| Abstract: | 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 compared directly with the Ca2+ pump characterized in detail in synaptosomal membrane vesicles (Gill, D. L., Grollman, E. F., and Kohn, L. D. (1981) J. Biol. Chem. 256, 184-192; Gill, D. L., Chueh, S. H., and Whitlow, C. L. (1984) J. Biol. Chem. 259, 10807-10813) which was proven by flux reversal studies to be derived from the neural plasma membrane (Gill, D. L. (1982) J. Biol. Chem. 257, 10986-10990). 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+ (Km = 0.28 microM), is dependent on both ATP (Km = 26 microM) 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 microM 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 microM) and an apparent 400-fold lower sensitivity to VO4(3-) (IC50 = 185 microM), 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 microM). 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(beta-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 microM. |
