assay, a protein methylesterase from Bacillus subtilis has been purified. A 1200-fold purification has been achieved by CM-Bio-Gel A, hydroxylapatite, and Bio-Gel P-60 column chromatography. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate the enzyme is a monomer of 41 000 in molecular weight. The enzyme is stabilized and activated by aqueous glycerol solutions. Methyl-accepting chemotaxis proteins (MCPs) serve as substrates for the enzyme. The enzyme requires divalent cation for activity, with maximum activity obtained at 1.1 mM Mg2+. The enzyme is most active at pH 7.5 and at 28 degrees C. Methylesterase has an apparent Km for methylated MCPs of about 10 nM.

Chemotaxis in Bacillus subtilis: effects of attractants on the level of methylation of methyl-accepting chemotaxis proteins and the role of demethylation in the adaptation process.

By performing in vivo methylation experiments and using highly resolving NaDodSO4-polyacrylamide gels, we have examined the effects of amino acid attractants on the methylation profile of Bacillus subtilis MCPs. Both increases and decreases have been found to occur in the level of methylation of these proteins. By using competition experiments and Conway diffusion cells, we have found that the demethylation event is correlated with the adaptation process. Gas chromatographic analysis indicates that methanol is evolved upon demethylation of these proteins. As more attractant receptors are titrated, corresponding increases in methanol evolution result. During this period of increased rate of methanol production, bacteria swim smoothly.

Sensory adaptation and deadaptation by Bacillus subtilis.

Cells of Bacillus subtilis, when tethered by using antiflagellar antibody, rotate briefly counterclockwise (swimming behavior) or clockwise (tumbling behavior) when amino acids are added or removed, respectively. "Dissociation constants" for attractant-binding site interactions, calculated from duration of the rotational response to addition of amino acids, agreed with those calculated for their removal and with previous values calculated from sensitivity capillary assays. The ratio of adaptation times for addition versus removal of attractant averaged 1.7, which differs greatly from the value of 50 for Escherichia coli.

Chemotaxis away from uncouplers of oxidative phosphorylation in Bacillus subtilis.

In a capillary assay, uncouplers of oxidative phosphorylation and inhibitors of electron transport are repellents for Bacillus subtilis. They also cause transient tumbling in naturally smooth swimming strains. Tumbling strains can be made to swim smoothly by addition of attractant and then immediately returned to tumbling by subsequent addition of repellent. Arsenate does not cause transient tumbling, suggesting that decrease in concentration of adenosine triphosphate does not cause tumbling and that adenosine triphosphate concentration does not govern tumbling frequency. Instead, the evidence suggests that diminution of the energized state of the membrane, or membrane potential, causes tumbling although the level of the energized state itself does not govern tumbling frequency.