| Abstract No.: | B-B2065 |
| Country: | Canada |
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| Title: | INVERTEBRATE SYNAPTIC CALCIUM CHANNEL LACK A SYNAPTIC PROTEIN BINDING SITE
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| Authors/Affiliations: | 1 Patrick McCamphill*; 1 Phuc Pham; 1 Allan Siu; 1 Kate Gardiner; 1 J. David Spafford;
1 University of Waterloo, ON, Canada
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| Content: | Introduction: Fast synaptic transmission is initiated by calcium influx into the presynaptic nerve terminal and is mediated by fusion of transmitter filled vesicles with the plasma membrane. Vesicle fusion has been shown to require a complex of synaptic proteins in close association with voltage gated calcium channels (VGCC). In vertebrate neurons, sequences in the cytoplasmic II-III loop of Cav2 channel, termed the synaptic SYNaptic protein PRotein interaction INTeraction site (synprint) are thought to be necessary for incorporation of channels into the vesicle fusion machinery and efficient synaptic transmission. However, recent reports of novel human Cav 2.2 channel variants lacking the synprint region and the discovery of an invertebrate calcium channel that still functions in synaptic transmission with out this domain, question the requirement of the synprint region in presynaptic targeting and synaptic transmission. While the mammalian II-III linker peptide of calcium channels will block synaptic transmission in some invertebrates, the snail II-III linker of LCav2 neither binds syntaxin1A in vitro nor does the snail II-III linker peptide interfere with Lymnaea synaptic transmission. These data suggest that the mammalian synaptic protein binding site is a specialization evolved for fine-tuning vertebrate, but not invertebrate synaptic transmission.
Objectives: To assess the functional consequences of inserting the rat synaptic protein binding site in the II-III linker of LCav2, the synaptic calcium channel of Lymnaea neurons.
Results: Western blots indicate that 3xHA tagged LCav2 wildtype and LCav2 with rat Cav2.2 synprint site channels express in HEK293T cells. In addition, Pull-down assays and co-immunoprecipitation studies demonstrate binding of Lymnaea GST-syntaxin 1A, GST-Snap-25 and GST-synaptotagmin 1 to the channel in vitro. Finally, the functional consequences of synprint binding to synaptic proteins are assessed through patch clamp electrophysiology.
Conclusions: This research is of broad importance in the field of synaptic physiology because calcium channels are essential players in the growth and development of neurons, synapse formation and synaptic communication. The functional relevance of synaptic proteins binding to calcium channels has come under question recently and these studies address this fundamental issue and shed new light on the properties of calcium channels and their associated protein binding domains.
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