Pivotal role of mitochondrial Na+-Ca2+ exchange in antigen receptor mediated Ca2+ signalling in DT40 and A20 B lymphocytes

Cytoplasmic Ca2+ concentration ([Ca2+](i)) increases upon activation of antigen-receptor in lymphocytes. Mitochondria have been suggested to regulate the [Ca2+](i) response, but the molecular mechanisms and the roles are poorly understood. To clarify them, we carried out a combination study of mathematical simulations and knockout or knockdown of NCLX, a gene candidate for themitochondrial Na+-Ca2+ exchanger (NCXmit), in B lymphocytes. A mathematical model of Ca2+ dynamics in B lymphocytes demonstrated that NCXmit inhibition reduces basal Ca2+ content of endoplasmic reticulum (ER) and suppresses B-cell antigen receptor (BCR)-mediated [Ca2+](i) rise. The predictions were validated in DT40 B lymphocytes of heterozygous NCLX knockout (NCLX+/-). In NCLX+/- cells, mitochondrial Ca2+ efflux via NCXmit was strongly decelerated, suggesting NCLX is a gene responsible for NCXmit in B lymphocytes. Consistent with the predictions, ER Ca2+ content declined and [Ca2+](i) hardly rose upon BCR activation in NCLX+/- cells. ER Ca2+ uptake was reduced to similar to 58% of the wild-type (WT), while it was comparable toWT whenmitochondrial respiration was disturbed. Essentially the same results were obtained by a pharmacological inhibition or knockdown of NCLX by siRNA in A20 B lymphocytes. Unexpectedly, ER Ca2+ leak was augmented and co-localization of mitochondria with ER was lower in NCLX+/- and NCLX silenced cells. Taken together, we concluded that NCLX is a key Ca2+ provider to ER, and that NCLX-mediated Ca2+ recycling between mitochondria and ER is pivotal in B cell responses to antigen.