Doctor of Philosophy (PhD), Wright State University, 2023, Biomedical Sciences PhD

Transient receptor melastatin 7 (TRPM7) functions both as an ion channel and a protein kinase. TRPM7 has been implicated in Mg2+ homeostasis, embryogenesis, cardiac automaticity, and immunity. The purpose of this research was to deepen our understanding of TRPM7 channel and kinase functions. To this end, we used two transgenic mouse models: the TRPM7 gain-of-function (GOF) and TRPM7 kinase-dead (KD) mice to address the consequences of increased channel activity and kinase inactivation, respectively. Global deletion of TRPM7 or the kinase domain alone are embryonic lethal, therefore, we used the TRPM7 GOF mouse to investigate germline transmission. We examined embryo development stages that follow placenta formation. We found that the GOF point mutation is post-placental lethal, and embryos at the specified developmental stages undergo intrauterine growth restriction (IUGR). The heterozygous GOF mice were viable. IUGR affects the development of organs such as the heart, brain and intestines. To ascertain the consequences of IUGR in our heterozygous GOF mice, we examined their body composition and behavior. We also examined how dysregulation of this channel is affected under hypomagnesemic conditions. Hypomagnesemic GOF mice were smaller than WT littermates and had lower lean mass. Low Mg2+ diet for at least two weeks reduced serum levels of Mg2+ roughly by half, and decreased viability of both WT and GOF mice. GOF and WT mice behaved similarly in working memory and anxiety tests, suggesting that brain development was not grossly impaired. TRPM7 protein is highly expressed in immune cells, where it was first identified, and we used the TRPM7 GOF and KD mouse models to address TRPM7 channel function in splenic macrophages. We measured the basal phagocytic activity of splenic macrophages in vitro. The phagocytic activity in KD mice was potentiated, pointing to suppressive effect of the kinase. Finally, we examined the roles of Ca2+ and Mg2+ in phagocytosis. We found tha (open full item for complete abstract)

Committee: J. Ashot Kozak Ph.D. (Advisor); Lucille E. Wrenshall M.D., Ph.D. (Committee Member); Hongmei Ren Ph.D. (Committee Member); David Cool Ph.D. (Committee Member); Weiwen Long Ph.D. (Committee Member) Subjects: Biology; Biomedical Research; Biophysics; Immunology; Physiology

Master of Science (MS), Wright State University, 2020, Physiology and Neuroscience

Humans are exposed daily to a variety of metals that can be harmful to our immune system. Although certain divalent metal cations are essential for numerous cellular functions and are critical trace elements in humans, the uptake mechanisms of these ions remain mostly unknown. Transient receptor potential melastatin 7 (TRPM7), which is expressed in a variety of human cell types, including lymphocytes and macrophages, conducts many divalent metal cations. TRPM7 channels are largely inactive under normal physiological conditions due to cytoplasmic magnesium acting as a channel inhibitor. Magnesium is a cofactor for many biochemical reactions. Low serum levels of magnesium, hypomagnesemia, can occur from increased magnesium loss from renal or gastrointestinal systems, redistribution of magnesium across the cell membranes, and decreased magnesium intake. Magnesium depletion allows both physiological and non-physiological divalent metal cations to enter through TRPM7, which is highly expressed in T-lymphocytes. Alterations to TRPM7 channel activity by channel blockers were found to affect the cell viability sequence. Through the use of Jurkat, a leukemic T-lymphocyte cell line which expresses high levels of TRPM7, HAP1 cells, and a TRPM7 kinase-dead mouse model, the entry of both physiological and non-physiological cations can be quantitated by measuring cell toxicity. A cell toxicity/viability assessment in Jurkat T-lymphocytes provided the sequence of Cd2+ > Zn2+ > Co2+ > Ni2+ > Mn2+ >> Sr2+ ≈ Ba2+ ≈ Ca2+ ≈ Mg2. Homeostatic mechanisms alter the effects of divalent metal cation entry and viability of T-lymphocytes, suggesting that TRPM7 in part contributes to metal ion entry.

Committee: Juliusz Ashot Kozak Ph.D. (Advisor); Christopher N. Wyatt Ph.D. (Committee Member); David R. Ladle Ph.D. (Committee Member) Subjects: Cellular Biology; Immunology; Pharmacology; Physiology

Master of Science (MS), Wright State University, 2019, Physiology and Neuroscience

Magnesium is an important divalent metal cation that is involved in numerous cellular functions. The details of cellular Mg2+ regulation, homeostasis and transport remain unclear. Magnesium transporter protein (MagT1) is a Mg2+ transporter and deficiency of this protein has been reported to lead to impaired Mg2+ influx and a decreased cytoplasmic [Mg2+]. Transient receptor potential melastatin 7 (TRPM7) is a ubiquitously expressed membrane protein containing a channel pore and a C-terminal alpha-type serine/threonine protein kinase domain. Importantly, TRPM7 channel is believed to conduct both Mg2+ and Ca2+. In the present study, we investigated if TRPM7 can be used as a bioassay of internal and external Mg2+ in Jurkat T cells. We have investigated the long term effects of Mg2+ changes on TRPM7 channel activity. Under physiological conditions, cytoplasmic Mg2+ concentrations of 0.1 – 0.3 mM are sufficient to inhibit the majority of TRPM7 channels. Extracellular Mg2+ blocks inward TRPM7 currents carried by monovalent cations. When the cytoplasmic Mg2+ concentration is reduced, TRPM7 channels open and produce an outwardly-rectifying current. We find that the extent of TRPM7 current activation can be used effectively to estimate changes of cytoplasmic Mg2+ concentration. Our findings can be extended to cell types other than Jurkat T cells.

Committee: J. Ashot Kozak Ph.D. (Advisor); Mauricio Di Fulvio Ph.D. (Committee Member); Kathrin L. Engisch Ph.D. (Committee Member) Subjects: Biology; Cellular Biology; Neurosciences; Physiology

Doctor of Philosophy (PhD), Wright State University, 2018, Biomedical Sciences PhD

The characteristic feature of ubiquitously expressed transient receptor potential melastatin 7 (TRPM7) protein is the presence of a C-terminal atypical kinase domain fused to a cation channel domain. Even though this protein has been implicated in a number of physiological and pathological processes, the mechanisms regulating TRPM7 channel activity are poorly understood. In the present study, regulation of TRPM7 channel by its kinase activity was investigated using the TRPM7 kinase-dead knock-in mouse (KD mouse). These mice have a point mutation Lys1646Arg in the kinase region abrogating the kinase activity. Examining TRPM7 channel activity in KD peritoneal macrophages revealed that TRPM7 kinase activity is not required for channel activity yet may reduce the number of channels functioning in an intact cell. Thus, kinase activity is dispensable for the formation of functional TRPM7 channels, in agreement with previous studies done in overexpression systems. T lymphocytes isolated from KD mice showed reduced blastogenesis, proliferation and cytokine expression upon activation, potentially due to defective up-regulation of Ca2+ and Mg2+ dependent pathways. The up-regulation of the primary calcium entry pathway in activated T cells, the store-operated calcium entry, and calcium elevations in response to T-cell receptor ligation were impaired in KD T cells. TRPM7 protein levels were increased in both WT and KD mouse T cells upon activation. Additionally, KD mice exhibited splenomegaly and increased extramedullary hematopoiesis, along with a modest pre-activation of T cells. The present work provides insights into the physiological role of TRPM7 kinase in regulating its channel activity and immune cell function.

Committee: J. Ashot Kozak Ph.D. (Advisor); Gerald M. Alter Ph.D. (Committee Member); Dan R. Halm Ph.D. (Committee Member); F. Javier Alvarez-Leefmans Ph.D. (Committee Member); Kathrin Engisch Ph.D. (Committee Member) Subjects: Immunology; Physiology