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

Takuya Kishimoto *†, Ting-Ting Liu *†, Yasunori Ninomiya †, Hiroshi Takagi ‡, Tohru Yoshioka ‡, Graham C. R. Ellis-Davies §, Yasushi Miyashita *† and Haruo Kasai *

1. The ion selectivities of the Ca²⁺ sensors for the two components of exocytosis in rat phaeochromocytoma (PC12) cells were examined by measurement of membrane capacitance and amperometry. The cytosolic concentrations of metal ions were increased by photolysis of caged-Ca²⁺ compounds and measured with low-affinity indicators benzothiazole coumarin (BTC) or 5-nitrobenzothiazole 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 the 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 investigated, including Cd²⁺ (median effective concentration, 18 pM), Mn²⁺ (500 nM), Co²⁺ (900 nM), Ca²⁺ (8 µM), Sr²⁺ (180 µM), Ba²⁺ (280 µM) and Mg²⁺ (> 5 mM). In contrast, the fast component of exocytosis was activated by Cd²⁺ (26 pM), Mn²⁺ (620 nM), Ca²⁺ (24 µM) and Sr²⁺ (320 µM), but was only slightly increased by Ba²⁺ (> 2 mM) and Co²⁺ and not at all by Mg²⁺.
4. The fast component, but not the slow component, was competitively blocked 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 selective 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 Å (1 Å = 0.1 nm) being effective.
5. These data support a role for synaptotagmin-phospholipid as the Ca²⁺ sensor for the exocytosis of large dense-core vesicles and they suggest that an additional Ca²⁺-sensing mechanism operates in the synchronous exocytosis of synaptic-like vesicles.

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