Quantitative Localization of Ca_v2.1 (P/Q-Type) Voltage-Dependent Calcium Channels in Purkinje Cells: Somatodendritic Gradient and Distinct Somatic Coclustering with Calcium-Activated Potassium Channels

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 Cav2.1 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 Cav2.1 distribution, scattered and clustered, were found in Purkinje cells. The scattered Cav2.1 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 Cav2.1 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 Cav2.1 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 Cav2.1 channels likely activates the two channels that limit the extent of depolarization.

Numbers of presynaptic Ca 2+ channel clusters match those of functionally defined vesicular docking sites in single central synapses

“…Because VGluT1 is selectively expressed in PF boutons in the molecular layer (43), these results confirm that these profiles originate from PF-MLI synapses. We also found that the presynaptic P-face of these synapses was flat, in contrast to the concave profile observed at PF-PC synapses (33); this difference provides an additional criterium to distinguish between PF-MLI and PF-PC synapses. Finally, we occasionally found GABAergic synapses on MLIs by double labeling for AZ proteins and VGAT (Fig.…”

“…Assuming that receptor density remains constant, as suggested by the finding of very high and homogenous AMPA receptor density at PF-MLI synapses (37) (30,31,33), we found that Ca v 2.1 channel labels were not distributed uniformly. The distribution of distances between any pair of particles within an AZ displayed a peak in the range between 0 nm and 50 nm (particularly evident in 4-wk data), indicating particle association within clusters, whereas beyond this range particle distances were distributed broadly ( Fig.…”

Variable priming of a docked synaptic vesicle

“…At typical synapses, docking and fusion take place at structurally specialized regions along the PM called active zones (9,10). Several lines of evidence suggest that the formation of the SNARE core complex occurs in the macromolecules composing the common active zone organelle, active zone material (AZM) (2,(11)(12)(13)(14), which is positioned near Ca 2+ channels concentrated in the PM at active zones (15)(16)(17). Influx of Ca 2+ through the channels after the arrival of a nerve impulse triggers fusion of the VM of docked SVs with the PM.…”

T-type channel blockade impairs long-term potentiation at the parallel fiber–Purkinje cell synapse and cerebellar learning

“…Unlike in the hippocampus, postsynaptic Ca 2+ signaling at PF-PC spines may not be dominated by ionotropic glutamatergic receptors, as postsynaptic N-methyl-D-aspartate receptors (NMDARs) are not prominently present at this site and AMPA receptors are predominantly impermeable for calcium ions (4,5). PCs bear different voltage-dependent Ca channels including P/Q-type (6)(7)(8) and T-type channels (9,10). The spines of PCs contain a high density of Ca V 3.1 T-type channels (11), which can be readily activated by typical bursts of PF activity that occur during sensory stimulation (12)(13)(14).…”