Abstract

The small-conductance, Ca2+-activated K+ (SK) channel subtype SK2 regulates the spike rate and firing frequency, as well as Ca2+ transients in Purkinje cells (PCs). To understand the molecular basis by which SK2 channels mediate these functions, we analyzed the exact location and densities of SK2 channels along the neuronal surface of the mouse cerebellar PCs using SDS-digested freeze-fracture replica labeling (SDS-FRL) of high sensitivity combined with quantitative analyses. Immunogold particles for SK2 were observed on post- and pre-synaptic compartments showing both scattered and clustered distribution patterns. We found an axo-somato-dendritic gradient of the SK2 particle density increasing 12-fold from soma to dendritic spines. Using two different immunogold approaches, we also found that SK2 immunoparticles were frequently adjacent to, but never overlap with, the postsynaptic density of excitatory synapses in PC spines. Co-immunoprecipitation analysis demonstrated that SK2 channels form macromolecular complexes with two types of proteins that mobilize Ca2+: CaV2.1 channels and mGlu1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to CaV2.1 channels, though they are not significantly co-clustered. These data demonstrate that SK2 channels located in different neuronal compartments can associate with distinct proteins mobilizing Ca2+, and suggest that the ultrastructural association of SK2 with CaV2.1 and mGlu1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs.

Highlights

  • Purkinje cells (PCs) are fundamental to the function in the cerebellum, as out of the different neuron populations they are the only ones projecting out of the cerebellar cortex (Altman and Bayer, 1997)

  • We show that SK2 is restricted to somato-dendritic domains of PCs, with the highest density in PC spines

  • We provide the first direct evidence that SK2 is absent from the postsynaptic density of excitatory parallel fiber (PF)-PC synapses, and always distributed in the extrasynaptic plasma membrane

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Summary

Introduction

Purkinje cells (PCs) are fundamental to the function in the cerebellum, as out of the different neuron populations they are the only ones projecting out of the cerebellar cortex (Altman and Bayer, 1997). SK2 channels are known to modulate Ca2+ transients and to influence spike-firing frequency in dendritic spines, as well as contributing to activity-dependent and compartment-specific plasticity in dendrites of PCs (Hosy et al, 2011; Ohtsuki et al, 2012; Grasselli et al, 2016). The way SK channels regulate these forms of plasticity most likely arises from their specific location along the neuronal surface, as well as from their interaction with different proteins mobilizing Ca2+. PCs do not express these receptors, suggesting that SK2 channels are associated with other group of molecules controlling cytosolic Ca2+ transients, including CaV2.1 channels (Womack and Khodakhah, 2003) and mGlu1α receptors (ref.) that show high levels of expression in PCs (Starr et al, 1991). The association between SK channels and the sources of Ca2+ for their activation in the cerebellar cortex remains unexplored

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