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 VO43- (IC50 = 185 μM), although the inhibitory effectiveness of VO43- is increased 37-fold by a 15-min preincubation of the permeabilized cells with VO43- 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.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|State||Published - 1985|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology