Dissecting the Ca2+ entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells

Abstract

Rotavirus infection modifies Ca2+ homeostasis provoking an increase in Ca2+ permeation, cytoplasmic Ca2+ concentration ([Ca2+]cyto), total Ca2+ pools and, a decrease of Ca2+ response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca2+ homeostasis is not yet known. It has been proposed that the increase in [Ca2+]cyto is the result of Ca2+ release from intracellular stores, thereby activating store-operated Ca2+ entry (SOCE). We studied the mechanisms involved in the changes of Ca2+ permeability of the plasma membrane elicited by rotavirus infection and NSP4 expression in Cos-7 cells loaded with fura-2 or fluo-4, using inhibitors and activators of different pathways. Total depletion of ER Ca2+ stores induced by thapsigargin or ATP was not able to elicit Ca2+ entry in mock-infected cells to the level attained with infection or NSP4-EGFP expression. The pathway induced by NSP4-EGFP expression or infection shows properties shared by SOCE: it can be inactivated by high [Ca2+]cyto, is permeable to Mn2+ and inhibited by La3+ and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca2+ entry is small and not modified by infection. The plasma membrane permeability to Ca2+ in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na+/Ca2+ exchanger (NCX), operating in its reverse mode. In conclusion, the expression of NSP4 in infected Cos-7 cells appears to activate the NCX in reverse mode and the SOCE pathway to induce increased Ca2+ entry.