SK2 Channels Associate With mGlu1α Receptors and CaV2.1 Channels in Purkinje Cells

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⁺: Ca_V2.1 channels and mGlu_1α receptors in the cerebellum. Freeze-fracture replica double-labeling showed significant co-clustering of particles for SK2 with those for Ca_V2.1 channels and mGlu_1α receptors. SK2 channels were also detected at presynaptic sites, mostly at the presynaptic active zone (AZ), where they are close to Ca_V2.1 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 Ca_V2.1 and mGlu_1α provides the mechanism that ensures voltage (excitability) regulation by distinct intracellular Ca²⁺ transients in PCs.

Keywords: calcium channel; cerebellum; electron microscopy; immunohistochemistry; mGlu receptor; potassium channel; synapse.

Figure 1

Subcellular localization of SK2 channels along the surface of Purkinje cells (PCs) using the SDS-digested freeze-fracturereplica labeling (SDS-FRL) technique. Immunolabeling is restricted to the plasma membrane P-face of PCs. The E-face of Bergmann glia (bg), cross-fractures (panel A) or E-faces of neurons (panel G) are free of any immunolabeling. (A–E) Immunoparticles for SK2 channels (10 nm gold) are scattered (arrows) and clustered (double arrows) in dendritic spines (S) and dendritic shafts (PC dendrite, DEN) of PCs. (G) The image shows a high-magnification of the replicated plasma membrane of an axon terminal (at), presumably from granule cells, and spines (s) in a E-face view, showing the co-localization of SK2 (10 nm immunogold) and SNAP25 (5 nm immunogold, white arrowheads). Within this presynaptic localization, SK2 immunoparticles were observed both along the active zone (AZ; double black arrowheads) and at extrasynaptic membranes (black arrowheads). (H) The image shows the P-face of an axon initial segment (AIS) co-labeled for the SK2 (10 nm, black arrowheads) and Nav1.6 (5 nm, white arrowheads) channels. Very low frequency of immunogold labeling for SK2 was detected along AIS. (F) The specificity of the antibody was confirmed in replicas of SK2 channel-knockout mice (SK2 KO) that were free of any immunolabeling. Scale bar: (A,D,F,G) 0.2 μm; (E,H) 0.5 μm.

Figure 2

Density gradient of SK2 immunoparticles in the surface of PCs. (A) Bar graph shows the density (mean ± SEM) of the SK2 channels in eight compartments of PCs. Density of SK2 immunoparticles increased from soma to dendritic spines (soma = 4.31 ± 0.13/μm²; 1/3 ML main dendrite = 12.69 ± 1.96/μm²; 2/3 ML main dendrite = 11.77 ± 0.81/μm²; 1/3 ML spiny branchlet dendrite = 30.67 ± 3.11/μm²; 2/3 ML spiny branchlet dendrite = 29.14 ± 2.69/μm²; 1/3 ML spines = 51.23 ± 2.78/μm²; 2/3 ML spines = 47.60 ± 2.69/μm²; Kruskal–Wallis test, pairwise Mann–Whitney U test and Dunns method, *p < 0.001). Immunoparticle density for SK2 channels on the AISs was not significantly different from the background (AIS = 0.64 ± 0.02/μm²; background = 0.62 ± 0.03/ μm²). ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer. (B) The graph shows the quantification for the number of SK2 particles per cluster in the spines, spiny branchlets (Sp Br D) and main dendrites (M Den). Approximately 76% of clusters in dendritic spines and spiny branchlets and 81% in main dendrites were in the range of 3–8 immunoparticles. (C) Cumulative probability plots of SK2 to SK2 NND. Real and simulated SK2 are shown by solid and dotted lines, respectively. AZ, active zone; AT, axon terminal. The EM image is an example that show random simulation of SK2 immunoparticles in a dendritic spine. Red, real SK2; Yellow, simulated SK2; blue, real Cav2.1.

Figure 3

SK2 channels are excluded from the postsynaptic membrane specializations in PC spines. Electron micrographs showing immunoparticles for SK2 in the molecular layer of the cerebellar cortex, as detected using the post-embedding immunogold (panels A,B) and the SDS-FRL (panels C,D) techniques in the adult mice. (A,B) Using the post-embedding immunogold method, immunoparticles for SK2 were always detected outside the synaptic specialization and only detected both at perisynaptic and extrasynaptic sites (arrows) of PC spines (s) establishing excitatory synapses with parallel fiber terminals (pf). (C,D) Using the highly sensitive SDS-FRL technique, immunoparticles for SK2 (10 nm, black arrowheads) did not co-cluster with particles for Gluδ2 that is expressed selectively at PF-PC synapses (5 nm, white arrowheads), demonstrating the absence of SK2 from the PSD of excitatory synapses between PC spines and PFs. Scale bars: (A,B) 0.2 μm; (C,D) 0.1 μm.

Figure 4

Co-immunoprecipitation of mGlu_1α receptor and SK2 and Cav2.1 channels from mouse cerebellum. Solubilized cerebellar membrane extracts underwent immunoprecipitation analysis using rabbit control IgG (2 μg, lane 1); rabbit anti-Cav2.1 (2 μg, lane 2); rabbit anti-mGlu_1α (2 μg, lane 3) and rabbit anti-SK2 (2 μg, lane 4). Immunoprecipitates (IP) were investigated by SDS-PAGE and immunoblotted using a rabbit anti-Ca_V2.1 (1 μg/ml), rabbit anti-mGlu_1α (1 μg/ml) and guinea-pig anti-SK2 (1 μg/ml). Immunoreactive bands were detected as we have described in detail in the experimental procedures.

Figure 5

Co-localization of SK2 channels with Ca_V2.1 channels and mGlu_1α receptor in PC spines. Electron micrographs obtained in the molecular layer of the cerebellar cortex showing double labeling for SK2 (10 nm, red arrowheads) and Ca_V2.1 (5 nm, blue arrowheads; panels A,B), double labeling for SK2 (10 nm, red arrowheads) and mGlu_1α (5 nm, blue arrowheads; panels C,D), and double labeling for mGlu_1α (10 nm, red arrowheads) and Ca_V2.1 (5 nm, blue arrowheads; panels E,F). In dendritic spines of PCs, SK2 immunoparticles (red arrowheads) co-clustered with those for Ca_V2.1 and mGlu_1α (blue arrows). In addition, mGlu_1α immunoparticles (red arrowheads) co-clustered with those for Ca_V2.1 (blue arrowheads). at, axon terminals. Scale bars: (A–F) 0.2 μm.

Figure 6

Co-localization of SK2 channels with Ca_V2.1 channels and mGlu_1α receptor in PC dendritic shafts. Electron micrographs obtained in the molecular layer of the cerebellar cortex showing immunoparticles for SK2, as detected using the SDS-FRL technique. (A,B) Double labeling for SK2 (10 nm) and mGlu_1α (5 nm) showing their co-clustering in the P-face of PC dendritic shafts (Den). The black box in panel (A) demarcates the dendritic area shown at higher magnification in panel (B). Clusters of immunoparticles for the two proteins are delineated by black ellipses. (C,D) Double labeling for SK2 (10 nm) and Ca_V2.1 (5 nm) showing their co-clustering in dendritic spines of PCs. The black box in panel (C) demarcates the dendritic area shown at higher magnification in panel (D). Clusters of immunoparticles for the two proteins are delineated by black ellipses. cf, cross-fracture of dendritic spines; E, E-face. Scale bars: (A–D) 0.2 μm.

Figure 7

Spatial relationship between SK2, Ca_V2.1 and mGlu_1α in PCs. (A) Example showing random simulation of immunoparticles for Ca_V2.1 in a dendritic spine. (B) Example showing random simulation of immunoparticles for Ca_V2.1 in a dendritic shaft. Red, real SK2; blue, real Cav2.1; green, simulated Cav2.1. (C) Cumulative probability plot of NND from SK2 to Ca_V2.1 particles in dendrites (blue) and spines (red). Solid: real NND between SK2 and Ca_V2.1; Dashed: simulated NND. The SK2 to Ca_V2.1 NNDs were significantly smaller than the random NNDs in dendritic shafts and spines (p < 0.05, pairwise Mann–Whitney U test, numbers of profiles analyzed for spines and dendrites should be indicated). (D) Example showing random simulation of immunoparticles for mGlu_1α in a dendritic spine. (E) Example showing random simulation of immunoparticles for mGlu_1α in a dendritic shaft. Red, real SK2; blue, real mGlu_1α; green, simulated mGlu_1α. (F) Cumulative probability plot of NND from SK2 to mGlu_1α particles in dendrites (blue) and spines (red). Solid: real NND between SK2 and mGlu_1α; Dashed NND: simulated. The SK2 to mGlu_1α NNDs were significantly smaller than the random NNDs in dendritic shafts and spines (p < 0.05, pairwise Mann–Whitney U test, numbers of profiles analyzed for spines and dendrites should be indicated). (G,H) Examples showing random simulation of immunoparticles for Ca_V2.1 in dendritic spines. Red, real mGlu_1α; blue, real Cav2.1; green, simulated Cav2.1. (I) Cumulative probability plot of NND between Ca_V2.1 and mGlu_1α particles in spines (blue). Solid: real NND between mGlu_1α and Ca_V2.1; Dashed: simulated. The mGlu_1α to Ca_V2.1 NNDs were significantly smaller than the random NNDs in dendritic spines (p < 0.05, pairwise Mann–Whitney U test, number of profiles analyzed should be indicated).

Figure 8

Co-localization of SK2 and Ca_V2.1 channels in the presynaptic AZ of ATs. Electron micrographs obtained in the molecular layer of the cerebellar cortex showing the P-face and cross-fractured face of ATs (ax), which were identified by presence of synaptic vesicles (white arrowheads), as well as active zones (az) recognized by the concave shape of the P-face and the accumulation of IMPs. (A,B) Immunoparticles for SK2 (10 nm particles, red arrowheads) were found within the AZ co-clustering with immunoparticles for Ca_V2.1 (5 nm particles, blue arrowheads). A few immunoparticles for SK2 were also observed along the extrasynaptic site (double arrowheads) of ATs. Scale bars: (A,B) 0.2 μm. (C) NNDs from SK2 to Ca_V2.1 and in AZs. The NNDs from SK2 to real and simulated Ca_V2.1 immunoparticles were generated using the same numbers of Ca_V2.1 immunoparticles in the same compartment areas, showing no significant differences (p > 0.05, Kruskal-Wallis test). Insert is an example showing simulation of Cav2.1 immunoparticles in an AZ. Scale bar: 0.2 μm. (D) A high variability in the number of SK2 (range: 3–21) and Ca_V2.1 (range: 4–24) immunoparticles was found at the AZs. Box chart shows 5th, 25th, 75th and 95th percentiles and median (bar). (E) Histogram showing the densities of SK2 and Ca_V2.1 immunoparticles at the AZ and extrasynaptic sites of ATs. The density of both SK2 and Ca_V2.1 immunoparticles was significantly larger at the AZs (SK2 = 104.10 ± 32.87/ μm²; Ca_V2.1 = 125.50 ± 26.61/μm²) than at extrasynaptic sites (Extra; SK2 = 47.24 ± 22.81/ μm²; Ca_V2.1 = 14.95 ± 5.93/μm²; Kruskal–Wallis test, pairwise Mann–Whitney U test and Dunns method, *p < 0.001).