Measurement and Analysis of Calcium-Dependent Exocytosis in Giant Excised Membrane Patches

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Measurement and Analysis of Calcium-Dependent Exocytosis in Giant Excised Membrane Patches

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Title: Measurement and Analysis of Calcium-Dependent Exocytosis in Giant Excised Membrane Patches
Author: Wang, Tzu-Ming
Abstract: 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.
URI: http://hdl.handle.net/2152.5/503
Date: 2008-09-19

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