Molecular background of Orai1 accumulation in the immunological synapse

Abstract

Abstract CRAC channels have a vital role in the activation of T lymphocytes, through their calcium influx. CRAC is composed of the ER-membrane resident STIM1 (stromal interaction molecule 1) and the plasma membrane Orai1 pore-forming subunit. Both are accumulated at the interface of the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). CRAC channelopathies due to mutations in either Orai1 or STIM1 are characterized by severe combined immunodeficiency (SCID)-like disease, autoimmunity, muscular hypotonia, and ectodermal dysplasia. The down-regulation of actin-binding protein expression (e.g. HS1), which may be responsible for the immobilization of ion channels, hampers Ca2+-signaling and IS formation of T lymphocytes. We hypothesized that actin-binding proteins modulate the trafficking of the CRAC channels to the IS, which is crucial for the Ca2+-signaling. By means of GST-affinity assay we could show that HS1, which is an actin-regulatory adaptor protein, binds to the N-terminus of the Orai1. N-terminal truncated Orai1 channels (ΔN1: 1–74, ΔN2: 1–88) were created and expressed in Jurkat cells. Then Orai1 channel expressing Jurkat cells were conjugated to CD3/CD28 beads as an APC and Orai1 polarization was determined: the Orai1-ΔN1 mutant similar to the wild-type Orai1 redistributed to the IS but the Orai1-ΔN2 did not. Furthermore, the HS1 protein did not show IS-accumulation in cells expressing the Orai1-ΔN2 subunits unlike for those transfected with the Orai1-ΔN1 or the wild-type constructs. Overall, these data indicate that Orai1-HS1 interaction could contribute to the anchoring of the Orai1 in the IS and can affect the Ca2+-signaling upon T cell activation.