Rayner, Sharon Louise; (1999) Development and characterization of gold-labelled anti-GABAA receptor subunit-specific antibodies for direct subcellular localization studies. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

GABAA receptors are fast-acting, ligand-gated C1- ion channels. There are currently 17 GABAA receptor genes, which are classified according to the amino acid sequence similarities of their products, i.e. the α1-α6, β1-β3, γ1-γ3, δ, ε, π and p1-p2 GABAA receptor subunits. These subunits are thought to coassemble in different pentameric combinations to form functional receptors with distinct properties. Insights into the functional properties of receptor subtypes may be obtained by studying their respective subcellular localizations using subunit-specific antibodies. However, quantification is difficult at synapses because of both antibody accessibility through the post-synaptic density and the large size of the complex formed for detection by conventional secondary antibody methodology. In this thesis, these problems have been addressed by the development of techniques permitting the direct labelling of whole antibody molecules together with their Fab' fragments, using monomaleimido Nanogold (1.4 nm). Thus, anti-mGluR1α, anti- GABAA receptor α1 1-14 Cys and anti- GABAA receptor α6 1-15 Cys antibodies were affinity-purified, and the whole IgG and Fab' fragments were labelled with monomaleimido Nanogold, following reduction with mercaptoethylamine. Labelled antibodies were characterized with respect to the efficiency of monomaleimido Nanogold labelling, antibody specificity and antibody avidity. Immunocytochemical studies were carried out using the labelled anti-IgG and anti-Fab' antibodies. In the second part of this thesis, GABAA receptor subunit-specific antibodies were used to study the regulation of subunit expression in cerebellar granule cells in culture, following elevation of intracellular cAMP. It was found that the previously described increase and decrease in expression of GABAA receptor α1 and α6 subunits, respectively, was accompanied by a concomitant increase in β2 and decrease in β3 subunits.

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