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L. Curtis, R. Janz; Syntaxin 3B is a Binding Partner of the Cav1.4 L-Type Calcium Channel. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1014.
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© ARVO (1962-2015); The Authors (2016-present)
Photoreceptors and bipolar cells relay information to other retinal cells by continuously releasing neurotransmitter-filled vesicles at specialized synapses called ribbon synapses. This is in contrast to conventional synapses in the nervous system which release neurotransmitter phasically. Despite these differences in release properties, neurotransmitter release at both synapse types is a Ca2+- dependent process supported by presynaptic calcium channels. In addition to providing the calcium ions needed for neurotransmitter release, the presynaptic calcium channels have been shown to interact with the constituents of the synaptic vesicle docking/fusion complex (i.e. SNARE complex). These interactions are thought to play a role in the regulation of neurotransmitter release from neurons. Previous studies have focused on how members of the SNARE complex regulate calcium channels in conventional synapses. Little is known about the interplay between the constituents of the SNARE complex and the presynaptic calcium channels in retinal ribbon synapses. As such, this study seeks to analyze whether syntaxin 3B, a member of the SNARE complex in retinal ribbon synapses, is a binding partner of a calcium channel known to support neurotransmitter release from photoreceptor terminals, the Cav1.4 L-type channel.
Immunoprecipitation (IP)Syntaxin 3B was immunoprecipitated with an antibody which recognizes syntaxin 3 (A-B).Western blotting was used to analyze which proteins interact with syntaxin 3B.Expression of Syntaxin 3B and Cav1.4 in HEK293 cellsCells were transfected with syntaxin 3B (pCMV-synt. 3B) and Cav1.4 (pcDNA-1F, pcDNA - β2A, pcDNA-2Δ) expression vectors. After 48 hrs, the cells were collected and subjected to subcellular fractionation. The microsomal pellet was resuspended in 2X SDS Sample Buffer and an aliquot was run on an SDS-PAGE gel. The proteins were transferred to a nitrocellulose membrane and western blotting was used to analyze to presence of syntaxin 3B and 1F -Cav1.4 in the microsomal fraction.
1. When transfected with syntaxin 3B and Cav1.4 expression vectors, HEK293 cells expressed both proteins.2. Syntaxin 3B and the Cav1.4 channel were shown to biochemically interact.
1. Syntaxin 3B can interact with the Cav1.4 channel. The function of this interaction may be to ensure the Cav1.4 channel is in close proximity to vesicle release sites and/or syntaxin 3B may act as a modulator of the Cav1.4 channel to regulate vesicle release.2. HEK293 cells transfected with syntaxin 3B and the Cav1.4 channel could potentially be used for electrophysiological experiments to study the function of the interaction between syntaxin 3B and the Cav1.4 channel.
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