In this study we have used mature, primary cultured mouse cerebellar granule cells (CGCs) to initiate our studies on the mechanisms governing neuronal trafficking of GABA A receptors (GABARs). Initially the steady-state distribution of GAB-AR a1, a6, b2 and b3 subunits between the cell surface and cell interior was quanti®ed. Cell surface proteins were modi®ed with a membrane-impermeable cross-linking agent, bis(sulfosuccinimidyl)suberate (BS 3 ) or the proteolytic enzyme, chymotrypsin. The proportion of unmodi®ed (intracellular) and modi®ed (cell surface) subunits was quanti®ed by immunoblotting. We found that 51% of a6, 74% of a1, and 83% of b2/3 were expressed at the cell surface, thus identifying a sizeable intracellular pool of a6 in contrast to the low levels of intracellular a1 and b2/3. Chronic activation of protein kinase A (PKA) in CGCs in vitro, post-transcriptionally up-regulated expression of a1, b2 and b3 but not a6. This was paralleled by an increase in the BZ-S subtype of4513 binding sites. GABAR a1 was increased at the cell surface and in the cell interior, b2 was increased almost exclusively at the cell surface whilst b3 was increased almost exclusively in the cell interior. The intracellular pool of a6 was not affected. Thus, GABAR subunits are subject to differentially regulated traf®cking, affording yet greater scope for GABAR diversity and plasticity.
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