Fig. 4.

CIRL supports both Ca²⁺ entry and the sustained elevation of intracellular Ca²⁺elicited by α-latrotoxin. A, HEK293 cells were transfected with plasmids for green fluorescent protein (GFP) and either pCDR7 (CIRL) or pCMVneo by Lipofectamine, as described. After 2 d the cells were incubated with the indicated concentrations of α-Ltx in physiological saline without Ca²⁺ or Mg²⁺ and with 0.2 mm EGTA for 4 min. The incubation with toxin was followed by a 6 min incubation in PSS containing 2.2 mm Ca²⁺, 0.5 mm Mg²⁺, and 1 μCi/ml⁴⁵Ca²⁺. The cells were rinsed immediately three times with PSS without⁴⁵Ca²⁺, and the amount of⁴⁵Ca²⁺ in the cells was determined by liquid scintillation spectrometry; n = 5 wells/group. B, C, Chromaffin cells were transfected with plasmids for GFP and either pCDR7 or pCMVneo, with GFP serving as a marker for the transfected cells. Cultures were loaded with 1 μm fura-2 AM and subsequently were perfused with a 10 μm concentration of a nicotinic agonist (dimethylphenylpiperazinium, DMPP), followed by the indicated concentrations of α-Ltx in PSS. The duration of each agonist application is indicated by a horizontal bar. Intracellular Ca²⁺ levels were obtained as described in Materials and Methods. Following are the mean resting Ca²⁺ levels: −CIRL, 56 ± 11 nm,n = 3; +CIRL, 45 ± 7 nm,n = 4.