Alteration of Ca²⁺Dependence of Neurotransmitter Release by Disruption of Ca²⁺Channel/Syntaxin Interaction

Abstract

Presynaptic N-type calcium channels interact with syntaxin and synaptosome-associated protein of 25 kDa (SNAP-25) through a binding site in the intracellular loop connecting domains II and III of the ␣ 1 subunit. This binding region was loaded into embryonic spinal neurons of Xenopus by early blastomere injection. After culturing, synaptic transmission of peptide-loaded and control cells was compared by measuring postsynaptic responses under different external Ca 2ϩ concentrations. The relative transmitter release of injected neurons was reduced by ϳ25% at physiological Ca 2ϩ concentration, whereas injection of the corresponding region of the L-type Ca 2ϩ channel had virtually no effect. When applied to a theoretical model, these results imply that 70% of the formerly linked vesicles have been uncoupled after action of the peptide. Our data suggest that severing the physical interaction between presynaptic calcium channels and synaptic proteins will not prevent synaptic transmission at this synapse but will make it less efficient by shifting its Ca 2ϩ dependence to higher values.

The C2b Domain of Synaptotagmin Is a Ca2+–Sensing Module Essential for Exocytosis

“…It was suggested that inhibition was due to uncoupling syntaxin from Ca 2+ channels (Mochida et al 1996; Rettig et al 1997). However, chronic application of synprint (Rettig et al 1997) can disrupt synaptotagmin–AP-2 interactions (Zhang et al 1994; Chapman et al 1998), resulting in a loss in the total number of vesicles (Jorgensen et al 1995). Use of permeable PC12 cells circumvents a number of these problems since Ca 2+ -triggered secretion does not depend upon Ca 2+ channels nor upon endocytosis.…”

Dissection, Culture, and Analysis of <em>Xenopus laevis</em> Embryonic Retinal Tissue

“…25. Once in the Dissection Plate, pull back the ectoderm overlying the retina (or optic vesicle for the embryos stage [26][27][28][29][30] (Figure 2F and 2G).…”

“…For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,[19][20][21][22][24][25][26][27][28][29][30][31][32][33] . Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,[27][28][29][30][33][34][35][36][37][38][39] . Xenopus laevis, a classic model system for the study of early neural development 19,27,29,[31][32][40][41][42] , serves as a particularly suitable system for retinal primary cell culture 10,38,[43][44][45] .…”

“…Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,[27][28][29][30][33][34][35][36][37][38][39] . Xenopus laevis, a classic model system for the study of early neural development 19,27,29,[31][32][40][41][42] , serves as a particularly suitable system for retinal primary cell culture 10,38,[43][44][45] .Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43 . In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,[44][45] .…”

A Nitric Oxide/Cyclic GMP‐Dependent Protein Kinase Pathway Alters Transmitter Release and Inhibition by Somatostatin at a Site Downstream of Calcium Entry

“…When active, the NO-PKG pathway is hypothesized to reduce the Ca2+ sensitivity and, when pharmacologically blocked, to increase the Ca2+ sensitivity. Individual proteins associated with the secretory machinery have been shown to be phosphorylated by a variety of kinases, and some of these phosphorylation events have been shown to alter the Ca2+ dependence of transmitter release (Trudeau et al, 1996(Trudeau et al, , 1998Rettig et al, 1997;Yokoyama et al, 1997). Furthermore, intracellular signaling cascades can alter transmitter release downstream of Ca2' entry (Dale and Kandel, 1989;Man-Son-Hing et al, 1989;Przywara et a]., 1991).…”