Ion selectivities of the Ca(2+) sensors for exocytosis in rat phaeochromocytoma cells.
1. The ion selectivities of the Ca(2+) 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-Ca(2+) compounds and measured with low-affinity indicators benzothiazole coumarin (BTC) or 5-nitrobenzothiazole coumarin (BTC-5N). 2. The Ca(2+)-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 Ca(2+) concentration of > 0.1 mM. 3. The slow component of exocytosis was activated by all metal ions investigated, including Cd(2+) (median effective concentration, 18 pM), Mn(2+) (500 nM), Co(2+) (900 nM), Ca(2+) (8 microM), Sr(2+) (180 microM), Ba(2+) (280 microM) and Mg(2+) (> 5 mM). In contrast, the fast component of exocytosis was activated by Cd(2+) (26 pM), Mn(2+) (620 nM), Ca(2+) (24 microM) and Sr(2+) (320 microM), but was only slightly increased by Ba(2+) (> 2 mM) and Co(2+) and not at all by Mg(2+). 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 Ca(2+) 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 A (1 A = 0.1 nm) being effective. 5. These data support a role for synaptotagmin--phospholipid as the Ca(2+) sensor for the exocytosis of large dense-core vesicles and they suggest that an additional Ca(2+)-sensing mechanism operates in the synchronous exocytosis of synaptic-like vesicles.
Kishimoto
T
T
Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan. takuya@nips.ac.jp
Liu
T T
TT
Ninomiya
Y
Y
Takagi
H
H
Yoshioka
T
T
Ellis-Davies
G C
GC
Miyashita
Y
Y
Kasai
H
H
D000818:Animals; D002118:Calcium / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004594:Electrophysiology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D007477:Ions; D008670:Metals / Q000494:pharmacology; D015388:Organelles / Q000502:physiology; D016716:PC12 Cells / Q000502:physiology*; D051381:Rats; D012964:Sodium / Q000494:pharmacology
D007477:Ions; D008670:Metals; D012964:Sodium; D002118:Calcium