Differential association of GABA(B) receptors with their effector ion channels in Purkinje cells

Metabotropic GABA(B) receptors mediate slow inhibitory effects presynaptically and postsynaptically through the modulation of different effector signalling pathways. Here, we analysed the distribution of GABA(B) receptors using highly sensitive SDS-digested freeze-fracture replica labelling in mouse cerebellar Purkinje cells. Immunoreactivity for GABA(B1) was observed on presynaptic and, more abundantly, on postsynaptic compartments, showing both scattered and clustered distribution patterns. Quantitative analysis of immunoparticles revealed a somato-dendritic gradient, with the density of immunoparticles increasing 26-fold from somata to dendritic spines. To understand the spatial relationship of GABA(B) receptors with two key effector ion channels, the G protein-gated inwardly rectifying K(+) (GIRK/Kir3) channel and the voltage-dependent Ca(2+) channel, biochemical and immunohistochemical approaches were performed. Co-immunoprecipitation analysis demonstrated that GABA(B) receptors co-assembled with GIRK and Ca(V)2.1 channels in the cerebellum. Using double-labelling immunoelectron microscopic techniques, co-clustering between GABA(B1) and GIRK2 was detected in dendritic spines, whereas they were mainly segregated in the dendritic shafts. In contrast, co-clustering of GABA(B1) and Ca(V)2.1 was detected in dendritic shafts but not spines. Presynaptically, although no significant co-clustering of GABA(B1) and GIRK2 or Ca(V)2.1 channels was detected, inter-cluster distance for GABA(B1) and GIRK2 was significantly smaller in the active zone than in the dendritic shafts, and that for GABA(B1) and Ca(V)2.1 was significantly smaller in the active zone than in the dendritic shafts and spines. Thus, GABA(B) receptors are associated with GIRK and Ca(V)2.1 channels in different subcellular compartments. These data provide a better framework for understanding the different roles played by GABA(B) receptors and their effector ion channels in the cerebellar network.