Abstract
It is generally accepted that voltage-gated Ca2+ channels, CaV, regulate Ca2+ homeostasis in excitable cells following plasma membrane depolarization. Here, we show that the Ca2+ protein α1D of CaV1.3 channel is overexpressed in colorectal cancer biopsies compared to normal tissues. Gene silencing experiments targeting α1D reduced the migration and the basal cytosolic Ca2+ concentration of HCT116 colon cancer cell line and modified the cytosolic Ca2+ oscillations induced by the sodium/calcium exchanger NCX1/3 working in its reverse mode. Interestingly, NCX1/3 regulated membrane potential of HCT116 cells only when α1D was silenced, and blocking NCX1/3 increased cytosolic Ca2+ concentration and cell migration. However, membrane depolarization did not induce an increase in intracellular Ca2+. Patch-clamp experiments clearly showed that the inward Ca2+ current was absent. Finally, flow cytometry and immunofluorescence studies showed that α1D protein was localized at the plasma membrane, in cytosol and cell nuclei. Altogether, we uncover a novel signaling pathway showing that α1D is involved in the regulation of Ca2+ homeostasis and cell migration by a mechanism independent of its plasma membrane canonical function but that involved plasma membrane Na+/Ca2+ exchanger.
Highlights
Voltage-gated Ca2+ channels (CaV) are activated by membrane depolarization and mediate Ca2+ influx in response to action potentials and subthreshold depolarizing signals
We observed an influence of pT stages on α1D staining suggesting a role of α1D in the cell migration/invasion known to play a pivotal role in metastatic process (Fig. 1C)
Our data have demonstrated that α1D protein regulates migration/invasion of HCT116 cells mainly through its non-canonical activity because: i) we did not detect any voltage-gated inward Ca2+currents in these cells using patch -clamp technique irrespective of the clamp configurations or the experimental conditions and ii) depolarization with high external K+ solutions did not increase the [Ca2+]c in contrast to what had been observed in excitable cells
Summary
Voltage-gated Ca2+ channels (CaV) are activated by membrane depolarization and mediate Ca2+ influx in response to action potentials and subthreshold depolarizing signals. The mechanisms implied in the metastatic process are not yet completely elucidated, it is clear that the degradation of the extracellular matrix and the cellular migration, both regulated by Ca2+ channels[14], play a pivotal role in this process It has not been determined if the regulation of the biology of cancer cells by α1D protein subunit depends on its plasma membrane canonical function. We investigated the role of the α1D protein of CaV1.3 in the migration of the non-excitable and epithelial cancer cells HCT116, its contribution in intracellular Ca2+ regulation and we raised the question on its role as a channel in these cells. Alpha 1D protein regulates the migration and invasion of HCT116 colon cancer cells and its intracellular Ca2+ concentration by a mechanism that does not depend on its plasma membrane canonical function but that involves plasma membrane NCX1/3 exchanger and endoplasmic reticulum (ER) Ca2+ release
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