A novel synaptic junction preparation for the identification and characterization of cleft proteins

Abstract

Identification of synaptic cleft components has been hampered by the lack of a suitable preparation enriched in synaptic junctions devoid of adjoining peripheral membranes. Prior strategies for the isolation of synaptic junctions, relying on detergents for the removal of peripheral membranes, resulted in substantial loss of membranes lining the cleft. Here, a novel, detergent-free method is described for the preparation of a synaptic junction (SJ) fraction, using phospholipase A2. Limited digestion of synaptic plasma membrane (SPM) fraction with phospholipase A2 followed by centrifugation over a sucrose cushion results in selective removal of membranes peripheral to the cleft while junctional membranes remain relatively intact as observed by electron microscopy. Enrichment in synaptic junctional structures and loss of membranes peripheral to the junctional area are further verified by demonstrating enrichment in PSD-95 and loss in mGluR5, respectively. The SJ fraction is enriched in neuroligins and neurexins, in agreement with immuno-electron microscopy data showing their selective localization to the junctional area. Among additional cell adhesion molecules tested, N-cadherin and specific isoforms of the SynCAM and SALM families also show marked enrichment in the SJ fraction, suggesting preferential localization at the synaptic cleft while others show little enrichment or decrease, suggesting that they are not restricted to or concentrated at the synaptic cleft. Treatment of the SJ fraction with glycosidases results in electrophoretic mobility shifts of all cell adhesion molecules tested, indicating glycosylation at the synaptic cleft. Biochemical and ultrastructural data presented indicate that the novel synaptic junction preparation can be used as a predictive tool for the identification and characterization of the components of the synaptic cleft.

Introductory Chapter: Ion Channels

“…Alterations in the electrical potential of presynaptic neurons initiate the release of neurotransmitters from the vesicles in the synaptic cleft [13]. These chemicals move toward the postsynaptic cells through the diffusion and occupy their specific receptor sites on membranes and generate the electrical potential by opening ion channels [14]. Removal of neurotransmitters from the synaptic cleft is essential to avoid any effect on the nearby cells [14][15][16].…”

“…These chemicals move toward the postsynaptic cells through the diffusion and occupy their specific receptor sites on membranes and generate the electrical potential by opening ion channels [14]. Removal of neurotransmitters from the synaptic cleft is essential to avoid any effect on the nearby cells [14][15][16]. Cell signaling by neurotransmitters is far more adaptable and versatile as compared to the gap junctions [17].…”

“…In most of the ion channels, the passage across the pores is governed by a "gate." The gate may be opened or closed in response to different factors such as: electrical signals, chemical signals, temperature, and the mechanical force [14,15,18]. In summary, ion channels are the integral membrane proteins which are usually present as assemblies of many subunit proteins [16,19].…”