The Journal of General Physiology
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Published online June 18, 2008
doi:10.1085/jgp.200709950
The Journal of General Physiology, Vol. 132, No. 1, 51-65
The Rockefeller University Press, 0022-1295 $30.00
© 2008 Wang et al.
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ARTICLE

 Ca-dependent Nonsecretory Vesicle Fusion in a Secretory Cell



Tzu-Ming Wang and Donald W. Hilgemann

Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390

Correspondence to Donald Hilgemann: donald.hilgemann{at}utsouthwestern.edu

We have compared Ca-dependent exocytosis in excised giant membrane patches and in whole-cell patch clamp with emphasis on the rat secretory cell line, RBL. Stable patches of 2–4 pF are easily excised from RBL cells after partially disrupting actin cytoskeleton with latrunculin A. Membrane fusion is triggered by switching the patch to a cytoplasmic solution containing 100–200 µM free Ca. Capacitance and amperometric recording show that large secretory granules (SGs) containing serotonin are mostly lost from patches. Small vesicles that are retained (non-SGs) do not release serotonin or other substances detected by amperometry, although their fusion is reduced by tetanus toxin light chain. Non-SG fusion is unaffected by N-ethylmaleimide, phosphatidylinositol-4,5-bis-phosphate (PI(4,5)P2) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P2, PI(3)P, and PI(4)P antibodies. In patch recordings, but not whole-cell recordings, fusion can be strongly reduced by ATP removal and by the nonspecific PI-kinase inhibitors wortmannin and adenosine. In whole-cell recording, non-SG fusion is strongly reduced by osmotically induced cell swelling, and subsequent recovery after shrinkage is then inhibited by wortmannin. Thus, membrane stretch that occurs during patch formation may be a major cause of differences between excised patch and whole-cell fusion responses. Regarding Ca sensors for non-SG fusion, fusion remains robust in synaptotagmin (Syt) VII–/– mouse embryonic fibroblasts (MEFs), as well as in PLC{delta}1, PLC {delta}1/{delta}4, and PLC{gamma}1–/– MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (KD ~71 µM) than expected for these C2 domain–containing proteins. In summary, we find that non-SG membrane fusion behaves and is regulated substantially differently from SG fusion, and we have identified an ATP-dependent process that restores non-SG fusion capability after it is perturbed by membrane stretch or cell dilation.

Abbreviations used in this paper: CAPS, calcium-dependent activator protein for secretion; DAG, diacylglycerol; DC, direct-current; IP3, inositol triphosphate; MEF, mouse embryonic fibroblast; NEM, N-ethylmaleimide; NMG, N-methyl-D-glucamine; NSF, NEM-sensitive factor; PI(3)K, PI(3) kinase; PI(3)P, phosphatidylinositol 3-phosphate; PI(4,5)P2, phosphatidylinositol-4,5-bis-phosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; SG, secretory granule; Syt, synaptotagmin; TeTx, tetanus toxin.


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