Transmitter release face Ca²⁺ channel clusters persist at isolated presynaptic terminals

Abstract

Ca(2+) influx through N-type Ca(2+) channels (CaV2.2) is known to be critical for transmitter release at many synapses. These channels are known to be localized to transmitter release sites, but their anchoring mechanism remains unknown. Recent studies have demonstrated that presynaptic organization is subject to interactions with the postsynaptic cell or the intervening extracellular matrix. We used a previously described high-affinity antibody against the N-type Ca(2+) channels, Ab571, to localize Ca(2+) channel clusters at the release face of an isolated giant calyx-type synapse to test whether the maintenance of these clusters requires an intact extracellular matrix or contact with the postsynaptic cell. Because the number of Ca(2+) channel clusters was unchanged after extracellular matrix dispersal or nerve terminal isolation, we conclude that presynaptic transmitter release face Ca(2+) clusters can be maintained independently of extracellular influences. Our results suggest that a presynaptic molecular scaffold is responsible for the maintenance of release site Ca(2+) channel clusters.

Independent Regulation of Basal Neurotransmitter Release Efficacy by Variable Ca2+ Influx and Bouton Size at Small Central Synapses

“…The predominant occurrence of VGCCs within the AZ [31],[33]–[35],[51] implies that AP-evoked is directly related to the local Ca 2+ concentration transients at the vesicular release sites. This further argues that the observed co-variation of and could be a direct consequence of inter-synaptic heterogeneity of local Ca 2+ influx at the AZ.…”

The presynaptic CaV2.2 channel–transmitter release site core complex

“…We scored each CaV2.2 punctum along the transmitter release‐face profile (Sun et al ., 2006) for its pattern of staining relative to that for the costained antibody according to four general types (Fig. 3, Ai–iv): CaV2.2 puncta that were ‘isolated from’, ‘abut’, ‘partially overlap’, or ‘superimpose’ binding partner staining.…”

“…As spatial resolution in the z ‐axis is typically lower than that in the x ‐ or y ‐axes, it is probable that the number of partially overlapping puncta are overestimated at the expense of the number of abutting clusters. Due to their low density on the release face (Sun et al ., 2006), random fully superimposed clusters should be rare and should not materially affect the count. The numbers of clusters in each category were counted for each staining partner and were expressed as a percentage of the whole (Fig.…”

“…This is a near‐ideal experimental model as virtually all the calcium channels are CaV2.2‐type (Stanley & Atrakchi, 1990; Stanley, 1991; Yawo & Chuhma, 1994) and the terminal can be fully isolated on a coverslip permitting imaging at close to the resolution limits for the standard light microscope (Stanley & Mirotznik, 1997; Mirotznik et al ., 2000; Li et al ., 2004). This feature permits a distinction of TRS calcium‐channel clusters and release site‐associated proteins from those in non‐TRS areas, either within the terminal or on the transmitter release face (Li et al ., 2004; Khanna et al ., 2006b; Khanna et al ., 2006c; Sun et al ., 2006). We use quantitative immunocytochemistry (Li et al ., 2004; Bolte & Cordelieres, 2006) to test whether the staining intensities of the channel and the partner protein covary, as expected for two proteins that are parts of a common complex.…”

Slow chemical transmission between dorsal root ganglion neuron somata

“…After enzyme treatment the ganglia were washed with warmed and CO 2 ‐equilibrated Minimal Essential Medium and were gently triturated with a standard 200‐μL plastic pipette tip. Tissue slices were prepared from previously untreated, freshly dissociated ganglia immediately frozen using standard methods as described (Sun et al. , 2006) and sectioned at 12 μm.…”