Photopharmacological control of TRPC3/6 channels in T-cells

Changes in calcium within the cell are known to be essential for the proper function of all cells, including immune cells. Calcium channels are a particularly well-suited molecular target for influencing the immune system therapeutically. The clinical benefit of modulation of immune system by targeting ion channels depends on the cell specificity as well as the spatial and temporal precision of the pharmacological intervention. Calcium channels called TRPC (transient receptor potential canonical) are present on the cell surface in special immune cells and in certain disease processes. Recently, in our lab we developed a series of molecules which can activate/inactivate TRPC channels type 3 and 6. These molecules, known as Optobenzimidazole (OptoBI) photoswitches, can be switched on by UV light and switched off by blue light. This results in the possibility of a previously unattained precision of the pharmacological modulation in terms of temporal and spatial limitation. Having this new generation of TRPC activating/inhibiting molecules at hand, we now aim to utilize this novel approach to investigate the function of TRPC3/6 channels in mouse lymphocytes. In the work package 1 we plan to, on the basis of these new photoswitchable molecules, develop a new therapeutic concept. For this purpose, the possibilities for controlling the function of lymphocytes via photopharmacological influencing of TRPC3/6 channels in a mouse model will be investigated. The newly available TRPC3 / 6 activators/inhibitors will be then tested with regard to their possible therapeutic benefit and their application will be further developed. Our second aim (work package 2) is to, by using the tools developed in the first package, pinpoint specific lymphocyte populations in which TRPC3/6 channels play significant role. Cells will be isolated from the spleen of mice which are genetically engineered to express different levels of TRPC channels. Calcium signals related to TRPC channels will be analyzed in detail. The third aim of this proposal (work package 3) is to test TRPC3/6 photoswitches in vivo in a murine psoriasis model of skin inflammation. In the end of this project, we expect to better understand calcium signals within the highly complex immunological network. Identification of so far not known signaling pathways in lymphocytes by using photoswitchable molecules could provide new therapeutic targets and drug candidates in immune diseases.

Calcium signaling is a central regulator of immune cell behavior and plays a decisive role in inflammation and immunemediated diseases. Targeting calcium channels therefore represents an attractive strategy for modulating immune responses with high precision. Among these channels, transient receptor potential canonical (TRPC) channels, in particular TRPC3 and TRPC6, have emerged as important but still incompletely understood players in immune regulation. This project combined advanced calcium signaling approaches with in vivo disease modeling to explore the role of TRPC3 and TRPC6 channels in inflammation. Using a murine model of psoriasislike skin inflammation, we examined how the absence of TRPC3 or TRPC6 affects immune cell responses in a diseaserelevant context. Analysis of spleenderived immune cells revealed that defined immune cell populations are altered in TRPC3 and TRPC6deficient mice during inflammatory conditions, pointing to a functional role of these channels in shaping systemic immune responses. In parallel, the project generated detailed mechanistic insight into TRPC3dependent calcium signaling. Using innovative, genetically encoded calcium indicators targeted directly to TRPC3, we characterized highly localized calcium signals at the channel microenvironment. The data demonstrate that TRPC3 operates within dynamic signaling microdomains in close functional interaction with inositol 1,4,5trisphosphate receptors (IP3Rs). Calcium entry through TRPC3 was shown to influence IP3Rmediated calcium release, thereby finetuning the strength, timing, and pattern of intracellular calcium signals. This work provides a mechanistic basis for how TRPC3 channels can confer specificity to calciumdependent immune signaling pathways. Overall, this project bridges molecular calcium signaling mechanisms with immune regulation in an in vivo inflammatory model. By linking channellevel signaling events to immune cell behavior in disease, the work establishes a strong foundation for future efforts aimed at selectively targeting TRPC channels to modulate immune responses with high spatial and temporal precision.