Immunolocalization of the acid-sensing ion channel 2a in the rat cerebellum

The acid-sensing ion channels (ASICs) are members of the DEG/ENaC superfamily of Na+ channels. Acid-gated cation currents have been detected in neurons from multiple regions of the brain including the cerebellum, but little is known about their molecular identity and function. Recently, one of ASICs (ASIC1a) was implicated in synaptic plasticity. In this study we examined the subcellular distribution of ASIC2a in rat cerebellum by immunostaining and confocal microscopy. Monoclonal antibodies for labeling of defined brain structures, for example, astroglia, Purkinje cell dendrites, nuclei, and presynaptic terminals were used for colocalization analyses. In the gray matter, the anti-ASIC2a antibody intensively stained dendrite branches of Purkinje cells evenly distributed throughout the entire molecular layer (ML). In the granule cell layer (GL), anti-ASIC2a antibody stained synaptic glomeruli. Neuronal localization of ASIC2a was confirmed by lack of co-staining with glial fibrillary acidic protein. Anti-ASIC2a staining in the ML colocalized with metabotropic glutamate receptor 1alpha (mGluR1alpha) in Purkinje cell dendrites and dendritic spines. Both proteins, mGluR1alpha and ASIC2a, were enriched in a crude synaptic membrane fraction prepared from cerebellum, suggesting synaptic expression of these proteins. Dual staining with anti-syntaxin 1A and anti-ASIC2a antibodies demonstrates characteristic complementary distribution of two proteins in both ML and GL. Because syntaxin 1A localized in presynaptic membranes and synaptic vesicles, complementary distribution with ASIC2a suggests postsynaptic localization of ASIC2a in these structures. This study shows specific localization of ASIC2a in both Purkinje and granule cell dendrites of the cerebellum and enrichment of ASIC2a in a crude cerebellar synaptic membrane fraction. The study is the first report of synaptic localization of ASIC2a in the CNS. The synaptic localization of ASIC2a in the cerebellum makes this channel a candidate for a role in motor coordination and learning.