The voltage-gated Ca2+ channel is the Ca2+ sensor of fast neurotransmitter release.
Previously it demonstrated that in the absence of Ca2+ entry, evoked secretion occurs neither by membrane depolarization, induction of [Ca2+]i rise, nor by both combined (Ashery, U., Weiss, C., Sela, D., Spira, M. E., and Atlas, D. (1993). Receptors Channels 1:217-220.). These studies designate Ca2+ entry as opposed to [Ca2+]i rise, essential for exocytosis. It led us to propose that the channel acts as the Ca+ sensor and modulates secretion through a physical and functional contact with the synaptic proteins. This view was supported by protein-protein interactions reconstituted in the Xenopus oocytes expression system and release experiments in pancreatic cells (Barg, S., Ma, X., Elliasson, L., Galvanovskis, J., Gopel, S. O., Obermuller, S., Platzer, J., Renstrom, E., Trus, M., Atlas, D., Streissnig, G., and Rorsman, P. (2001). Biophys. J; Wiser, O., Bennett, M. K., and Atlas, D. (1996). EMBO J 15:4100-4110; Wiser, O., Trus, M.. Hernandez, A., Renström, E., Barg, S., Rorsman. P., and Atlas, D. (1999). Proc. Natl. Acad. Sci. U.S.A. 96:248-253). The kinetics of Ca(v)1.2 (Lc-type) and Ca(v)2.2 (N-type) Ca2+ channels were modified in oocytes injected with cRNA encoding syntaxin 1A and SNAP-25. Conserved cysteines (Cys271, Cys272) within the syntaxin 1A transmembrane domain are essential. Synaptotagmin 1, a vesicle-associated protein, accelerated the activation kinetics indicating Ca(v)2.2 coupling to the vesicle. The unique modifications of Ca(v)1.2 and Ca(v)2.2 kinetics by syntaxin 1A, SNAP-25, and synaptotagmin combined implied excitosome formation, a primed fusion complex of the channel with synaptic proteins. The Ca(v)1.2 cytosolic domain Lc(753-893), acted as a dominant negative modulator, competitively inhibiting insulin release of channel-associated vesicles (CAV), the readily releasable pool of vesicles (RRP) in islet cells. A molecular mechanism is offered to explain fast secretion of vesicles tethered to SNAREs-associated Ca2+ channel. The tight arrangement facilitates the propagation of conformational changes induced during depolarization and Ca2+-binding at the channel, to the SNAREs to trigger secretion. The results imply a rapid Ca2+-dependent CAV (RRP) release, initiated by the binding of Ca2+ to the channel, upstream to intracellular Ca2+ sensor thus establishing the Ca2+ channel as the Ca2+ sensor of neurotransmitter release.
Atlas
D
D
Department of Biological Chemistry, Hebrew University of Jerusalem, Israel. datlas@vms.huji.ac.il
Wiser
O
O
Trus
M
M
D000818:Animals; D000954:Antigens, Surface / Q000378:metabolism; D015220:Calcium Channels / Q000378:metabolism*; D020746:Calcium Channels, L-Type / Q000378:metabolism; D020013:Calcium Signaling / Q000502:physiology*; D002135:Calcium-Binding Proteins; D002490:Central Nervous System / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D005260:Female; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008959:Models, Neurological; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D009865:Oocytes; D011247:Pregnancy; D017434:Protein Structure, Tertiary / Q000502:physiology; D013570:Synaptic Membranes / Q000378:metabolism; D009435:Synaptic Transmission / Q000502:physiology*; D013572:Synaptic Vesicles / Q000378:metabolism; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050857:Synaptotagmins; D050827:Syntaxin 1; D014982:Xenopus laevis
D000954:Antigens, Surface; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D002135:Calcium-Binding Proteins; C419385:L-type calcium channel alpha(1C); D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D018377:Neurotransmitter Agents; D050825:Synaptosomal-Associated Protein 25; D050863:Synaptotagmin I; D050827:Syntaxin 1; D050857:Synaptotagmins