guanosine-triphosphate and 2-amino-4-phosphonobutyric-acid

The cDNA encoding hmGluR6, appended with a 15-amino acid antibody epitope (1D4), was transiently transfected in COS-7 cells. The receptor was purified from COS cell membranes using an antibody affinity column. The purified receptor was then reconstituted into lipid vesicles, and its ability to activate either transducin, the rod photoreceptor-specific GTP-binding protein, or the alpha subunit of Go was assayed in vitro using a guanosine 5'-3-O-(thio)triphosphate binding assay. Activation of both transducin and Go was observed. The rate of Go activation was 18-fold greater than the rate of transducin activation. This indicates that the coupling of mGluR6 to Go is more efficient and suggests that Go may be involved in coupling to mGluR6 in ON-bipolar cells.

Topics: Amino Acid Sequence; Aminobutyrates; Animals; Anticonvulsants; Cattle; COS Cells; Excitatory Amino Acid Agonists; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Humans; Molecular Sequence Data; Rats; Receptors, Metabotropic Glutamate; Transducin; Transfection

Depolarizing bipolar cells (DBCs) of the retina are the only neurons in the vertebrate central nervous system known to be hyperpolarized by the neurotransmitter glutamate. Both glutamate and its analogue L-2-amino-4-phosphonobutyrate (APB) hyperpolarize DBCs by decreasing membrane conductance. Furthermore, glutamate responses in DBCs slowly decrease during whole-cell recording, suggesting that the response involves a second messenger system. Here we report that intracellular cyclic GMP or GTP activates a membrane conductance that is suppressed by APB, resulting in an enhanced APB response. In the presence of GTP-gamma-S, APB causes an irreversible suppression of the conductance. Inhibitors of G-protein activation or phosphodiesterase activity decrease the APB response. Thus, the DBC glutamate receptor seems to close ion channels by increasing the rate of cGMP hydrolysis by a G protein-mediated process that is strikingly similar to light transduction in photoreceptors.

Topics: 1-Methyl-3-isobutylxanthine; Aminobutyrates; Animals; Cell Membrane; Cyclic GMP; Electric Conductivity; Glutamates; Glutamic Acid; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; Photoreceptor Cells; Receptors, Glutamate; Receptors, Neurotransmitter; Retina; Second Messenger Systems; Thionucleotides; Urodela

The effects of purine nucleotides on the binding of DL-[3H] 2-amino-4-phosphonobutyrate (DL-[3H] APB) to rat brain membranes were investigated. Certain guanine nucleotides, especially cyclic GMP and GTP, were found to be potent inhibitors of binding. Kinetic studies revealed that both cyclic GMP and GTP acted to decrease receptor affinity without affecting significantly binding site density. These endogenous substances may therefore play an important role in the regulation of excitatory amino acid receptor function.

Topics: Aminobutyrates; Animals; Brain; Cyclic GMP; Female; Guanosine Triphosphate; In Vitro Techniques; Kinetics; Male; Purine Nucleotides; Rats; Rats, Inbred Strains; Receptors, Glutamate; Receptors, Neurotransmitter; Synaptic Membranes; Tritium