Ion selectivities of the Ca2+ sensors for exocytosis in rat phaeochromocytoma CELLS

1. The ion selectivities of the Ca2+ sensors for the two components of exoc ytosis in rat phaeochromocytoma (PC12) cells were examined by measurement o f membrane capacitance and amperometry. The cytosolic concentrations of met al ions were increased by photolysis of caged-Ca2+ compounds and measured w ith low-affinity indicators benzothiazole coumarin (BTC) or 5-nitrobenzothi azole coumarin (BTC-5N). 2. The Ca2+-induced increases in membrane capacitance comprised two phases with time constants of 30-100 ms and 5 s. Amperometric events reflecting th e exocytosis of large dense-core vesicles occurred selectively in the slow phase, even with increases in the cytosolic Ca2+ concentration of > 0.1 mM. 3. The slow component of exocytosis was activated by all metal ions investi gated, including Cd2+ (median effective concentration, 18 pM), Mn2+ (500 nM .), Co2+ (900 nM), Ca2+ (8 muM), Sr2+ (180 muM), Ba2+ (280 muM) and Mg2+ (> 5 mM). In contrast, the fast component of exocytosis was activated by Cd2 (26 pM), Mn2+ (620 nM), Ca2+ (24 muM) and Sr2+ (320 muM), but was only sli ghtly increased by Ba2+ (> 2 mM) and Co2+ and not at all by Mg2+. 4. The fast component, but not the slow component, was competitively blocke d by Na+ (median effective concentration, 44 mM) but not by Li+, K+ or Cs+. Thus, the Ca2+ sensor for the fast component of exocytosis is more selecti ve than is that for the slow component; moreover, this selectivity appears to be based on ionic radius, with cations with radii of 0.84 to 1.13 Angstr om (1 Angstrom = 0.1. nm) being effective. 5. These data support a role for synaptotagmin-phospholipid as the Ca2+ sen sor for the exocytosis of large dense-core vesicles and they suggest that a n additional Ca2+-sensing mechanism operates in the synchronous exocytosis of synaptic-like vesicles.