Project description:GABA(C) receptors (GABA(C)Rs) are widely expressed in the mammalian subcortical visual system, particularly in the retina and superior colliculus (SC). GABA(C)Rs are composed of specific rho1-3 subunits the expression of which varies among visual structures. Thus rho1 subunits are most abundant in retina, and their loss eliminates GABA(C)R expression and function. In the SC, rho2 subunit expression may be equal to or stronger than rho1 subunit expression; however, results across studies vary considerably. To more directly assess the expression of GABA(C)R subunits, we characterized inhibition in the SC of wild-type (WT) and GABA(C) rho1 Null mice that lack expression of GABA(C) rho1 subunits. We used whole cell patch-clamp recordings and evaluated GABA(C)R-mediated modulation of electrically evoked post synaptic currents using either agonists or antagonists in WT mice. In GABA(C) rho1 Null stratum griseum superficiale (SGS) cells, inhibitory postsynaptic currents were shorter in duration and their excitatory postsynaptic currents (EPSCs) were longer, indicating that a slow GABA(C)R-mediated inhibitory component was reduced in each case. In contrast to retina, GABA(C)R-mediated currents in the SC were altered but not eliminated in GABA(C) rho1 Null mice. In the majority of SC cells in GABA(C) rho1 Null mice, GABA(C)R activation could still be induced to alter EPSC peak amplitudes in putative interneurons and in many projection neurons. These results, compared with previously published data, indicate a fundamental difference between retina and SC in the control of GABA(C)R expression and subunit composition.
Project description:Highly fluorescent CdSe quantum dots (qdots) can serve as a platform for tethering multiple copies of a receptor-targeted ligand, affording study of how the level of multivalency affects receptor binding. We previously showed that qdots conjugated with long PEG chains terminated by muscimol, a known GABA(C) agonist, exhibit specific binding to the surface membrane of GABA(C) receptor-expressing Xenopus oocytes. The present report addresses the effect of varying the number, i.e., valency, of muscimol- (M-) terminated PEG chains attached to the qdot on binding of the resulting conjugate to GABA(C) receptors. M-PEG-qdots of differing muscimol valency were prepared by conjugating AMP-CdSe/ZnS qdots with muscimol-terminated and methylamine-terminated PEG chains in proportions designed to yield varying percentages of muscimol-terminated chains among the total approximately 150-200 chains bound to the qdot. The investigated valencies represented 0%, approximately 25%, approximately 50%, and 100% loading with muscimol (preparations termed M-PEG-qdot0, M-PEG-qdot25, M-PEG-qdot50, and M-PEG-qdot100, respectively. Binding of a given conjugate to surface membranes of GABA(C) receptor-expressing oocytes was analyzed by quantitative fluorescence microscopy following defined incubation with approximately 30 nM of the conjugate. With 5-20 min incubation, the fluorescence signal resulting from incubation with M-PEG-qdot25 exceeded, by approximately 6-fold, the fluorescence level obtained with M-PEG-qdot preparations that lacked muscimol-terminated chains (M-PEG-qdot0). M-PEG-qdot50 yielded a net signal roughly similar to that of M-PEG-qdot25, and that produced by M-PEG-qdot100 exceeded, by approximately 30-50%, those for M-PEG-qdot25 and M-PEG-qdot50. The time course of changes in oocyte surface membrane fluorescence resulting from the introduction of and removal of M-PEG-qdots in the medium bathing the oocyte indicated only a modest dependence of both binding and wash-out kinetics on muscimol valency. The results demonstrate a dependence of the binding activity of the M-PEG-qdot conjugates on muscimol valency, presumably reflecting higher GABA(C) avidity and/or affinity of the muscimol at high valency, and provide insight on the interactions of membrane receptor proteins with qdot conjugates containing multiple copies of a receptor-targeting ligand.
Project description:To generate monoclonal antibodies to the human ρ1 GABA(C) receptor, a ligand-gated chloride ion channel that is activated by the neurotransmitter γ-aminobutyric acid (GABA), we recovered the immunoglobulin variable heavy chain (V(H)) and light chain (V(L)) regions of a guinea pig immunized with a 14-mer peptide segment of the N-terminal extracellular domain of the ρ1 subunit. Oligonucleotide primers were designed and used to amplify the V(H) and V(L) regions of guinea pig RNA by the reverse transcriptase polymerase chain reaction. The amplified and cloned V(H) and V(L) regions were transferred together into a phagemid vector, yielding a library of 5×10(6) members, which displayed chimeric fragments of antigen binding (Fabs) with guinea pig variable and human constant regions fused to protein III of M13 bacteriophage. Through affinity selection of this phage-display library with the biotinylated 14-mer peptide segment of GABA(C), we isolated four different antibody fragments that bound specifically to the immunogenic peptide. Phage particles displaying two of these antibodies, but not negative controls, bound selectively to the surface of neuroblastoma cells expressing the ρ1 GABA(C) receptor. Such antibody fragments will be useful in future studies involving targeting of specific neural tissues that express the GABA(C) receptor.