Ca-dependent nonsecretory vesicle fusion in a secretory cell

Tzu Ming Wang, Donald W. Hilgemann

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

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 PLCδ1, PLC δ1/δ4, and PLCγ1-/- 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.

Original languageEnglish (US)
Pages (from-to)51-65
Number of pages15
JournalJournal of General Physiology
Volume132
Issue number1
DOIs
StatePublished - Jul 2008

Fingerprint

Synaptotagmins
Membrane Fusion
Membranes
Cell Fusion
Secretory Vesicles
Patch-Clamp Techniques
Serotonin
Fibroblasts
Adenosine Triphosphate
Tetanus Toxin
2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
Ethylmaleimide
Neomycin
Exocytosis
Phosphatidylinositols
Actin Cytoskeleton
Phosphatidylinositol 3-Kinases
Adenosine
Dilatation
Proteins

ASJC Scopus subject areas

  • Physiology

Cite this

Ca-dependent nonsecretory vesicle fusion in a secretory cell. / Wang, Tzu Ming; Hilgemann, Donald W.

In: Journal of General Physiology, Vol. 132, No. 1, 07.2008, p. 51-65.

Research output: Contribution to journalArticle

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