Synaptotagmin 1 is required for vesicular Ca2+/H+‐antiport activity

• Published in: Journal of neurochemistry 2013-07, Vol.126 (1), p.37-46
• Main Authors: Cordeiro, Joao Miguel, Boda, Bernadett, Gonçalves, Paula P., Dunant, Yves
• Format: Article
• Language: English
• Subjects: Antiporters - antagonists & inhibitors; Antiporters - metabolism; Brain Chemistry - drug effects; Ca2+/H+‐antiport; Calcium - metabolism; Calcium - pharmacology; Cation Transport Proteins - antagonists & inhibitors; Cation Transport Proteins - metabolism; Cellular biology; Clone Cells; Enzyme Inhibitors - pharmacology; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Hydrogen - metabolism; Hydrogen-Ion Concentration; Ionomycin - pharmacology; Macrolides - pharmacology; Neurochemistry; Neurotransmitters; PC12 Cells; PC12 pheochromocytoma cells; Peripheral Nervous System - cytology; Peripheral Nervous System - drug effects; Peripheral Nervous System - metabolism; synaptic vesicles; Synaptic Vesicles - drug effects; Synaptic Vesicles - metabolism; synaptotagmin; Synaptotagmin I - genetics; Synaptotagmin I - physiology; Transfection; Vesicle-Associated Membrane Protein 1 - antagonists & inhibitors; Vesicle-Associated Membrane Protein 1 - immunology
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/jnc.12278

A low‐affinity Ca2+/H+‐antiport was described in the membrane of mammalian brain synaptic vesicles. Electrophysiological studies showed that this antiport contributes to the extreme brevity of excitation‐release coupling in rapid synapses. Synaptotagmin‐1, a vesicular protein interacting with membranes upon low‐affinity Ca2+‐binding, plays a major role in excitation‐release coupling, by synchronizing calcium entry with fast neurotransmitter release. Here, we report that synaptotagmin‐1 is necessary for expression of the vesicular Ca2+/H+‐antiport. We measured Ca2+/H+‐antiport activity in vesicles and granules of pheochromocytoma PC12 cells by three methods: (i) Ca2+‐induced dissipation of the vesicular H+‐gradient; (ii) bafilomycin‐sensitive calcium accumulation and (iii) pH‐jump‐induced calcium accumulation. The results were congruent and highly significant: Ca2+/H+‐antiport activity is detectable only in acidic organelles expressing functional synaptotagmin–1. In contrast, synaptotagmin‐1‐deficient cells – and cells where transgenically encoded synaptotagmin‐1 was acutely photo‐inactivated – were devoid of any Ca2+/H+‐antiport activity. Therefore, in addition to its previously described functions, synaptotagmin‐1 is involved in a rapid vesicular Ca2+ sequestration through a Ca2+/H+ antiport. Synaptotagmin‐1, an abundant protein attached to synaptic vesicles, is necessary for the expression of the vesicular Ca2+/H+ antiport. This low‐affinity antiport was previously described using isolated mammalian brain vesicles and its function was analysed in rapid cholinergic transmission. Therefore, in addition to its previously described functions, we here show that synaptotagmin‐1 is involved in a rapid vesicular Ca2+ sequestration.