The B cell receptor-induced calcium flux involves a calcium mediated positive feedback loop

The B cell receptor (BCR)-elicited calcium flux results in activation of mature B cells. We have recently shown that the adaptor protein Swiprosin-1/EFhd2 (EFhd2) amplifies the BCR-induced calcium flux in B cell lines. EFhd2 is a calcium binding adaptor protein with two predicted EF-hands. Here we asked whether these domains are functional and control its function. Using a blot-overlay assay with radioactive calcium we show that both EF-hands of EFhd2 have an intrinsic capacity to bind calcium. Equilibrium centrifugation confirmed that EFhd2 binds 2 calcium ions, with an apparent Kd of 110 μM. Point mutations revealed that the conserved residues E116 and E152, which reside in the canonical calcium binding loop in EF-hands 1 and 2, are essential for calcium binding by EFhd2. These mutations as well as deletion of the EF-hands, in particular EF-hand 1, abolished the ability of EFhd2 to restore BCR-induced calcium signaling in EFhd2-deficient WEHI231 cells. N-terminal deletions, but not C-terminal deletions, acted similarly. Thus, the N-terminal part of EFhd2 as well as calcium binding to its EF-hands control the intracellular calcium concentration in response to BCR stimulation in WEHI231 cells. Hence, EFhd2 regulates the BCR-elicited calcium flux through a calcium-dependent positive feedback mechanism in WEHI231 cells.