A key role for Mg2+ in TRPM7's control of ROS levels during cell stress

TRPM7 is one of only two vertebrate ion channels to contain both ion channel and kinase domains [1, 2]. The other bifunctional channel is TRPM6, mutations in which cause familial hypomagnesemia with secondary hypocalcemia (HSH), an autosomal-recessive disease characterized by low serum Mg2+ accompanied by hypocalcemia [3, 4]. Whereas TRPM6 is expressed mainly in the kidney and the intestine [3, 5], TRPM7 is expressed in nearly all cell types [1, 2]. Evidence points to a pivotal role for TRPM7 in cellular Mg2+ homeostasis. Scharenberg and colleagues revealed that TRPM7-deficient DT40 B-cells exhibited Mg2+ deficiency and growth arrest [6, 7]. Supplementation of growth medium with extracellular Mg2+ or expression of the Mg2+-transporter SLC41A2 (solute carrier family 41 member 2) in TRPM7-deficient cells restored the ability of TRPM7-deficient DT40 cells to proliferate [8]. In mice, homozygous deletion of TRPM7 resulted in early embryonic lethality [9]. Early developmental arrest caused by loss of the channel-kinase in mice appears to be related to the channel’s ability to permeate Mg2+, as depletion of TRPM7 in Xenopus laevis embryos produced a disruption in convergent-extension cell movements during gastrulation that could be prevented by Mg2+ supplementation as well as by expression of the Mg2+-transporter SLC41A2 [10]. Later in development, other physiological functions have been ascribed to the channel-kinase, including skeletogenesis and melanophore maturation, kidney and pancreatic development, synaptic vesicle fusion, and thymopoiesis [9, 11–15]. The pleiotropic phenotypes caused by loss of the channel-kinase is likely due to TRPM7’s bifunctional nature as well as to the channel’s ability to permeate multiple species of divalent cations [16]. The best illustration of this comes from studies of the channel’s role in cell death. TRPM7 appears to be playing a major role in a cell’s response to cell stress. The first and perhaps most striking example of the channel’s influence on this process comes from the collective work by Tymianski, MacDonald and colleagues [17–19]. Their studies revealed that TRPM7 constitutes a Ca2+-permeable nonselective cation conductance (IOGD) that becomes activated by reactive oxygen/nitrogen species to promote Ca2+ overload and anoxic death in cultured cortical neurons subjected to oxygen glucose deprivation (OGD) [17]. Suppressing TRPM7 expression using small interfering RNA (siRNA) reduced the ischemia-induced current, decreased Ca2+ uptake and increased cell viability [17]. Using intrahippocampal injections of adeno-associated viral vectors packaged with short hairpin RNA specific for TRPM7, a subsequent study by Sun and colleagues provided in vivo evidence that regional TRPM7 suppression provides a comparable level of protection against brain ischemia [18]. Importantly, depletion of the channel had no negative effect on animal survival, dendritic morphology, neuronal excitability or synaptic plasticity [18]. In addition to its contribution to Ca2+ overload during OGD, TRPM7 is also required for Zn2+-induced neuronal cell death, indicating that permeation of Ca2+ and Zn2+ both contribute to the TRPM7 channel’s ability to mediate cell death in neurons [20]. More recently, knockdown of TRPM7 in hippocampal neurons has been shown to reduce the increase in intracellular Mg2+ levels detected following OGD, suggesting that conduction of Mg2+ by the channel during ischemia may also be contributing to neuronal cell death [21]. Consistent with the notion that conduction of multiple ions are involved in TRPM7’s ability to mediate cell death, overexpression of TRPM7 in human embryonic kidney (HEK-293) cells increased Mg2+ and Ca2+ influx, which led to increased production of reactive oxygen species (ROS) and nitric oxide (NO) production [22]. The resultant oxidative stress caused by overexpression of the channel in turn activated the stress-activated protein kinases p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), which caused loss of cell adhesion and increased cell death [22, 23]. Conversely, depletion of TRPM7 in HEK-293 cells was protective against many forms of cell stress, including the apoptosis inducer doxorubicin, translation inhibitor cycloheximide, and broad kinase inhibitor staurosporine [23]. To further uncover how TRPM7 affects the cellular response to stress we have employed a stable TRPM7-knockdown Swiss 3T3 fibroblast line (M7shRNA6 cells), which we previously used to investigate the mechanisms by which TRPM7 controls cell motility [24]. M7shRNA6 cells exhibit defects in the ability to form lamellipodia and migrate directionally, which can be rescued by re-expression of TRPM7 as well as by expression of the Mg2+ transporter SLC41A2 [24]. In the present study, we show that depletion of TRPM7 from fibroblasts lowered intracellular Mg2+, rendered cells more resistant to apoptotic stimuli and lowered the concentration of cellular ROS, which can all be reversed by re-expression of TRPM7, as well as by expression of the Mg2+ transporter SLC41A2. These results indicate that Mg2+ is not only playing a key role in TRPM7’s ability to control cell survival, but is also critical to the regulation of cellular ROS levels, both in the absence and presence of cell stress.