Enhancement of the Ca²⁺-triggering steps of native membrane fusion via thiol-reactivity

• Published in: Journal of chemical biology 2009-03, Vol.2 (1), p.27-37
• Main Authors: Furber, Kendra L, Brandman, David M, Coorssen, Jens R
• Format: Article
• Language: English
• Subjects: Biochemistry; Biological and Medical Physics; Biophysics; calcium; Cell Biology; Chemistry; Chemistry and Materials Science; exocytosis; Membrane fusion; Membranes; Original; Original Article; Pharmacology/Toxicology; Physical Chemistry; strontium; Thiol reactivity; Vesicle
• ISSN: 1864-6166; 1864-6158; 1864-6166
• DOI: 10.1007/s12154-008-0013-3

Ca²⁺-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca²⁺ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca²⁺ sensitivity of both CV-plasma membrane and CV-CV fusion. If Sr²⁺, a weak Ca²⁺ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca²⁺, suggesting that IA acts at the Ca²⁺-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency.