A direct entry model for capacitative calcium entry

We studied the phenomenon of capacitative calcium entry (CCE) - the phenomenon of intracellular store refilling where the amount of calcium influx is inversely proportional to the endoplasmic reticulum (ER) store with an aim at providing experimental support for the direct entry model of CCE. The direct entry model postulates that any decrease in the ER [Ca2+] creates a gradient for refilling of ER through a mass-action mechanism without traversing the cytosol. A kinetic difference indicating that, during CCE, the cytosolic appearance of Ca2+ is preceded by ER refilling was observed. A second and more Ca2+ specific ER targeted fluorescent dye was used to reproduce thapsigargin-insensitive ER entry. Reloading of the ER was observed even in presence of cytosolic Ca2+ chelator BAPTA-AM [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. The ER Ca2+ chelator TPEN [N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene diamine] was able to initiate CCE and also exhibited the susceptibility of being extracted out of the cells by prior addition of other metal ions. The effectiveness of low concentrations of lanthanum as a specific blocker of CCE was investigated. The phenomenon of non-capacitative calcium entry was explored and its difference from CCE was established. It was also observed that the net direction of the entirety of the non-capacitative entry is not towards the ER. After excitable (L6 skeletal muscle cell line) and non-excitable (Jurkat) cells, direct entry form the extracellular to ER space was also observed in a nerve cell line. The utility and universality of the direct entry model in explaining capacitative calcium entry was discussed.