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Molecular mechanism of calcium entry in the immune system

Final Report Summary - CAV IN IMMUNE SYSTEM (Molecular mechanism of calcium entry in the immune system)

Calcium signaling is an important step required for the various functions in virtually all the cells. In non-excitable cells (such as, T lymphocytes of the immune system) calcium is mostly required for gene expression. Although calcium is stored within specific intracellular organelles, the majority of calcium is acquired from the extracellular space from where it is transported through plasma membrane channels. In the past 10 years, efforts have been made towards identifying the genetic identity and mechanism of function of these plasma membrane channels.
The activation mechanism of calcium entry through plasma membrane channels in non-excitable cells, requires calcium release from intracellular stores at first hence these channels are generally called store-operated calcium (SOC) channels. In recent years, it became increasingly clear that store operated calcium entry (SOCE) is a multichannel and multi-step process, however the identity of all those plasma membrane channels involved was only recently discovered. Three types of calcium channels were described so far to be involved in calcium entry after TCR stimulation of T cells: ORAI, TrpV1 and IP3R.
During the course of our studies, we identified novel calcium channels, L type calcium channels (Cav1.1) α1S subunits, which we showed to be involved in calcium entry in response to T cell stimulation during the immune response. We also found that a giant scaffold protein, called AHNAK1, regulates their function during the immune response.

The objectives of the proposed research were to study the requirement for Cav1.1/AHNAK1 pathway during the immune response (proliferation, migration and cytokine production) as well as their mechanism of function.

Initially we investigated the involvement of the AHNAK1 in the response of T cells to chemokine stimulation, specifically Stromal Cell-Derived Factor-1 (SDF-1). We found that both chemokine stimulation, as well as TCR triggering, increased AHNAK1 protein expression in T cells. In addition, we showed that AHNAK1 is located in closely packed vesicles in naïve T cells and that short term stimulation via TCR or SDF-1 caused AHNAK1 to relocate to the membrane of the cells. Lastly, reduction of AHNAK1 protein expression in Jurkat T cells significantly reduced cell migration to SDF-1. Our results suggest that the AHNAK1 protein is essential for calcium entry after SDF-1 stimulation. Our studies suggest that AHNAK1 and Cav1.1 are used for calcium entry during activation, adhesion and migration of T cells.

Next, we studied the requirement for Cav1.1 channels during calcium entry after TCR stimulation. High expression levels of Cav1.1 channels were detected in activated T cells. Sequencing and cloning of Cav1.1 channel cDNA from T cells revealed that a single splice variant is expressed. This variant lacks exon 29, which encodes the linker region adjacent to the voltage sensor, but contains five new N-terminal exons that substitute for exons 1 and 2, which are found in the Cav1.1 muscle counterpart. Over expression studies using cloned T cell Cav1.1 in 293HEK cells, suggest that voltage sensing no longer controls the gating of these channels in T cells. Knockdown of Cav1.1 channels, in T cells abrogated calcium entry after TCR stimulation, suggesting that Cav1.1 channels are controlled by TCR signaling and that their underlying gating properties have been altered.
The results presented above are summarized in two manuscripts. The first manuscript about AHNAK1 is in preparation, and the second manuscript about Cav1.1 is currently under review.