AMPKα1-sensitivity of Orai1 and Ca(2+) entry in T - lymphocytes

• Published in: Cellular physiology and biochemistry Vol. 32; no. 3; pp. 687 - 698
• Main Authors: Bhavsar, Shefalee K, Schmidt, Sebastian, Bobbala, Diwakar, Nurbaeva, Meerim K, Hosseinzadeh, Zohreh, Merches, Katja, Fajol, Abul, Wilmes, Jan, Lang, Florian
• Format: Journal Article
• Language: English
• Published: Germany 2013
• Subjects: AMP-Activated Protein Kinases - deficiency; AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; Animals; Antibodies - immunology; Boron Compounds - pharmacology; Calcium - metabolism; Calcium Channels - chemistry; Calcium Channels - genetics; Calcium Channels - metabolism; CD3 Complex - immunology; CD3 Complex - metabolism; CD4-Positive T-Lymphocytes - drug effects; CD4-Positive T-Lymphocytes - immunology; CD4-Positive T-Lymphocytes - metabolism; CD8-Positive T-Lymphocytes - drug effects; CD8-Positive T-Lymphocytes - immunology; CD8-Positive T-Lymphocytes - metabolism; Cell Proliferation; Cells, Cultured; Fura-2 - chemistry; Membrane Glycoproteins - antagonists & inhibitors; Membrane Glycoproteins - genetics; Membrane Glycoproteins - metabolism; Mice; Mice, Knockout; ORAI1 Protein; Receptors, Antigen, T-Cell - metabolism; RNA Interference; RNA, Small Interfering - metabolism; Stromal Interaction Molecule 1; Thapsigargin - pharmacology
• ISSN: 1421-9778
• DOI: 10.1159/000354472

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.
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.
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.
AMPK downregulates Orai1 and thus SOCE in T-lymphocytes and thus participates in negative feed-back regulation of cytosolic Ca(2+) activity.