Double-immunolabelling analysis of GABAA receptor subunits in label-fracture replicas of cultured rat cerebellar granule cells

Abstract

We used double-immunolabelling with various size gold particles in label-fracture replicas to gain information on the subunit composition of GABAA receptors in clusters located in the plasma membrane of cultured cerebellar granule cells. We hypothesize that if two subunits colocalize in the same GABAA receptor, the density of the label of any given subunit decreases when another subunit of the same receptor complex is prelabelled with an antibody linked to a gold particle of larger size. Following this hypothesis, the interpretation of our results allows to suggest that a consistent receptor colocalization of beta 2/3 with alpha 1 or alpha 6 subunits may be operative in cerebellar granule cells. The decrease in the density of the second labelling in double-labelling experiments using the alpha 1 and gamma 2 antibodies suggests that these subunits colocalize in the same receptor complex, this may be also true for the alpha 6 and gamma 2 subunits. When one prelabels the delta subunit the intensity of labelling of the alpha 1 subunit fails to change but that of the alpha 6 subunit decreases significantly, suggesting a consistent colocalization of alpha 6 and delta but not of alpha 1 and delta. Finally, when one prelabels the alpha 1 subunit the intensity of labelling for the alpha 6 subunit fails to change, suggesting that these subunits do not colocalize in the same receptor complex. We conclude that in the GABAA receptors expressed in granule cells the alpha 1 subunit is preferentially colocalized with the beta 2-3 and gamma 2 subunits, while the alpha 6 subunit is preferentially colocalized with the beta 2-3 and either the delta or the gamma 2 subunit. This conclusion is supported by parallel experiments in which the sequence of labelling is inverted.