Calcium buffering and protection from excitotoxic cell death by exogenous calbindin-D28k in HEK 293 cells

Calbindin-D28k (CaBP) is a calcium-binding protein found in specific neuronal populations in the mammalian brain that, as a result of its proposed calcium-buffering action, may protect neurons against potentially harmful increases in intracellular calcium. We have stably transfected HEK 293 cells with recombinant human CaBP in order to determine the influence of this protein upon transient increases in intracellular ionic calcium concentration ([Ca2+](i)) induced either by transient transfection of the NR1 and NR2A subunits of the N-methyl-D-aspartate (NMDA) receptor and brief exposure to glutamate, photolysis of the caged calcium compound NP-EGTA, or exposure to the Ca2+-ionophore 4-Br-A23187. The presence of CaBP did not significantly reduce the peak [Ca2+](i) stimulated by glutamate activation of NMDA receptors but significantly prolonged the recovery to baseline values. Flash photolysis of NP-EGTA in control cells resulted in an almost instantaneous increase in [Ca2+](i) followed by a bi-exponential recovery to baseline values. In cells stably expressing CaBP, the peak [Ca2+](i) levels were not statistically different from the controls, however, there was a significant prolongation of the initial portion of the slow recovery phase. In cells exposed to 4-Br-A23187, the presence of CaBP significantly reduced the rate of rise of [Ca2+](i), reduced the peak response, slowed the rate of recovery, and reduced the depolarization of mitochondria, In studies of delayed, Ca2+-dependent cell death, CaBP transfected cells exhibited enhanced survival 24 h after a 1-h exposure to 200 muM NMDA. However, necrotic cell death observed after the first 6 h was not prevented by the presence of CaBP. These results provide direct evidence for a Ca2+-buffering effect of CaBP which serves to limit Ca2+ entry and the depolarization of mitochondria, thereby protecting cells from death mediated most likely by apoptosis. (C) 2001 Harcourt Publishers Ltd.