Differential cell surface expression of GABAA receptor alpha1, alpha6, beta2 and beta3 subunits in cultured mouse cerebellar granule cells influence of cAMP-activated signalling.

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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 GABAA receptors (GABARs). Initially the steady-state distribution of GABAR alpha1, alpha6, beta2 and beta3 subunits between the cell surface and cell interior was quantified. Cell surface proteins were modified with a membrane-impermeable cross-linking agent, bis(sulfosuccinimidyl)suberate (BS3) or the proteolytic enzyme, chymotrypsin. The proportion of unmodified (intracellular) and modified (cell surface) subunits was quantified by immunoblotting. We found that 51% of alpha6, 74% of alpha1, and 83% of beta2/3 were expressed at the cell surface, thus identifying a sizeable intracellular pool of alpha6 in contrast to the low levels of intracellular alpha1 and beta2/3. Chronic activation of protein kinase A (PKA) in CGCs in vitro, post-transcriptionally up-regulated expression of alpha1, beta2 and beta3 but not beta6. This was paralleled by an increase in the BZ-S subtype of [3H]Ro154513 binding sites. GABAR alpha1 was increased at the cell surface and in the cell interior, beta2 was increased almost exclusively at the cell surface whilst beta3 was increased almost exclusively in the cell interior. The intracellular pool of alpha6 was not affected. Thus, GABAR subunits are subject to differentially regulated trafficking, affording yet greater scope for GABAR diversity and plasticity.