TRPM6 kinase activity regulates TRPM7 trafficking and inhibits cellular growth under hypomagnesic conditions.

The channel kinases TRPM6 and TRPM7 are both members of the melastatin-related transient receptor potential (TRPM) subfamily of ion channels and the only known fusions of an ion channel pore with a kinase domain. TRPM6 and TRPM7 form functional, tetrameric channel complexes at the plasma membrane by heteromerization. TRPM6 was previously shown to cross-phosphorylate TRPM7 on threonine residues, but not vice versa. Genetic studies demonstrated that TRPM6 and TRPM7 fulfill non-redundant functions and that each channel contributes uniquely to the regulation of Mg(2+) homeostasis. Although there are indications that TRPM6 and TRPM7 can influence each other's cellular distribution and activity, little is known about the functional relationship between these two channel-kinases. In the present study, we examined how TRPM6 kinase activity influences TRPM7 serine phosphorylation, intracellular trafficking, and cell surface expression of TRPM7, as well as Mg(2+)-dependent cellular growth. We found TRPM7 serine phosphorylation via the TRPM6 kinase, but no TRPM6 serine phosphorylation via the TRPM7 kinase. Intracellular trafficking of TRPM7 was altered in HEK-293 epithelial kidney cells and DT40 B cells in the presence of TRPM6 with intact kinase activity, independently of the availability of extracellular Mg(2+), but TRPM6/7 surface labeling experiments indicate comparable levels of the TRPM6/7 channels at the plasma membrane. Furthermore, using a complementation approach in TRPM7-deficient DT40 B-cells, we demonstrated that wild-type TRPM6 inhibited cell growth under hypomagnesic cell culture conditions in cells co-expressing TRPM6 and TRPM7; however, co-expression of a TRPM6 kinase dead mutant had no effect-a similar phenotype was also observed in TRPM6/7 co-expressing HEK-293 cells. Our results provide first clues about how heteromer formation between TRPM6 and TRPM7 influences the biological activity of these ion channels. We show that TRPM6 regulates TRPM7 intracellular trafficking and TRPM7-dependent cell growth. All these effects are dependent upon the presence of an active TRPM6 kinase domain. Dysregulated Mg(2+)-homeostasis causes or exacerbates many pathologies. As TRPM6 and TRPM7 are expressed simultaneously in numerous cell types, understanding how their relationship impacts regulation of Mg(2+)-uptake is thus important knowledge.

The channel-kinase TRPM7, revealing the untold story of Mg(2+) in cellular signaling.

Ion homeostasis dysregulations have severe effects on human health, impairing the effectiveness and appropriateness of major cellular events, including immune responses. The adverse effects of Mg(2+) deficiency on cellular physiology are well known and documented, but mechanistic insights into Mg(2+) sensitive signal transduction are still lacking. TRPM7 and its sister channel TRPM6 stand out as the only known fusions of an ion pore with a Ser/Thr kinase domain. Both channels are permeable to divalent cations and are central regulators of Mg(2+) homeostasis. One crucial aspect of TRPM7 function we have extensively studied is the relationship between its ion channel portion and its C-terminal Ser/Thr kinase domain. The modulation of ion channels by phosphorylation through exogenous kinases is common, however the covalent bound between the TRPM7 channel and its kinase suggests a novel kind of link between ion-entry and signal transduction events. Current knowledge supports a reciprocal "two-way street" model where TRPM7-kinase modulates ion transport function through Ser/Thr phosphorylation, and in turn, channel gating and ionic conditions in close proximity to the pore regulate TRPM7-kinase mediated signaling. We have shown that TRPM7 acts as a sensor of Mg(2+)-availability, adjusting key cellular functions such as the rate of cellular protein translation to the Mg(2+) nutritional status. Since molecular mechanisms controlling rates of protein translation are critical for cell growth and division in response to nutrient availability, this could have relevance for example for therapies targeted at molecules shaping the cancerous translational apparatus. In our quest to understand the biology of Mg(2+) in the context of immune responses, we found that TRPM7 associates with, and phosphorylates phospholipase C gamma 2 (PLCγ2), a pivotal molecule in the signaling pathway following B-cell receptor (BCR) activation. This contributes to the Mg(2+)-dependent modulation of the Ca(2+) response elicited by BCR ligation, and provides the first molecular pathway underlying the Mg(2+)-sensitivity of immune responses. Expanding our knowledge about the modulation of immunoreceptor signaling in response to Mg(2+) availability could allow for the development of unexplored strategies for therapeutic intervention in autoimmune diseases, immunodeficiencies, and lymphoma.

The role of Mg2+ in immune cells.

The physiological and clinical relevance of Mg(2+) has evolved over the last decades. The molecular identification of multiple Mg(2+) transporters (Acdp2, MagT1, Mrs2, Paracellin-1, SLC41A1, SLC41A2, TRPM6 and TRPM7) and their biophysical characterization in recent years has improved our understanding of Mg(2+) homeostasis regulation and has provided a basis for investigating the role of Mg(2+) in the immune system. Deletions and mutations of Mg(2+) transporters produce severe phenotypes with more systemic symptoms than those seen with Ca(2+) channel deletions, which tend to be more specific and less profound. Deficiency of the Mg(2+) permeable ion channels TRPM6 or TRPM7 in mice is lethal at embryonic day 12.5 or at day 6.5, respectively, and, even more surprisingly, chicken DT40 B cells lacking TRPM7 die after 24-48 h. Recent progress made in Mg(2+) research has helped to define underlying mechanisms of two hereditary diseases, human Hypomagnesemia (TRPM6 deletion) and X-chromosomal immunodeficiency (MagT1 deletion), and has revealed a potential new role for Mg(2+) as a second messenger. Future elucidation of human Mg(2+) transporters (Mrs2, SLC41A1, SLC41A2, TRPM7) expressed in immunocytes, beyond MagT1 and TRPM6, will widen our knowledge about the potential role of Mg(2+) in the activation of the immune response.

A-kinase anchoring protein 150 expression in a specific subset of TRPV1- and CaV 1.2-positive nociceptive rat dorsal root ganglion neurons.

Modulation of phosphorylation states of ion channels is a critical step in the development of hyperalgesia during inflammation. Modulatory enhancement of channel activity may increase neuronal excitability and affect downstream targets such as gene transcription. The specificity required for such regulation of ion channels quickly occurs via targeting of protein kinases and phosphatases by the scaffolding A-kinase anchoring protein 79/150 (AKAP79/150). AKAP79/150 has been implicated in inflammatory pain by targeting protein kinase A (PKA) and protein kinase C (PKC) to the transient receptor potential vanilloid 1 (TRPV1) channel in peripheral sensory neurons, thus lowering threshold for activation of the channel by multiple inflammatory reagents. However, the expression pattern of AKAP150 in peripheral sensory neurons is unknown. Here we identify the peripheral neuron subtypes that express AKAP150, the subcellular distribution of AKAP150, and the potential target ion channels in rat dorsal root ganglion (DRG) slices. We found that AKAP150 is expressed predominantly in a subset of small DRG sensory neurons, where it is localized at the plasma membrane of the soma, axon initial segment, and small fibers. Most of these neurons are peripherin positive and produce C fibers, although a small portion produce Aδ fibers. Furthermore, we demonstrate that AKAP79/150 colocalizes with TRPV1 and Ca(V) 1.2 in the soma and axon initial segment. Thus AKAP150 is expressed in small, nociceptive DRG neurons, where it is targeted to membrane regions and where it may play a role in the modulation of ion channel phosphorylation states required for hyperalgesia.