Browsing by Subject "Calcium Signaling"
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Item Measurement and Analysis of Calcium-Dependent Exocytosis in Giant Excised Membrane Patches(2008-09-19) Wang, Tzu-Ming; Hilgemann, Donald W.Ca2+-dependent exocytosis was studied in both excised and whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Capacitance and amperometric recordings show that secretory granules (SGs) containing serotonin are mostly lost from excised patches. Small vesicles that are retained (non-SGs) do not contain substances detected by amperometry. Non-SG fusion is reduced by tetanus toxin light chain treatment, however, it is unaffected by N-ethylmaleimide, implying that SNARE cycling is not required for non-SG fusion in excised patches. Although non-SG fusion is ATP-dependent and blocked by PI-kinase inhibitors, wortmannin and adenosine, the dependency is not neutralized by the PI(3)-kinase inhibitor LY294002, PI(4,5)P2 ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, and PI(4,5)P2, PI(3)P and PI(4)P antibodies etc. In whole-cell recording, non-SG fusion is strongly reduced by osmotically-induced cell swelling, and subsequent recovery after shrinkage is inhibited by wortmannin, indicating that membrane stretch occurring during patch formation may be a major cause of the ATP-dependency in excised patches. Syt7 and several PLCs are not required for non-SG fusion because fusion remains robust in mouse embryonic fibroblasts deficient of Syt7, PLC(delat)1, PLC(delta)1/(delta)4, or PLC(gamma)1. Furthermore, the Ca2+ dependence of non-SG fusion reflects a lower Ca2+ affinity (KD ~71 uM) than expected for these C2-domain-containing proteins. I also developed a program for measuring and analyzing membrane capacitance. The program uses either sine waves or square waves to estimate cell parameters. Phase-sensitive detection is utilized in both cases. For square wave perturbation, either integrated charges or direct current trace is used for calculating cell parameters. Other functions like digital filtering, pulse stimulation, offline phase angle adjustment, baseline subtraction, and data normalization are also implemented. In summary, using the software I developed, non-SG fusion were characterized and found to be regulated substantially differently from SG fusion. An ATP-dependent process is probably required for restoring non-SG fusion capability after it is perturbed by membrane stretch and dilation.Item Regulation of SNARE-Mediated Synaptic Vesicle Release by Synaptotagmins and Complexins(2010-11-02) Kaeser-Woo, Yea Jin; Südhof, Thomas C.In the brain, neurons communicate with each other by synaptic transmission. This process includes release of neurotransmitter from vesicles in the presynaptic neuron into the synaptic cleft, and sensing of these neurotransmitters by the postsynaptic neuron with specific receptors. Long-lasting changes in the strength of synaptic contacts between neurons in the human brain, a process that is referred to as long-term synaptic plasticity, are the cellular correlates that underlie learning and memory. Synaptic transmission is initiated when an action potential arrives at the presynaptic terminal, and induces Ca2+ influx through voltage-gated Ca2+ channels. The SNARE (Soluble N-ethylmaleimide-sensitive-factor Attachment Protein Receptors) complex is the core component of the fusion machinery in the presynaptic terminal, as it forms a physical bridge between the vesicular membrane and the presynaptic target membrane that delivers the force to fuse the two membranes. Additional presynaptic proteins are required to activate or suppress neurotransmitter release which allows the presynaptic neuron to tightly control and regulate the process of neurotransmitter release. Among these proteins are synaptotagmins and complexins, two protein families that directly interact with the SNARE complex, and that are interdependent to each other in regulating SNARE-mediated synaptic vesicle release: complexin clamps neurotransmitter release until synaptotagmin is recruited by Ca2+ influx, and then it activates SNARE-mediated fusion process together with synaptotagmin. Here I describe the prospective in vivo function of synaptotagmin 12, a novel isoform of synaptotagmin, which lacks the typical Ca2+ binding residues of synaptotagmin, but instead contains a unique sequence motif which can be phosphorylated by cAMP-dependent protein kinase A. By using gene targeting method, I directly examined whether phosphorylation of synaptotagmin 12 is involved in presynaptic forms of long-term plasticity. In parallel, I developed a structure-function approach to functionally dissect how individual domains of complexin contribute to its dichotomic functions of clamping and activating neurotransmitter release. With this approach, we focused on how the accessory alpha-helix of complexin participates in SNARE-mediated synaptic fusion.Item Study of Presynaptic Calcium Channels and a Novel Calcium Channel Calhm1(2010-11-02) Ozkan, Emin D.; Bezprozvanny, IlyaCalcium signaling is essential for all cellular processes. In brain these processes include basic synaptic transmission, modification of established synapses, elimination of synapses as well as elimination of whole neurons by way of apoptosis. In this thesis, we have studied two of these processes. One process is synaptic targeting of presynaptic calcium channels. We have tested the hypothesis that synaptic targeting of presynaptic calcium channels depends on carboxy terminal interactions with mint and CASK proteins. To this end we have used mice mutant for P/Q-type calcium channel. Our experiments show that a P/Q-type calcium channel without its carboxy terminal can partially contribute to synaptic transmission. Secondly we have studied the role of Calhm1 gene in modulating calcium signaling pathways. For this purpose we have used overexpression of Calhm1 gene in HEK293 cells as well as in dissociated neuronal cultures. Our experiments show that Calhm1 modulates calcium signaling in HEK293 cell and in certain neurons. Calhm1 overexpression also modulates spontaneous synaptic transmission.Item [UT Southwestern Medical Center News](2008-11-25) McKenzie, Aline