Anoctamin 9 determines Ca²⁺ signals during activation of T-lymphocytes

Activation of T-cells is initiated by an increase in intracellular Ca²⁺, which underlies positive and negative regulation. Because the phospholipid scramblase and ion channel ANO9 (TMEM16J) was shown previously to regulated Ca²⁺ signals in renal epithelial cells, we asked whether ANO9 demonstrates a similar regulation in T-cells.

We used measurements of the intracellular Ca²⁺ concentration to examine the effects of ANO9 on intracellular Ca²⁺ signaling and demonstrated expression of ANO9 and its effects on cellular and molecular parameters.

ANO9 was found to be expressed in human lymphocytes, including the Jurkat T-lymphocyte cell line and mouse lymphocytes. ANO9 has been shown to affect intracellular Ca²⁺ signals in renal epithelial cells. Here we demonstrate the essential role of ANO9 during initiation of intracellular Ca²⁺ signals in Jurkat T-cells and isolated mouse lymphocytes. ANO9 is essential for the initial rise in intracellular Ca²⁺ due to influx of extracellular Ca²⁺ through store-operated ORAI1 Ca²⁺ entry channels. ANO9 is indispensable for T-cell function, independent on whether cells are activated by stimulation of the T-cell receptor with CD3-antibody or by PMA/phytohemagglutinin.

Upon activation of T-cells and formation of the immunological synapse, ANO9 recruits the Ca²⁺-ATPase (PMCA) to the plasma membrane, which is supported by the scaffolding protein discs large 1 (DLG1). PMCAs maintain low Ca²⁺ levels near ORAI1 channels thereby suppressing Ca²⁺-inhibition of ORAI1 and thus retaining store-operated Ca²⁺ entry (SOCE). It is suggested that ANO9 has a role in interorganelle communication and regulation of cellular protein trafficking, which probably requires its phospholipid scramblase function.