Roles of ion channels in immune cells

The Editorial on the Research Topic - Roles of Ion Channels in Immune Cell

Ion channel expression in human melanoma samples. in silico identification and experimental validation of molecular targets

Expression of 328 ion channel genes was investigated, by in silico analysis, in 170 human melanoma samples and controls. Ninety-one members of this gene-family (i.e., about 28%) show a significant (p 0.90 and p 90% in most cases). Such five genes (namely, SCNN1A, GJB3, KCNK7, GJB1, KCNN2) are novel potential melanoma markers or molecular targets, never previously related to melanoma. The “druggable genome” analysis was then carried out. Miconazole, an antifungal drug commonly used in clinics, is known to target KCNN2, the best candidate among the five identified genes. Miconazole was then tested in vitro in proliferation assays; it dose-dependently inhibited proliferation up to 90% and potently induced cell-death in A-375 and SKMEL-28 melanoma cells, while it showed no effect in control cells. Moreover, specific silencing of KCNN2 ion channel was achieved by siRNA transfection; under such condition miconazole strongly increases its anti-proliferative effect. In conclusion, the present study identified five ion channels that can potentially serve as sensitive and specific markers in human melanoma specimens and demonstrates that the antifungal drug miconazole, known to target one of the five identified ion channels, exerts strong and specific anti-melanoma effects in vitro

Ruling out pyridine dinucleotides as true TRPM2 channel activators reveals novel direct agonist ADP-ribose-2'-phosphate

Transient receptor potential melastatin 2 (TRPM2), a Ca(2+)-permeable cation channel implicated in postischemic neuronal cell death, leukocyte activation, and insulin secretion, is activated by intracellular ADP ribose (ADPR). In addition, the pyridine dinucleotides nicotinamide-adenine-dinucleotide (NAD), nicotinic acid-adenine-dinucleotide (NAAD), and NAAD-2'-phosphate (NAADP) have been shown to activate TRPM2, or to enhance its activation by ADPR, when dialyzed into cells. The precise subset of nucleotides that act directly on the TRPM2 protein, however, is unknown. Here, we use a heterologously expressed, affinity-purified-specific ADPR hydrolase to purify commercial preparations of pyridine dinucleotides from substantial contaminations by ADPR or ADPR-2'-phosphate (ADPRP). Direct application of purified NAD, NAAD, or NAADP to the cytosolic face of TRPM2 channels in inside-out patches demonstrated that none of them stimulates gating, or affects channel activation by ADPR, indicating that none of these dinucleotides directly binds to TRPM2. Instead, our experiments identify for the first time ADPRP as a true direct TRPM2 agonist of potential biological interest

Multiple molecular mechanisms form a positive feedback loop driving amyloid β42 peptide-induced neurotoxicity via activation of the TRPM2 channel in hippocampal neurons

Emerging evidence supports an important role for the ROS-sensitive TRPM2 channel in mediating age-related cognitive impairment in Alzheimer’s disease (AD), particularly neurotoxicity resulting from generation of excessive neurotoxic Aβ peptides. Here we examined the elusive mechanisms by which Aβ₄₂ activates the TRPM2 channel to induce neurotoxicity in mouse hippocampal neurons. Aβ₄₂-induced neurotoxicity was ablated by genetic knockout (TRPM2-KO) and attenuated by inhibition of the TRPM2 channel activity or activation through PARP-1. Aβ₄₂-induced neurotoxicity was also inhibited by treatment with TPEN used as a Zn²⁺-specific chelator. Cell imaging revealed that Aβ₄₂-induced lysosomal dysfunction, cytosolic Zn²⁺ increase, mitochondrial Zn²⁺ accumulation, loss of mitochondrial function, and mitochondrial generation of ROS. These effects were suppressed by TRPM2-KO, inhibition of TRPM2 or PARP-1, or treatment with TPEN. Bafilomycin-induced lysosomal dysfunction also resulted in TRPM2-dependent cytosolic Zn²⁺ increase, mitochondrial Zn²⁺ accumulation, and mitochondrial generation of ROS, supporting that lysosomal dysfunction and accompanying Zn²⁺ release trigger mitochondrial Zn²⁺ accumulation and generation of ROS. Aβ₄₂-induced effects on lysosomal and mitochondrial functions besides neurotoxicity were also suppressed by inhibition of PKC and NOX. Furthermore, Aβ₄₂-induced neurotoxicity was prevented by inhibition of MEK/ERK. Therefore, our study reveals multiple molecular mechanisms, including PKC/NOX-mediated generation of ROS, activation of MEK/ERK and PARP-1, lysosomal dysfunction and Zn²⁺ release, mitochondrial Zn²⁺ accumulation, loss of mitochondrial function, and mitochondrial generation of ROS, are critically engaged in forming a positive feedback loop that drives Aβ₄₂-induced activation of the TRPM2 channel and neurotoxicity in hippocampal neurons. These findings shed novel and mechanistic insights into AD pathogenesis

Az ischaemiás agykárosodásban szerepet játszó TrpM2 kationcsatorna szerkezet-funkció vizsgálata = Structure-function studies of the TrpM2 cation channel involved in ischaemic brain damage.

Megállapítottuk, hogy a csatornát 4 Ca2+ ion kötődése aktiválja. Az aktiváció a Monod-Wymann-Changeux mechanizmust követi, 1 Ca2+ kötődése ~33-szorosra, a 4 ion összesen ~10^6-szorosra, növeli a nyitott-csukott egyensúlyi állandót. A Ca2+ kötőhelyei a kaputól intracelluláris irányban találhatók egy védett üregben, közel a pórus nyílásához. A nyitott póruson beáramló Ca2+ telítésben tartja az aktiváló helyeket, ezért intakt sejtekben, ADPR jelenlétében, egy rövid Ca2+ szignál is elnyújtott TRPM2 aktivitást válthat ki. Megállapítottuk, hogy az ADPR nagy affinitással (K1/2=1uM) aktivál, de az ADPR-hidrolízisnek a csatorna csukódásában játszott szerepét nem tudtuk tisztázni. Az ADPRázok konzervált ""Nudix-box"" motívuma (REFXEE) a TRPM2 NUDT9-H doménjében atípusos (RILRQE). A NUDT9 enzimben az EF->IL mutáció 1%-ára csökkenti, az EE->KK mutáció felfüggeszti az ADPRáz aktivitást. Létrehoztunk egy ""inaktív"" QE->KK és egy ""hiperaktív"" IL->EF TRPM2 mutánst, de e 4 egyedi és 2 dupla mutáció egyike sem befolyásolta az ADPR iránti affinitást illetve a csukódási sebességet. Intakt sejtekben az AMP gátolta, míg a H2O2, a ciklikus ADPR (cADPR), és a nikotinsav-adenin-dinukleotid-foszfát (NAADP) aktiválták a TRPM2-t, és fokozták ADPR iránti érzékenységét. Izolált membrán patch-ben megállapítottuk, hogy a H2O2, az AMP, és a cADPR közvetlenül nem hatnak a TRPM2-re, míg az NAADP és az NAAD kis affinitású parciális agonisták. Tehát intakt sejtekben e modulátorok hatásai közvetettek. | We have revealed that the channel is activated by binding of 4 Ca2+ ions, following the Monod-Wymann-Changeux mechanism. Binding of 1 Ca2+ increases the closed-open equilibrium constant by ~33-fold, the 4 ions altogether by ~10^6-fold. The Ca2+ binding sites are found intracellularly of the gate, in a protected crevice, near the pore entrance, and are kept saturated by Ca2+ flowing through the open pore. Thus, in intact cells, in the presence of ADPR, a single brief Ca2+ spark can elicit prolonged TRPM2 channel activity. We have shown that ADPR activates the channel with high affinity (K1/2=1 uM), but could not clarify the role of ADPR hydrolysis in channel closure. The conserved ADPRase ""Nudix-box"" motif (REFXEE) is atypical in the NUDT9-H domain of TRPM2 (RILRQE). The EF->IL mutation decreases ADPRase activity of the NUDT9 enzyme to ~1%, while the EE->KK mutation completely abolishes it. We constructed an ""inactive"" QE?KK and a ""hiperactive"" IL->EF TRPM2 mutant, but neither of the 4 single and 2 double mutants affected ADPR affinity or channel closing rate. In intact cells AMP inhibits, while H2O2, cyclic ADPR (cADPR), and nicotinic acid-adenin-dinucleotide-phosphate (NAADP) activate TRPM2, and enhance its sensitivity to ADPR. We have found that direct application of H2O2, AMP, and cADPR in isolated patches does not affect the channels, while NAADP and NAAD are low-affinity partial agonists. Thus, in intact cells the effects of these modulators are indirect

Role of TRPM2 in H2O2-induced cell apoptosis in endothelial cells

Melastatin-like transient receptor potential channel 2 (TRPM2) is an oxidant-sensitive and cationic non-selective channel that is expressed in mammalian vascular endothelium. Here we investigated the functional role of TRPM2 channels in hydrogen peroxide (H(2)O(2))-induced cytosolic Ca(2+) ([Ca(2+)](i)) elavation, whole-cell current increase, and apoptotic cell death in murine heart microvessel endothelial cell line H5V. A TRPM2 blocking antibody (TM2E3), which targets the E3 region near the ion permeation pore of TRPM2, was developed. Treatment of H5V cells with TM2E3 reduced the [Ca(2+)](i) rise and whole-cell current change in response to H(2)O(2). Suppressing TRPM2 expression using TRPM2-specific short hairpin RNA (shRNA) had similar inhibitory effect. H(2)O(2)-induced apoptotic cell death in H5V cells was examined using MTT assay, DNA ladder formation analysis, and DAPI-based nuclear DNA condensation assay. Based on these assays, TM2E3 and TRPM2-specific shRNA both showed protective effect against H(2)O(2)-induced apoptotic cell death. TM2E3 and TRPM2-specific shRNA also protect the cells from tumor necrosis factor (TNF)-alpha-induced cell death in MTT assay. In contrast, overexpression of TRPM2 in H5V cells resulted in an increased response in [Ca(2+)](i) and whole-cell currents to H(2)O(2). TRPM2 overexpression also aggravated the H(2)O(2)-induced apoptotic cell death. Downstream pathways following TRPM2 activation was examined. Results showed that TRPM2 activity stimulated caspase-8, caspase-9 and caspase-3. These findings strongly suggest that TRPM2 channel mediates cellular Ca(2+) overload in response to H(2)O(2) and contribute to oxidant-induced apoptotic cell death in vascular endothelial cells. Down-regulating endogenous TRPM2 could be a means to protect the vascular endothelial cells from apoptotic cell death.published_or_final_versio

Reciprocal regulation of actin cytoskeleton remodelling and cell migration by Ca2+ and Zn2+: role of TRPM2 channels

Cell migration is a fundamental feature of tumour metastasis and angiogenesis. It is regulated by a variety of signalling molecules including H2O2 and Ca2+. Here, we asked whether the H2O2-sensitive transient receptor potential melastatin 2 (TRPM2) Ca2+ channel serves as a molecular link between H2O2 and Ca2+. H2O2-mediated activation of TRPM2 channels induced filopodia formation, loss of actin stress fibres and disassembly of focal adhesions, leading to increased migration of HeLa and prostate cancer (PC)-3 cells. Activation of TRPM2 channels, however, caused intracellular release of not only Ca2+ but also of Zn2+. Intriguingly, elevation of intracellular Zn2+ faithfully reproduced all of the effects of H2O2, whereas Ca2+ showed opposite effects. Interestingly, H2O2 caused increased trafficking of Zn2+-enriched lysosomes to the leading edge of migrating cells, presumably to impart polarisation of Zn2+ location. Thus, our results indicate that a reciprocal interplay between Ca2+ and Zn2+ regulates actin remodelling and cell migration; they call for a revision of the current notion that implicates an exclusive role for Ca2+ in cell migration

Cyclic ADP ribose is a novel regulator of intracellular Ca 2+ oscillations in human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine. However, the cellular biology of these cells is not fully understood. The present study characterizes the cyclic ADP-ribose (cADPR)-mediated Ca 2+ signals in human MSCs and finds that externally applied cADPR can increase the frequency of spontaneous intracellular Ca 2+ (Ca 2+ i) oscillations. The increase was abrogated by a specific cADPR antagonist or an inositol trisphosphate receptor (IP3R) inhibitor, but not by ryanodine. In addition, the cADPR-induced increase of Ca 2+ i oscillation frequency was prevented by inhibitors of nucleoside transporter or by inhibitors of the transient receptor potential cation melastatin-2 (TRPM2) channel. RT-PCR revealed mRNAs for the nucleoside transporters, concentrative nucleoside transporters 1/2 and equilibrative nucleoside transporters 1/3, IP3R1/2/3 and the TRPM2 channel, but not those for ryanodine receptors and CD38 in human MSCs. Knockdown of the TRPM2 channel by specific short interference RNA abolished the effect of cADPR on the Ca 2+ i oscillation frequency, and prevented the stimulation of proliferation by cADPR. Moreover, cADPR remarkably increased phosphorylated extracellular-signal-regulated kinases 1/2 (ERK1/2), but not Akt or p38 mitogen-activated protein kinase (MAPK). However, cADPR had no effect on adipogenesis or osteogenesis in human MSCs. Our results indicate that cADPR is a novel regulator of Ca 2+ i oscillations in human MSCs. It permeates the cell membrane through the nucleoside transporters and increases Ca 2+ oscillationviaactivation of the TRPM2 channel, resulting in enhanced phosphorylation of ERK1/2 and, thereby, stimulation of human MSC proliferation. This study delineates an alternate signalling pathway of cADPR that is distinct from its well-established role of serving as a Ca 2+ messenger for mobilizing the internal Ca 2+ stores. Whether cADPR can be used clinically for stimulating marrow function in patients with marrow disorders remains to be further studied. © 2011 The Authors © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.postprin