Ca^sup 2+^ regulates T-cell receptor activation by modulating the charge property of lipids

Ionic protein-lipid interactions are critical for the structure and function of membrane receptors, ion channels, integrins and many other proteins1-7. However, the regulatory mechanism of these interactions is largely unknown. Here we show that Ca^sup 2+^ can bind directly to anionic phospholipids and thus modulate membrane protein function. The activation of T-cell antigen receptor-CD3 complex (TCR), a key membrane receptor for adaptive immunity, is regulated by ionic interactions between positively chargedCD3ε/ζ cytoplasmic domains (CD3^sub CD^) and negatively charged phospholipids in the plasma membrane1,8-10. Crucial tyrosines are buried in the membrane and are largely protected from phosphorylation in resting T cells. It is not clear how CD3^sub CD^ dissociates from the membrane in antigen-stimulated T cells. The antigen engagement of even a single TCR triggers a Ca^sup 2+^ influx11 and TCR-proximal Ca^sup 2+^ concentration is higher than the average cytosolic Ca^sup 2+^ concentration12. Our biochemical, live-cell fluorescence resonance energy transfer and NMR experiments showed that an increase in Ca^sup 2+^ concentration induced the dissociation of CD3^sub CD^ from the membrane and the solvent exposure of tyrosine residues. As a consequence, CD3 tyrosine phosphorylation was significantly enhanced by Ca^sup 2+^ influx. Moreover, when compared with wild-type cells, Ca^sup 2+^ channel-deficient T cells had substantially lower levels of CD3 phosphorylation after stimulation. The effect of Ca^sup 2+^ on facilitating CD3 phosphorylation is primarily due to the charge of this ion, as demonstrated by the fact that replacing Ca^sup 2+^with the non-physiological ion Sr^sup 2+^ resulted in the same feedback effect. Finally, ^sup 31^P NMR spectroscopy showed that Ca^sup 2+^ bound to the phosphate group in anionic phospholipids at physiological concentrations, thus neutralizing the negative charge of phospholipids. Rather than initiating CD3 phosphorylation, this regulatory pathway of Ca^sup 2+^ has a positive feedback effect on amplifying and sustaining CD3 phosphorylation and should enhance T-cell sensitivity to foreign antigens. Our study thus provides a new regulatory mechanism of Ca^sup 2+^ to T-cell activation involving direct lipid manipulation. [PUBLICATION ABSTRACT]