Dissecting the Ca²⁺ entry pathways induced by rotavirus infection and NSP4-EGFP expression in Cos-7 cells.
Rotavirus infection modifies Ca(2+) homeostasis provoking an increase in Ca(2+) permeation, cytoplasmic Ca(2+) concentration ([Ca(2+)](cyto)), total Ca(2+) pools and, a decrease of Ca(2+) response to agonists. These effects are mediated by NSP4. The mechanism by which NSP4 deranges Ca(2+) homeostasis is not yet known. It has been proposed that the increase in [Ca(2+)](cyto) is the result of Ca(2+) release from intracellular stores, thereby activating store-operated Ca(2+) entry (SOCE). We studied the mechanisms involved in the changes of Ca(2+) 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 Ca(2+) stores induced by thapsigargin or ATP was not able to elicit Ca(2+) 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 [Ca(2+)](cyto), is permeable to Mn(2+) and inhibited by La(3+) and the SOC inhibitor 2-aminoethoxydiphenyl borate (2-APB). Contribution of the agonist-operated channels (AOCs) to Ca(2+) entry is small and not modified by infection. The plasma membrane permeability to Ca(2+) in rotavirus infected or NSP4-EGFP expressing cells is also blocked by KB-R7943, an inhibitor of the plasma membrane Na(+)/Ca(2+) 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 Ca(2+) entry.
Laboratorio de Fisiología Gastrointestinal, Instituto Venezolano de Investigaciones Científicas, Apartado 21827, Caracas 1020A, Venezuela.
Yuleima
Y
Díaz
Franshelle
F
Peña
Olga Carolina
OC
Aristimuño
Lorena
L
Matteo
Marisela
M
De Agrela
Maria Elena
ME
Chemello
Fabian
F
Michelangeli
Marie Christine
MC
Ruiz
D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism; D002463:Cell Membrane Permeability; D002522:Chlorocebus aethiops; D005456:Fluorescent Dyes / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D012401:Rotavirus / Q000472:pathogenicity*; D019831:Sodium-Calcium Exchanger / Q000378:metabolism; D014118:Toxins, Biological / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D002413:Cations, Divalent; D005456:Fluorescent Dyes; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D019831:Sodium-Calcium Exchanger; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D002118:Calcium