| Summary: | Calbindin-D28k (CaBP) is a 28 kD calcium-binding protein found in specific neuronal populations in the mammalian brain. The hypothesized Ca²⁺-buffering action of CaBP is the basis of suggestions that this protein may serve to protect neurons against cell death mediated by large or prolonged increases in intracellular free Ca²⁺ concentration. However, to date, there is little direct evidence to support this hypothesis. To address this question directly, we have examined Ca²⁺-buffering by CaBP in stably transfected HEK 293 and HeLa cell lines. A variety of methods were employed to induce calcium transients, including transfection of NMD A receptors followed by activation with glutamate. In all experiments there was evidence of CaBP-mediated Ca²⁺-buffering. Moreover, when NMDAR transfected cells were exposed to excitotoxic concentrations of glutamate, cells expressing CaBP exhibited enhanced survival over controls. CaBP was unable to prevent acute necrotic cell death but significantly protected cells from delayed, presumably apoptotic cell death. To examine the potential influence of CaBP upon intracellular Ca²⁺-oscillations, stably-transfected HeLa cells were treated with histamine, while measuring intracellular Ca²⁺. The observation that CaBP flattened the profile of component Ca²⁺ peaks, coupled with data from HEK cell lines, provides unequivocal evidence that CaBP can act to buffer increases in intracellular Ca²⁺. Utilizing a novel method for resolving intracellular Ca²⁺ waves, it was found that transfection with CaBP, or loading with artificial Ca²⁺ buffers, attenuated the velocity of Ca²⁺ waves. The scope of attenuation appeared to be a function of the buffer binding kinetics. The rate of Ca²⁺-binding by CaBP was apparently too slow to influence Ca²⁺- interaction between the closely situated IP₃ receptors in initiation sites, where the faster on rate buffer BAPTA exerted a significant effect. However, both CaBP and BAPTA had significant effects upon events which were more distal to the source of Ca²⁺ release, including effects between the more sparsely distributed IP₃ receptors involved in the propagation of Ca²⁺ waves, and global changes in Ca²⁺. In view of the fact that Ca²⁺ waves and oscillations have been shown to modulate neuron development and gene expression, it is possible that the effects of CaBP may include influencing these processes. === Medicine, Faculty of === Cellular and Physiological Sciences, Department of === Graduate
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