AMPKα1-sensitivity of orai1 and Ca 2+ entry in T - Lymphocytes

Abstract

Background/Aims: T-lymphocyte activation and function critically depends on Ca 2+ signaling, which is regulated by store operated Ca 2+ entry (SOCE). Human and mouse T lymphocytes express AMP activated kinase AMPKα1, which is rapidly activated following elevation of cytosolic Ca 2+ concentration ([Ca 2+ ] i ) by treatment of the cells with Ca 2+ ionophore or following inhibition of endosomal Ca 2+ ATPase with thapsigargin. AMPK is further activated by triggering of the T cell antigen receptor (TCR). The present study explored whether AMPK influences Ca 2+ entry and Ca 2+ -sensitive regulation of T-lymphocyte function. Methods: T-lymphocytes were isolated and cultured from AMPKα1-deficient (ampk -/- ) mice and from their wildtype (ampk +/+ ) littermates. The phenotype of the cells was analysed by flow cytometry, [Ca 2+ ] i estimated from Fura-2 fluorescence, SOCE from increase of [Ca 2+ ] i following thapsigargin treatment (1 μM), and cell function analysed by measuring cytokine secretion and western blotting. Results: Expression of surface markers in CD4 + and CD8 + T-cells were similar in ampk -/- and ampk +/+ T-lymphocyte blasts. Moreover, total STIM1 protein abundance was similar in ampk -/- and ampk +/+ T-lymphocyte blasts. However, Orai1 cell membrane protein abundance was significantly higher in ampk -/- than in ampk +/+ T-lymphocyte blasts. SOCE and increase of [Ca 2+ ] i following TCR activation by triggering TCR with anti-CD3 and cross-linking secondary antibody were both significantly more pronounced in ampk -/- than in ampk +/+ T-lymphocyte blasts. The difference of Ca 2+ entry between ampk -/- and ampk +/+ T-lymphocytes was abrogated by Orai1 inhibitor 2-aminoethoxydiphenyl borate (2-APB, 50 μM). Proliferation of unstimulated ampk -/- lymphocytes was higher than proliferation of ampk +/+ T-lymphocytes, a difference reversed by Orai1 silencing. Conclusions: AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca 2+ activity. © 2013 S. Karger AG, Basel.