Inhibitory and excitatory axon terminals share a common nano-architecture of their Cav2.1 (P/Q-type) Ca2+ channels

Abstract

Tuning of the time course and strength of inhibitory and excitatory neurotransmitter release is fundamental for the precise operation of cortical network activity and is controlled by Ca2+ influx into presynaptic terminals through the high voltage-activated P/Q-type Ca2+ (Cav2.1) channels. Proper channel-mediated Ca2+-signaling critically depends on the topographical arrangement of the channels in the presynaptic membrane. Here, we used high-resolution SDS-digested freeze-fracture replica immunoelectron microscopy together with automatized computational analysis of Cav2.1 immunogold labeling to determine the precise subcellular organization of Cav2.1 channels in both inhibitory and excitatory terminals. Immunoparticles labeling the pore-forming α1 subunit of Cav2.1 channels were enriched over the active zone of the boutons with the number of channels (3–62) correlated with the area of the synaptic membrane. Detailed analysis showed that Cav2.1 channels are non-uniformly distributed over the presynaptic membrane specialization where they are arranged in clusters of an average five channels per cluster covering a mean area with a diameter of about 70 nm. Importantly, clustered arrangement and cluster properties did not show any significant difference between GABAergic and glutamatergic terminals. Our data demonstrate a common nano-architecture of Cav2.1 channels in inhibitory and excitatory boutons in stratum radiatum of the hippocampal CA1 area suggesting that the cluster arrangement is crucial for the precise release of transmitters from the axonal boutons.

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

“…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.…”

“…To define a cluster, we set a threshold value of 40 nm for the distance between particles belonging to the same cluster. This value was calculated as equal to μ + 3σ, where μ and σ are the mean (18.7 nm) and SD (7.1 nm) obtained for a single Gaussian fit of a 2-wk nearest neighbor distance distribution; it is very close to the value of 42 nm used previously by Althof et al (30). Fig.…”

Variations in Ca2+Influx Can Alter Chelator-Based Estimates of Ca2+Channel–Synaptic Vesicle Coupling Distance

“…This recording configuration is convenient because the ATP concentration is known and endogenous EF-hand mobile buffers have been dialyzed (Müller et al, 2007). The resting free [Ca 2ϩ ] was set to 50 nM, the Ca 2ϩ diffusion coefficient (D Ca ) to 220 m 2 /s (Allbritton et al, 1992), the endogenous fixed buffer (EFB) binding ratio to 40 (Helmchen et al, 1997), the EFB concentration to 4 mM, the EFB K D to 100 M, and an EFB forward binding rate constant (k on ) to 1 ϫ 10 8 M Ϫ1 sec Ϫ1 (Nakamura et al, 2015). We included 200 M free ATP in the simulation, assuming the presence of 2 mM ATP (total concentration) and 3 mM Mg 2ϩ (calculation using Maxchelator RRID:SCR_000459).…”

Surface dynamics of voltage-gated ion channels

“…20,46,71,118 Investigations of the release probability, channel distribution, as well as evoked calcium signals, point to a clustered distribution of presynaptic calcium channels. 4,66,103 The distance of VGCC clusters and synaptic vesicles varies between different synapses 7,46,71,135 and implies different channel arrangements in the different synapses. 103,117 Bearing lateral diffusion in mind, several options for modulation of synaptic release probability and short-term plasticity emerge.…”