Effects of TRPM7 Kinase Inactivation in Macrophages
Transient Receptor Potential Melastatin 7 (TRPM7) is a unique protein possessing ion channel and serine/threonine kinase domains and functionality. Our earlier studies demonstrated that it is highly expressed in immune cells, such as T lymphocytes and macrophages. Deletion of full TRPM7 or the kinase domain is embryonic lethal in mice. Inactivating the kinase by introducing a kinase-dead (KD) point mutation, however, results in viable animals. We previously characterized KD mutant mice and found that their T cells had a defect in proliferation and reduced store-operated calcium entry (SOCE). KD mice displayed moderate splenomegaly and ectopic hematopoiesis. In the present study, we have investigated the consequences of global TRPM7 kinase inactivation in macrophages. TRPM7 channel activity was measured by patch-clamp electrophysiology and found to be normal in KD mouse macrophages. Specific cell populations were characterized by flow cytometry. Macrophages isolated from KD mice expressed F4/80, a marker found on splenic red pulp macrophages, high levels of leukocyte adhesion and migration marker CD11b, and C-type lectin receptor marker CD209b. KD mouse macrophages also expressed high levels of the marginal metallophilic marker CD169. The higher expression levels of these markers may indicate that KD macrophages have altered phagocytic properties. Therefore, phagocytosis was quantified using latex beads, fluorescent bioparticles and opsonized red blood cells and compared to WT mouse cells. Since calcium signaling is thought to be important for Fc-receptor-mediated phagocytosis we investigated intracellular Ca 2+ levels in macrophages isolated from WT and KD mice. Calcium entry was measured using the ratiometric dye FURA-2 and found to be smaller than in T cells. Macrophages isolated from KD mice showed no significant change in calcium entry magnitude compared to their WT littermates. We are currently investigating the relationship between calcium entry and phagocytic function in macrophages.
& Kozak, J. A.
(2020). Effects of TRPM7 Kinase Inactivation in Macrophages. Biophysical Journal, 118 (3), 415A, Supplement 1.