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

Authors


Corresponding author T. Kishimoto: Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan. Email: takuya@nips.ac.jp

Abstract

  • 1The 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).
  • 2The 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.
  • 3The 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+.
  • 4The 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.
  • 5These 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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