Research Interest:
Receptor mediated oscillations or sustained increases in cytosolic free calcium are a ubiquitous signaling mechanism in immune cells. The crucial role of calcium in this context is illustrated by the severe immunodeficiencies in patients with genetic defects in molecules involved in Ca2+-homeostasis regulation in immune cells. These signals are generated both by release of calcium from intracellular stores (mainly from the ER) and by entry of extracellular calcium. Calcium entry can be induced by the stimulation of receptors such as the B-cell (BCR) or T-cell receptor (TCR), or by non-receptor mediated events, such as stress due to oxidants or elevated temperatures. Whereas the molecular events leading to the first phase of cytoplasmic calcium elevation through depletion of the intracellular stores are better understood, less is known about the mechanisms underlying calcium entry from the outside. Molecular characterization of multiple ion channel families in the past two decades led to the surprising finding that immune cells, like excitable cells such as neurons, also have a great variety of different ion channels to their disposal.

Our research group is focusing on various aspects of calcium signaling and more generally ion homeostasis regulation in immune cells. To this aim, we use a combinatory approach including genetics, biochemistry, and biophysical characterization of ion entry pathways in lymphocytes as well as cells of the myleoid lineage.

Selected projects:
In an effort to identify new potential ion channel candidates, using a bioinformatic screen, we identified, cloned and completed a primary characterization of several members of a novel family of cation-channel (recently renamed TRPM) expressed in the immune context. Three members of this family show a feature unique among known channels, the presence of an active enzymatic domain, located at their C-terminus (“chanzymes”).

The ADP-ribose and oxidant gated TRPM2 channel in immune cells and lung inflammation - TRPM2 contains a so called NUDIX domain showing ADP-ribose (ADPR) hydrolase activity. Based on this observation, we hypothesized that ADPR might also be involved in channel regulation and could show in patch-clamp experiments that TRPM2 is a calcium permeable channel intracellularly gated by ADPR, a metabolite of NAD-metabolism.

One circumstance under which ADPR might be produced is oxidative stress, as known consequences include NAD depletion and breakdown in the mitochondrial subcompartment, as well as in the nucleus. In experiments using the fluorescent calcium indicator dye Fura-2 to monitor cytoplasmic calcium concentration changes, only HEK293 cells overexpressing TRPM2 show cytoplasmic calcium elevation following oxidative stress through external application of H2O2. These results support the idea of TRPM2 and ADPR as part of a novel signaling pathway directly linking oxidative stress to calcium entry from the extracellular space via the ADPR gating of the TRPM2 channels located in the plasma membrane.

In order to define the biological function of TRPM2, we study the signaling properties of ADPR and related compounds. Research projects include the characterization of ADPR-metabolism and its regulation using biochemical, cellular, molecular and genetic approaches to measure spatial and temporal patterns of ADPR-production in immune cells. Furthermore, we have started extensive structure-function relationship studies of TRPM2, as well as analyses of TRPM2 in vivo expression in immunocytes.
Another project focuses on understanding the role of TRPM2 in the lung. In particular, we would like to investigate the role of TRPM2 in the context of the healthy lung in comparison to the lung of cystic fibrosis or asthma patients.

Mg2+-homeostasis regulation in the immune system - As opposed to calcium the “signaling ion”, magnesium has commonly be described in literature as a “maintenance ion”. Despite the abundance and ubiquitous use of Mg2+ in all forms of life, and the well documented, crucial role of this cation in cellular physiology, extremely little is known about molecular components and mechanisms acting to regulate Mg2+-homeostasis, especially in vertebrates. Although there is still controversy whether cytosolic Mg2+ functions as a second messenger, over the last two decades, a growing number of studies in various cellular systems could demonstrate that Mg2+ homeostasis is a very dynamic process, with multiple and complex forms of regulation that probably plays an underestimated role in managing cell function and metabolism. It appears that TRPM7 is an essential Mg2+ uptake and sensing pathway, and therefore a key-component of these cellular processes.

Like TRPM2, TRPM7 (as well as its closest relative TRPM6) is also a chanzyme, since it is the unique fusion of an ion channel with a kinase domain. We have shown that TRPM7 is a Ca2+/Mg2+ permeable channel whose activity is modulated by intracellular Mg2+ and Mg-nucleotides. Furthermore, the lethal phenotype caused by the inducible homozygous deletion of TRPM7 in the DT40 chicken B-lymphocytes can be totally reverted by supplementing the growth medium with mM concentrations of Mg2+. Amazingly, this phenotype is extremely similar to that of patients suffering from an inherited form of Hypomagnesemia who have been shown to bear mutations in TRPM6, the closest relative of TRPM7.

Ongoing projects in our lab aim to better understand the signaling network(s) underlying cellular Mg2+-homeostasis regulation, with a strong focus on the unique structural feature of TRPM7, its kinase domain. We have constructed a series of cell lines stably and inducibly overexpressing TRPM7 kinase mutants, as well as isolated cytoplasmic TRPM7 domains. We are studying the effects of these mutations on channel function as well as on cellular physiology in the TRPM7-deficient DT40 cells. Our working hypothesis is that the TRPM7 channel and kinase activities are functionally linked, with the kinase modulating channel function, and the channel influencing kinase activity. In turn, the kinase might coordinate intracellular signaling pathways with the Mg2+-status of the cell. We are therefore also interested in identifying potential substrates of the TRPM7 kinase. Furthermore, we are investigating the effect of TRPM7-deficiency and Mg2+-homeostasis deregulation on B-cell receptor signaling.

Selected Publications:

• Schmitz C, Dorovkov MV, Zhao X, Davenport BJ, Ryazanov AG, Perraud AL: The channel kinases TRPM6 and TRPM7 are functionally nonredundant, J Biol Chem 280(45):37763-71, 2005.
• Perraud A.-L., Takanishi CL, Shen B, Kang S, Smith MK, Schmitz C, Knowles HM, Ferraris D, Li W, Zhang J, Stoddard BL, Scharenberg AM.: Accumulation of free ADP-ribose from mitochondria mediates oxidative stress-induced gating of TRPM2 cation channels, J Biol Chem 280(7):6138-48, 2005.
• Perraud AL, Knowles H, Schmitz C: Novel aspects of ion homeostasis regulation in Immunocytes:The TRPM ion channels and their potential role in modulating the immune response, Mol Immunol, 41(6-7):657-73, 2004.
• Schmitz C*, Perraud AL *, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, Scharenberg AM: Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7, Cell 114(2):191-200, 2003.
• Launay P *, Fleig A *, Perraud AL, Scharenberg, AM, Penner R, Kinet JP: TRPM4 Is a Ca2+-Activated Nonselective Cation Channel Mediating Cell Membrane Depolarization, Cell 109(3): 397-407, 2002.
• Perraud AL*, Fleig A*, Dunn CA*, Bagley LA, Launay P, Schmitz C, Stokes A, Zhu Q, Bessman MJ, Penner R, Kinet JP, and Scharenberg AM: ADP-ribose gating of the calcium-permeable LTRPC2 channel revealed by Nudix motif homology, Nature 411(6837): 595-599, 2001.
• Nadler MJS*, Hermosura MC*, Inabe K*, Perraud AL*, Zhu Q, Stokes A, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, and Fleig A: LTRPC7 is a Mg•ATP-regulated divalent cation channel required for cell viability, Nature 411(6837): 590-595, 2001.

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