SK2 Channels Associate With mGlu Receptors and CaV2.1 Channels in Purkinje Cells

Rafael Luján, Carolina Aguado, Francisco Ciruela, Xavier Morató Arus, Alejandro Martín-Belmonte, Rocío Alfaro-Ruiz, Jesús Martínez-Gómez, Luis de la Ossa, Masahiko Watanabe, John Adelman, Ryuichi Shigemoto, Yugo Fukazawa

Research output: Contribution to journalArticle

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 mGlu receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for CaV2.1 channels and mGlu 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 mGlu provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca2+ transients in PCs.

Original languageEnglish (US)
Article number311
JournalFrontiers in Cellular Neuroscience
Volume12
DOIs
StatePublished - Sep 19 2018

Fingerprint

Purkinje Cells
Macromolecular Substances
Post-Synaptic Density
Calcium-Activated Potassium Channels
Dendritic Spines
Carisoprodol
Immunoprecipitation
Synapses
Cerebellum
Cluster Analysis
Proteins
Spine
voltage-dependent calcium channel (P-Q type)

Keywords

  • Calcium channel
  • Cerebellum
  • Electron microscopy
  • Immunohistochemistry
  • MGlu receptor
  • Potassium channel
  • Synapse

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience

Cite this

Luján, R., Aguado, C., Ciruela, F., Arus, X. M., Martín-Belmonte, A., Alfaro-Ruiz, R., ... Fukazawa, Y. (2018). SK2 Channels Associate With mGlu Receptors and CaV2.1 Channels in Purkinje Cells. Frontiers in Cellular Neuroscience, 12, [311]. https://doi.org/10.3389/fncel.2018.00311

SK2 Channels Associate With mGlu Receptors and CaV2.1 Channels in Purkinje Cells. / Luján, Rafael; Aguado, Carolina; Ciruela, Francisco; Arus, Xavier Morató; Martín-Belmonte, Alejandro; Alfaro-Ruiz, Rocío; Martínez-Gómez, Jesús; de la Ossa, Luis; Watanabe, Masahiko; Adelman, John; Shigemoto, Ryuichi; Fukazawa, Yugo.

In: Frontiers in Cellular Neuroscience, Vol. 12, 311, 19.09.2018.

Research output: Contribution to journalArticle

Luján, R, Aguado, C, Ciruela, F, Arus, XM, Martín-Belmonte, A, Alfaro-Ruiz, R, Martínez-Gómez, J, de la Ossa, L, Watanabe, M, Adelman, J, Shigemoto, R & Fukazawa, Y 2018, 'SK2 Channels Associate With mGlu Receptors and CaV2.1 Channels in Purkinje Cells', Frontiers in Cellular Neuroscience, vol. 12, 311. https://doi.org/10.3389/fncel.2018.00311
Luján R, Aguado C, Ciruela F, Arus XM, Martín-Belmonte A, Alfaro-Ruiz R et al. SK2 Channels Associate With mGlu Receptors and CaV2.1 Channels in Purkinje Cells. Frontiers in Cellular Neuroscience. 2018 Sep 19;12. 311. https://doi.org/10.3389/fncel.2018.00311
Luján, Rafael ; Aguado, Carolina ; Ciruela, Francisco ; Arus, Xavier Morató ; Martín-Belmonte, Alejandro ; Alfaro-Ruiz, Rocío ; Martínez-Gómez, Jesús ; de la Ossa, Luis ; Watanabe, Masahiko ; Adelman, John ; Shigemoto, Ryuichi ; Fukazawa, Yugo. / SK2 Channels Associate With mGlu Receptors and CaV2.1 Channels in Purkinje Cells. In: Frontiers in Cellular Neuroscience. 2018 ; Vol. 12.
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AU - Luján, Rafael

AU - Aguado, Carolina

AU - Ciruela, Francisco

AU - Arus, Xavier Morató

AU - Martín-Belmonte, Alejandro

AU - Alfaro-Ruiz, Rocío

AU - Martínez-Gómez, Jesús

AU - de la Ossa, Luis

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AB - 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.

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