Ca-dependent nonsecretory vesicle fusion in a secretory cell.
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 microM 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)P(2)) ligands, such as neomycin, a PI-transfer protein that can remove PI from membranes, the PI(3)-kinase inhibitor LY294002 and PI(4,5)P(2), 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 PLCdelta1, PLC delta1/delta4, and PLCgamma1-/- MEFs. Thus, Syt VII and several PLCs are not required. Furthermore, the Ca dependence of non-SG fusion reflects a lower Ca affinity (K(D) approximately 71 microM) 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.
Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Dallas, TX 75390, USA.
Tzu-Ming
TM
Wang
Donald W
DW
Hilgemann
D000241:Adenosine / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D045744:Cell Line, Tumor; D048429:Cell Size; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D008561:Membrane Fusion / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D051381:Rats; D050600:SNARE Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D022142:Secretory Vesicles / Q000502:physiology*; D050857:Synaptotagmins / Q000172:deficiency / Q000235:genetics; D022161:Transport Vesicles / Q000502:physiology; D010738:Type C Phospholipases / Q000172:deficiency / Q000235:genetics; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D050600:SNARE Proteins; D050857:Synaptotagmins; D000255:Adenosine Triphosphate; D010738:Type C Phospholipases; D000241:Adenosine; D005033:Ethylmaleimide; D002118:Calcium; D000077191:Wortmannin