Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin 28kDa . Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin 28kDa -null mutants. An unchanged rate of calcium transport was verified by measurements of 45 Ca incorporation into developing teeth in vivo. In enamelforming cells, the absence of calbindin 28kDa was not compensated by other cytosolic calcium-binding proteins as detectable by 45 Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.The active transport of calcium across cells holds widespread importance in medicine and biology, yet the underlying mechanisms remain unclear. Operating in many places (e.g. gut, kidney, placenta, teeth, bones, oviduct, lung, inner ear), active transport is used to control the amount of calcium in body fluids and so impacts on nutrition, biomineralization, fertility, respiration, and hearing (1, 2). Superior control is achieved by passaging calcium actively through cells rather than passively between them, but this comes at the risk of cytotoxicity should the ability to regulate intracellular calcium be overburdened.Mechanistically, active transport is considered in three steps: the entry of calcium to the cell, transit across it, and extrusion at the other side. The transit step has received the most attention over several decades, being considered rate-limiting and having key molecular players defined. However, recent molecular characterization of calcium entry channels has transformed the field by providing a new mechanistic focus for vitamin D-restricted transport (3, 4). With these advances reigniting interest in therapeutic applications, it is important to revisit what happens following calcium entry.The 30-year-old paradigm that calcium is ferried through cytosol by mobile calcium-binding proteins (calbindins) remains widely accepted (3-9). Calbindins are thought to facilitate the naturally poor diffusion of calcium in cytosol and simultaneously buffer calcium at safe concentrations. Comprehensively supporting this view, tight correlations between calbindin expression and vitamin D-dependent transport were found in intestine...

Recent attempts to understand the function of calbindin28kDa, a widely expressed calcium-binding protein, are confounded by uncertainties over its subcellular location. Using immunoblot analysis of rat brain subregions, we found that the proportion of particulate calbindin28kDa (24-43% of total) was independent of expression level and location. The association of calbindin28kDa with particulate structures appeared to be specific, since it persisted when soluble calbindin28kDa was sequestered by antibodies added before tissue disruption. Moreover, when exogenous calbindin28kDa was added during homogenisation of brain from calbindin28kDa-nullmutant mice, only 10% partitioned to the particulate fraction compared with 33% of endogenous calbindin28kDa in wildtype controls. Confocal microscopy showed that calbindin28kDa was predominantly extranuclear in all tissues analysed (i.e. various brain regions, isolated neurons, and dental enamel epithelium). Dual-label microscopy of neural dense particulate fractions confirmed the extranuclear location of calbindin28kDa and also showed that it partly colocalised with synaptosome and microtubule markers. Using sucrose step gradients, calbindin28kDa was separated from nuclei in parallel with synaptosome and endoplasmic reticulum markers. However, no association with the marker proteins (synaptophysin, ERp29, alpha/beta-tubulin) was detected by calbindin28kDa-immunoprecipitation analysis. Together these findings provide the first consistent picture that calbindin28kDa is located predominantly outside of the nucleus, irrespective of tissue type (neuronal vs. non-neuronal) and experimental approach (biochemical vs morphological). The evidence of a substantial, strong and specific association with insoluble cellular structures challenges the widely held view of calbindin28kDa as a mobile calcium buffer, and supports the existence of important alternative roles that involve target proteins.