An intracellular (ATP + Mg²⁺)-dependent calcium pump within the N1E-115 neuronal cell line

An intracellular (ATP + Mg²⁺)-dependent Ca²⁺ pumping mechanism has been identified and characterized within the cultured clonal neuroblastoma cell line N1E-115. Using cell suspensions treated with 0.005% saponin which selectively permeabilizes the plasma membrane in 95-98% of the cells, it was possible to show clearly that the intracellular Ca²⁺ pump mechanism is of non-plasma membrane origin and therefore can be computerized directly with the Ca²⁺ pump characterized in detail in synaptosomal membrane vesicles which was proven by flux reversal studies to be derived from the neural plasma membrane. The intracellular Ca²⁺ pump in N1E-115 cells is distinct from mitochondrial Ca²⁺ accumulation and is increased up to 8-fold higher as cells reach confluency. In similarity to the neural plasma membrane pump, the intracellular Ca²⁺ pump within N1E-115 cells has high affinity for Ca²⁺ (K(m) = 0.28 μM), is dependent on both ATP (K(m) = 26 μM) and either Mg²⁺ or Mn²⁺ which half-maximally activate Ca²⁺ pumping at 0.35 mM and 0.32 mM, respectively, and shows similar specificity for Sr²⁺ and Ba²⁺ which half-maximally inhibit Ca²⁺ transport at 50 μM and 1.5 mM, respectively. In contrast to the neural plasma membrane pump, the intracellular Ca²⁺ pump displays approximately 40-fold higher sensitivity to La³⁺ (IC₅₀ = 5 μM) and an apparent 400-fold lower sensitivity to VO₄^3- (IC₅₀ = 185 μM), although the inhibitory effectiveness of VO₄^3- is increased 37-fold by a 15-min preincubation of the permeabilized cells with VO₄^3- in the absence of ATP (apparent IC₅₀ = 5 μM). In further contrast to the neural plasma membrane Ca²⁺ pump, the intracellular pump within N1E-115 cells is stimulated more than 20-fold by oxalate (giving prolonged linear Ca²⁺ accumulation), is resistant to low saponin concentrations, and is not modified by calmodulin even after extensive treatment with ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid and/or calmodulin antagonist drugs. However, calmidazolium is effective in inhibiting the intracellular Ca²⁺ pump with an IC₅₀ of approximately 2 μM.