Sk2 channels associate with mGlu1α receptors and CaV2.1 channels in Purkinje cells Journal Article

Author(s): Luján, Rafæl; Aguado, Carolina; Ciruela, Francisco; Arus, Xavier M; Martín-Belmonte, Alejandro; Alfaro-Ruiz, Rocío; Martinez-Gomez, Jesus; de la Ossa, Luis; Watanabe, Masahiko; Adelman, John P; Shigemoto, Ryuichi; Fukazawa, Yugo
Article Title: Sk2 channels associate with mGlu1α receptors and CaV2.1 channels in Purkinje cells
Affiliation IST Austria
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.
Keywords: Immunohistochemistry; potassium channel; Cerebellum; synapse; Electron microscopy; calcium channel; MGlu receptor
Journal Title: Frontiers in Cellular Neuroscience
Volume: 12
ISSN: 1662-5102
Publisher: Frontiers Media  
Date Published: 2018-09-19
Start Page: Article number: 311
Copyright Statement: CC BY
DOI: 10.3389/fncel.2018.00311
Notes: This project/research has received funding from the European Union’s Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 720270 (Human Brain Project SGA1) to RL and RS. This work was supported by grants from the Spanish Ministerio de Economía Industria y Competitividad (BFU2015-63769-R) and Junta de Comunidades de Castilla-La Mancha (PPII-2014-005-P) to RL, the Spanish Ministry of Education and Science (TIN2016-77902-C3-1-P) to JM-G and LO and Ministerio de Economía Industria y Competitividad/ Instituto de Salud Carlos III (SAF2017-87349-R and PIE14/00034), the Catalan government (2017 SGR 1604), Fundació la Marató de TV3 (Grant 20152031), FWO (SBO-140028) to FC and Life Science Innovation Center (Research and Education Program for Life Science) at University of Fukui to YF.
Open access: yes (OA journal)