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. 2013 Feb 20;33(8):3668-78.
doi: 10.1523/JNEUROSCI.2921-12.2013.

Quantitative Localization of Cav2.1 (P/Q-type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering With Calcium-Activated Potassium Channels

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Quantitative Localization of Cav2.1 (P/Q-type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering With Calcium-Activated Potassium Channels

Dwi Wahyu Indriati et al. J Neurosci. .
Free PMC article

Abstract

P/Q-type voltage-dependent calcium channels play key roles in transmitter release, integration of dendritic signals, generation of dendritic spikes, and gene expression. High intracellular calcium concentration transient produced by these channels is restricted to tens to hundreds of nanometers from the channels. Therefore, precise localization of these channels along the plasma membrane was long sought to decipher how each neuronal cell function is controlled. Here, we analyzed the distribution of Ca(v)2.1 subunit of the P/Q-type channel using highly sensitive SDS-digested freeze-fracture replica labeling in the rat cerebellar Purkinje cells. The labeling efficiency was such that the number of immunogold particles in each parallel fiber active zone was comparable to that of functional channels calculated from previous reports. Two distinct patterns of Ca(v)2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Ca(v)2.1 had a somatodendritic gradient with the density of immunogold particles increasing 2.5-fold from soma to distal dendrites. The other population with 74-fold higher density than the scattered particles was found within clusters of intramembrane particles on the P-face of soma and primary dendrites. Both populations of Ca(v)2.1 were found as early as P3 and increased in the second postnatal week to a mature level. Using double immunogold labeling, we found that virtually all of the Ca(v)2.1 clusters were colocalized with two types of calcium-activated potassium channels, BK and SK2, with the nearest neighbor distance of ∼40 nm. Calcium nanodomain created by the opening of Ca(v)2.1 channels likely activates the two channels that limit the extent of depolarization.

Figures

Figure 1.
Figure 1.
Localization of Cav2.1 in the presynaptic active zone of parallel fiber boutons. A, The P-face and cross-fractured face of a parallel fiber bouton at P28 showing synaptic vesicles (arrows) and active zone indicated by concave shape of the P-face, high density of IMPs, and dimples (black arrowheads) representing vesicle exocytosis or endocytosis. PF boutons were identified with labeling for vGluT1 (10 nm particles, white arrowheads). B, Small clusters of immunogold particles for Cav2.1 (5 nm particles, arrowheads) were found within the active zone structure. C, High variability of number of Cav2.1 particles (range: 6–43) was found in PF boutons at P28. Box chart shows fifth, 25th, 75th, and 95th percentiles, median (bar), and mean (square). D, In the wild-type mice (Cav2.1+/+), clustered gold particles for Cav2.1 (10 nm) were found at PF presynaptic active zone (P-face) with opposing postsynaptic sites (E-face) labeled for AMPA receptors (5 nm). E, The immunogold labeling for Cav2.1 was abolished in the Cav2.1 KO mice (Cav2.1−/−), while AMPA receptor labeling (5 nm) remained. Scale bars: A, 500 nm; B–E, 200 nm.
Figure 2.
Figure 2.
Two distinct localization patterns of Cav2.1 in Purkinje cells. A, The molecular layer (ML), Purkinje cell layer (PL), and granule cell layer (GL) are identifiable under low magnification of a replica image. B, The P-face of soma and proximal dendrite of a PC labeled for Cav2.1 (5 nm). Immunogold particles for Cav2.1 were found concentrated within IMP clusters (red) and scattered throughout the plasma membrane (blue). The Cav2.1-labeled IMP clusters were diffusely distributed in the soma and proximal dendrite. C, D, Scattered Cav2.1 particles in soma (C) and distal dendrites (D) showing isolated particles (black arrowheads) and small aggregations (white arrowheads). E, F, Clusters of immunogold particles for Cav2.1 were associated with IMP clusters. G, The two distinct patterns of Cav2.1 localization, clustered and scattered (black arrowheads for isolated particles and a white arrowhead for a small aggregation), were also seen in wild-type mice. H, The labeling for Cav2.1 within and outside of IMP clusters was absent in Cav2.1 KO mice. Scale bars: A, 50 μm; B, 5 μm; C, D, 100 nm; E–H, 200 nm.
Figure 3.
Figure 3.
Perisynaptic localization of Cav2.1 and colocalization with mGluR1α in Purkinje cell spines. A, D, Low-magnification pictures showing spines protruding from PC dendrites. Areas in boxes are magnified in C and F. These areas show the P-face of PC spines. B, E, Corresponding locations to the boxed areas in A and D were searched on the complimentary replica. The E-face of the spines had IMP clusters indicative of excitatory postsynaptic structure. Gray lines indicate the boundaries of synapses. Note that the corresponding replicas appear as mirrored images, thus one of the pictures (showing the E-face of synapses) was flipped to allow straightforward comparison with the other (C, F, showing the P-face of the same synapse). In C and F, the boundaries of the synapse determined in the E-face replica (B, E) were projected onto the P-face. Labelings for mGluR1α (10 nm) and Cav2.1 (5 nm, arrowheads) were found colocalized near the boundary of the synapse. Scale bars: A, D, 500 nm; B, C, E, F, 100 nm.
Figure 4.
Figure 4.
Density gradient of the scattered Cav2.1 along Purkinje soma and dendrites. A, Density of the scattered Cav2.1 particles (including both isolated particles and those within small aggregations) increased from soma to distal dendrites. (soma = 5.81 ± 1.68 /μm2; primary dendrite = 8.53 ± 3.99 /μm2; secondary dendrite = 12.3 ± 4.01 /μm2; distal dendrite = 14.1 ± 4.06 /μm2; measured area = 700–900 μm2). B, Out of all scattered Cav2.1 particles, the density of isolated particles only was extracted. The density increase from the soma to distal dendrites was also observed for the isolated particles (soma = 3.49 ± 1.50 /μm2; primary dendrite = 4.86 ± 2.44 /μm2; secondary dendrite = 6.78 ± 2.26 /μm2; distal dendrite = 6.80 ± 2.41 /μm2; measured area = 700–900 μm2). C, The number of the small aggregation of Cav2.1 particles was counted and the density of such small aggregations was calculated. The density of the small aggregation also increased from soma to distal dendrites (soma = 59.0 ± 15.7/100 μm2; primary dendrite = 90.9 ± 51.8/100 μm2; secondary dendrite = 132.3 ± 48.9/100 μm2; distal dendrite = 159.6 ± 74.4 /μm2; measured area = 700–900 μm2, Kruskal–Wallis test, pairwise Mann–Whitney U test with Bonferroni correction). D, Developmental increase of the density of scattered Cav2.1 particles was seen at PC soma after the second postnatal week (P3 = 1.76 ± 0.78 /μm2; P7 = 1.68 ± 0.38 /μm2; P14 = 4.06 ± 0.82 /μm2; P28 = 5.81 ± 1.68 /μm2; adult = 5.34 ± 0.65 /μm2; measured area = 500–1000 μm2, ANOVA Tukey's test). *p < 0.05; **p < 0.01.
Figure 5.
Figure 5.
Developmental changes of P-face IMP clusters labeled for Cav2.1 in Purkinje cell soma. A–D, P-face IMP clusters labeled for Cav2.1 at P7 (A), P14 (B), P28 (C), and adult (D). E, F, Cumulative frequency plots for the IMP cluster size (E) and number of Cav2.1 particles within each IMP cluster (F). Significant differences in the IMP cluster size was observed between P7 and P14 and between P28 and adult. The number of Cav2.1 particles was significantly increased from P7 to P28, and decreased from P21 to adult (IMP cluster size: P7 = 0.060 ± 0.043 μm2; P14 = 0.101 ± 0.077 μm2; P21 = 0.097 ± 0.085 μm2; P28 = 0.103 ± 0.090 μm2; adult = 0.181 ± 0.171 μm2; number of Cav2.1 particles; P7 = 23.8 ± 21.0 particles; P14 = 42.4 ± 34.0 particles; P21 = 46.3 ± 42.0 particles; P28 = 38.9 ± 35.0 particles; adult = 29.4 ± 26.5 particles; Kruskal–Wallis, pairwise Mann–Whitney U test with Bonferroni correction). G, Positive correlation was found between the number of Cav2.1 particles and the IMP cluster size in all observed ages. The IMP cluster size and the number of Cav2.1 particles concurrently increased, thus the density (signified in the slope of the scattered plots) remained similar from P7 to P28 but significantly reduced in adult. H, Density of IMP clusters increased in PC soma during postnatal development (P3 = 3.53 ± 1.75 clusters/100 μm2; P7 = 4.51 ± 1.22 clusters/100 μm2; P14 = 6.72 ± 0.78 clusters/100 μm2; P28 = 8.12 ± 2.13 clusters/100 μm2; adult = 9.28 ± 2.35 clusters/100 μm2; ANOVA with Tukey's test). I, Cumulative frequency plots of NNDs of Cav2.1 particles within the IMP cluster (Real) and randomly distributed NNDs generated using the same numbers of particles and IMP cluster areas (Random). The real NND was significantly smaller than the random NND, indicating clustering of Cav2.1 within the IMP cluster. *p < 0.05, **p < 0.01, ***p = 0.000.
Figure 6.
Figure 6.
Calcium nanodomain formation with clustered Cav2.1, BK and SK2 channels in Purkinje cell soma and proximal dendrites. A, B, Single labeling for BK or SK2 channels in the PC soma showing concentrated particles within the P-face IMP clusters. C, D, Double labeling for Cav2.1 (5 nm, black arrowheads) and either BK (10 nm, white arrowheads) or SK2 (10 nm, white arrowheads) channels revealed that these KCa channels are colocalized with Cav2.1 in the IMP clusters at PC soma. E, F, Double labeling for Cav2.1 (5 nm) and either BK (10 nm) or SK2 (10 nm) in BK KO (E) or SK2 KO (F) mice, respectively. The labeling for BK and SK2 channels disappeared in the respective KO mice verifying the labeling specificity. Labeling for Cav2.1 persisted in the IMP clusters in both KO mice. Scale bar, 200 nm.
Figure 7.
Figure 7.
NNDs between Cav2.1 and BK or SK2 channels. A, B, NNDs between BK (A) or SK2 (B) and Cav2.1 particles within the P-face IMP clusters were analyzed. In both cases, median of NND was quite small (∼40 nm) suggesting calcium nanodomain formation. These NNDs (Real) were compared with randomly distributed NNDs (Random) generated using the same numbers of BK or SK2 particles same IMP cluster areas. The real NNDs were significantly smaller than the random NNDs for Cav2.1 and SK2 (p = 0.019, Man–Whitney U test, p = 0.001, Kolmogorov–Smirnov test) but not for Cav2.1 and BK.

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