Permeable Ion Channels Research Articles

OmpW of Caulobacter crescentus Functions as an Outer Membrane Channel for Cations.

Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.

Measurement of relative Ca²⁺ permeability during sustained activation of TRPV1 receptors.

Some cation permeable ligand-gated ion channels, including the capsaicin-sensitive TRPV1, have been reported to exhibit a time-dependent increase in permeability to large inorganic cations during sustained activation, a phenomenon termed "pore dilation." TRPV1 conducts substantial Ca(2+) entry, and it has been suggested that this channel undergoes a time-dependent change in Ca(2+) permeability relative to Na(+) (P Ca/P Na) that parallels pore dilation. However, our experiments employing whole cell patch clamp photometry and single channel recordings to directly measure relative Ca(2+) current in TRPV1 expressing HEK293 cells show that relative Ca(2+) influx remains constant for the duration of capsaicin-evoked channel activation. Further, we present evidence from patch clamp photometry experiments suggesting that sustained activation of Ca(2+) permeable ion channels in the voltage-clamp configuration leads to rapid saturation of the pipette Ca(2+) chelator, and that subsequent observed shifts in the current reversal potentials in the presence of extracellular Ca(2+) are likely due to intracellular accumulation of this ion and a movement of the Ca(2+) equilibrium potential (E Ca) towards zero. Finally, using an adapted reversal potential-based protocol in which cells are only exposed to Ca(2+) after sustained capsaicin exposure in the absence of added extracellular Ca(2+), we demonstrate that the calculated P Ca/P Na is unaffected by duration of TRPV1 activation. In conclusion, we find no evidence in support of a time-dependent change in P Ca/P Na for TRPV1. Our data further urges caution in estimating relative Ca(2+) permeability using reversal potentials, as there is a limited time window in which the cytosolic Ca(2+) chelator included in the patch pipette can prevent localised elevations in cytosolic free Ca(2+) and thus allow for an accurate estimate of this important channel permeability parameter.

Coulomb blockade model of permeation and selectivity in biological ion channels

Biological ion channels are protein nanotubes embedded in, and passing through, the bilipid membranes of cells. Physiologically, they are of crucial importance in that they allow ions to pass into and out of cells, fast and efficiently, though in a highly selective way. Here we show that the conduction and selectivity of calcium/sodium ion channels can be described in terms of ionic Coulomb blockade in a simplified electrostatic and Brownian dynamics model of the channel. The Coulomb blockade phenomenon arises from the discreteness of electrical charge, the strong electrostatic interaction, and an electrostatic exclusion principle. The model predicts a periodic pattern of Ca2+ conduction versus the fixed charge Qf at the selectivity filter (conduction bands) with a period equal to the ionic charge. It thus provides provisional explanations of some observed and modelled conduction and valence selectivity phenomena, including the anomalous mole fraction effect and the calcium conduction bands. Ionic Coulomb blockade and resonant conduction are similar to electronic Coulomb blockade and resonant tunnelling in quantum dots. The same considerations may also be applicable to other kinds of channel, as well as to charged artificial nanopores.

Epidermal TRPM8 channel isoform controls the balance between keratinocyte proliferation and differentiation in a cold-dependent manner

Deviation of the ambient temperature is one of the most ubiquitous stimuli that continuously affect mammals' skin. Although the role of the warmth receptors in epidermal homeostasis (EH) was elucidated in recent years, the mystery of the keratinocyte mild-cold sensor remains unsolved. Here we report the cloning and characterization of a new functional epidermal isoform of the transient receptor potential M8 (TRPM8) mild-cold receptor, dubbed epidermal TRPM8 (eTRPM8), which is localized in the keratinocyte endoplasmic reticulum membrane and controls mitochondrial Ca(2+) concentration ([Ca(2+)]m). In turn, [Ca(2+)]m modulates ATP and superoxide (O2(·-)) synthesis in a cold-dependent manner. We report that this fine tuning of ATP and O2(·-) levels by cooling controls the balance between keratinocyte proliferation and differentiation. Finally, to ascertain eTRPM8's role in EH in vivo we developed a new functional knockout mouse strain by deleting the pore domain of TRPM8 and demonstrated that eTRPM8 knockout impairs adaptation of the epidermis to low temperatures.

Regulation and roles of Ca2+ stores in human sperm.

[Ca2 +]i signalling is a key regulatory mechanism in sperm function. In mammalian sperm the Ca2 +-permeable plasma membrane ion channel CatSper is central to [Ca2 +]i signalling, but there is good evidence that Ca2 + stored in intracellular organelles is also functionally important. Here we briefly review the current understanding of the diversity of Ca2 + stores and the mechanisms for the regulation of their activity. We then consider the evidence for the involvement of these stores in [Ca2 +]i signalling in mammalian (primarily human) sperm, the agonists that may activate these stores and their role in control of sperm function. Finally we consider the evidence that membrane Ca2 + channels and stored Ca2 + may play discrete roles in the regulation of sperm activities and propose a mechanism by which these different components of the sperm Ca2 +-signalling apparatus may interact to generate complex and spatially diverse [Ca2 +]i signals.

Polycystin-2 (TRPP2) Regulation by Ca2+ Is Effected and Diversified by Actin-Binding Proteins

Calcium regulation of Ca2+-permeable ion channels is an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the transient receptor potential superfamily, is a nonselective cation channel with Ca2+ permeability. The molecular mechanisms associated with PC2 regulation by Ca2+ remain ill-defined. We recently demonstrated that PC2 from human syncytiotrophoblast (PC2hst) but not the in vitro translated protein (PC2iv), functionally responds to changes in intracellular (cis) Ca2+. In this study we determined the regulatory effect(s) of Ca2+-sensitive and -insensitive actin-binding proteins (ABPs) on PC2iv channel function in a lipid bilayer system. The actin-bundling protein α-actinin increased PC2iv channel function in the presence of cis Ca2+, although instead was inhibitory in its absence. Conversely, filamin that shares actin-binding domains with α-actinin had a strong inhibitory effect on PC2iv channel function in the presence, but no effect in the absence of cis Ca2+. Gelsolin stimulated PC2iv channel function in the presence, but not the absence of cis Ca2+. In contrast, profilin that shares actin-binding domains with gelsolin, significantly increased PC2iv channel function both in the presence and absence of Ca2+. The distinct effect(s) of the ABPs on PC2iv channel function demonstrate that Ca2+ regulation of PC2 is actually mediated by direct interaction(s) with structural elements of the actin cytoskeleton. These data indicate that specific ABP-PC2 complexes would confer distinct Ca2+-sensitive properties to the channel providing functional diversity to the cytoskeletal control of transient receptor potential channel regulation.

Abstract P2-07-05: A potential role for Janus protein tyrosine kinases in the regulation of epithelial-mesenchymal transition in a model of epidermal growth factor induced breast cancer epithelial-mesenchymal transition

Abstract Background. Epithelial-mesenchymal transition (EMT), a process whereby tumorigenic epithelial cells acquire an invasive and migratory phenotype, is an important event in the invasion-metastasis cascade. As such, intracellular signaling pathways involved in the regulation of EMT represent potential therapeutic targets in the treatment and prevention of invasive cancer subtypes. The calcium ion, a highly versatile intracellular messenger, plays an important role in processes important in tumorigenesis including invasion and metastasis, and altered calcium signaling has been identified in various cancers. We recently identified that activation of signal transducer and activator of transcription 3 (STAT3) and expression of specific EMT markers in the MDA-MB-468 cell line model of epidermal growth factor (EGF) induced EMT display some calcium dependence. While the calcium permeable ion channel TRPM7 was shown to partially regulate this STAT3 activation and vimentin expression, the precise mechanisms of their regulation are not yet fully understood. The aim of this research was to investigate the upstream intracellular signaling pathway involved in EGF stimulated STAT3 activation and the subsequent induction of EMT in this model. Methods. MDA-MB-468 basal-like breast cancer cells were pre-treated for 1 hr with the Janus protein tyrosine kinase (JAK) inhibitor, JAK inhibitor I (1 and 10 μM), or the Src family tyrosine kinase inhibitor, PP2 (0.1, 1 and 10 μM), followed by stimulation with EGF (50 ng/mL) for 10 or 20 min, and 24 h to assess effects on STAT3 activation and/or EMT marker expression, respectively. Total cellular protein was isolated following inhibitor treatment ± EGF stimulation, and the level of phosphorylated STAT3 (10 or 20 min) or vimentin protein expression (24 h), was analyzed using Western blotting. Cellular RNA was isolated following inhibitor treatment ± EGF stimulation and levels of vimentin mRNA (24 h) were assessed using real time RT-PCR. Results. Treatment of MDA-MB-468 breast cancer cells with JAK inhibitor I resulted in a significant decrease in EGF stimulated STAT3 phosphorylation, while inhibition of Src family tyrosine kinases with PP2 also significantly decreased EGF stimulated STAT3 phosphorylation. In addition to its effects on STAT3 phosphorylation, pre-treatment of MDA-MB-468 cells with JAK inhibitor I also appeared to decrease EGF-induced vimentin protein and mRNA expression, indicating a potential role for Janus protein tyrosine kinases in the induction of EMT in this model. Conclusions. Janus protein tyrosine kinase signaling appears to play a role in the regulation of STAT3 activation, and the induction of the EMT marker vimentin in the MDA-MB-468 cell line model of EGF-induced EMT. Future studies will focus on investigating the specific JAK family member(s) involved in the EGF-STAT3 signaling pathway, as well as the nexus between calcium and identified regulators of EGF stimulated STAT3 activation, and EMT marker expression, in this model of breast cancer EMT. Citation Format: Teneale A Stewart, Iman Azimi, Felicity M Davis, Erik W Thompson, Andrew J Brooks, Sarah J Roberts-Thomson, Gregory R Monteith. A potential role for Janus protein tyrosine kinases in the regulation of epithelial-mesenchymal transition in a model of epidermal growth factor induced breast cancer epithelial-mesenchymal transition [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-07-05.

Identification and in vitro pharmacological characterization of a novel and selective α7 nicotinic acetylcholine receptor agonist, Br-IQ17B.

Aim:Alpha7-nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca2+-permeable ion channel implicated in cognition and neuropsychiatric disorders. Activation of α7 nAChR improves learning, memory, and sensory gating in animal models. To identify novel α7 nAChR agonists, we synthesized a series of small molecules and characterized a representative compound, Br-IQ17B, N-[(3R)-1-azabicyclo[2,2,2]oct-3-yl]-5-bromoindolizine-2-carboxamide, which specifically activates α7 nAChR.Methods:Two-electrode voltage clamp (TEVC) recordings were primarily used for screening in Xenopus oocytes expressing human α7 nAChR. Assays, including radioisotope ligand binding, Western blots, whole-cell recordings of hippocampal culture neurons, and spontaneous IPSC recordings of brain slices, were also utilized to evaluate and confirm the specific activation of α7 nAChR by Br-IQ17B.Results:Br-IQ17B potently activates α7 nAChR with an EC50 of 1.8±0.2 μmol/L. Br-IQ17B is selective over other subtypes such as α4β2 and α3β4, but it blocks 5-HT3A receptors. Br-IQ17B displaced binding of the α7 blocker [3H]-MLA to hippocampal crude membranes with a Ki of 14.9±3.2 nmol/L. In hippocampal neurons, Br-IQ17B evoked α7-like currents that were inhibited by MLA and enhanced in the presence of the α7 PAM PNU-120596. In brain slice recordings, Br-IQ17B enhanced GABAergic synaptic transmission in CA1 neurons. Mechanistically, Br-IQ17B increased ERK1/2 phosphorylation that was MLA-sensitive.Conclusion:We identified the novel, potent, and selective α7 agonist Br-IQ17B, which enhances synaptic transmission. Br-IQ17B may be a helpful tool to understand new aspects of α7 nAChR function, and it also has potential for being developed as therapy for schizophrenia and cognitive deficits.

Functional expression of transient receptor potential channels in human endometrial stromal cells during the luteal phase of the menstrual cycle.

Are members of the transient receptor potential (TRP) channel superfamily functionally expressed in the human endometrial stroma? The Ca(2+)-permeable ion channels TRPV2, TRPV4, TRPC6 and TRPM7 are functionally expressed in primary endometrial stromal cells. Intercellular communication between epithelial and stromal endometrial cells is required to initiate decidualization, a prerequisite for successful implantation. TRP channels are possible candidates as signal transducers involved in cell-cell communication, but no fingerprint is available of the functional distribution of TRP channels in the human endometrium during the luteal phase of the menstrual cycle. Endometrial biopsy samples (previously frozen) from patients of reproductive age with regular menstrual cycles, who were undergoing diagnostic laparoscopic surgery for pain and/or infertility, were analysed. Samples were obtained from the menstrual (Days 1-5, n = 3), follicular (Days 6-14, n = 6), early luteal (Days 15-20, n = 5) and late luteal (Days 21-28, n = 5) phases. In addition, a total of 13 patient samples taken during the luteal phase were used to set up primary cell cultures for further experiments. Quantitative real-time PCR (qRT-PCR), immunocytochemistry, Fura2-based Ca(2+)-microfluorimetry and whole-cell patch clamp experiments were performed to study the functional expression pattern of TRP channels. Specific pharmacological agents, such as Δ(9)-tetrahydrocannabinol, GSK1016790A and 1-oleoyl-2-acetyl-glycerol, were used to functionally assess the expression of TRPV2, TRPV4 and TRPC6, respectively. Expression of TRPV2, TRPV4, TRPC1, TRPC4, TRPC6, TRPM4 and TRPM7 was detected at the mRNA level in endometrial biopsies (n = 19) and in primary endometrial stromal cell cultures obtained from patients during the luteal phase (n = 5) of the menstrual cycle. Messenger RNA levels of TRPV2, TRPC4 and TRPC6 were significantly increased (P < 0.01) in the late luteal phase compared with the early luteal phase. Immunocytochemistry experiments showed a positive staining for TRPV2, TRPV4, TRPC6 and TRPM7 in the plasma membrane and in the cytoplasm of primary endometrial stromal cells. Ca(2+)-microfluorimetry revealed significant increases (P < 0.001) in intracellular Ca(2+) levels when stromal cells were incubated with specific activators of TRPV2, TRPV4 and TRPC6. Further functional characterization was performed using whole-cell patch clamp experiments. Taken together, these data provide evidence for the functional activity of TRPV2, TRPV4, TRPC6 and TRPM7 channels in primary stromal cell cultures. Although mRNA levels are detected for TRPV6, TRPC1, TRPC4 and TRPM4, the limited supply of specific antibodies and lack of selective pharmacological agents restricted any additional analysis of these ion channels. Embryo implantation is a dynamic developmental process that integrates many signalling molecules into a precisely orchestrated programme. Our findings identified certain members of the TRP superfamily as candidate sensors in the epithelial-stromal crosstalk. These results are very helpful to unravel the signalling cascade required for successful embryo implantation. In addition, this knowledge could lead to new strategies to correct implantation failure and facilitate the development of novel non-hormonal contraceptives. This work was supported by grants from the Research Foundation-Flanders (G.0856.13N to J.V.), the Research Council of the KU Leuven (OT/13/113 to J.V. and T.D. and PF-TRPLe to T.V.) and by the Planckaert-De Waele fund (to J.V.). K.D.C. and K.H. are funded by the FWO Belgium. None of the authors have a conflict of interest.

Zinc-permeable ion channels: effects on intracellular zinc dynamics and potential physiological/pathophysiological significance.

Zinc (Zn(2+)) is one of the most important trace metals in the body. It is necessary for the normal function of a large number of protein s including enzymes and transcription factors. While extracellular fluid may contain up to micromolar Zn(2+), intracellular Zn(2+) concentration is generally maintained at a subnanomolar level; this steep gradient across the cell membrane is primarily attributable to Zn(2+) extrusion by Zn(2+) transporting systems. Interestingly, systematic investigation has revealed that activities, previously believed to be dependent on calcium (Ca(2+)), may be partially mediated by Zn(2+). This is also supported by new findings that some Ca(2+)-permeable channels such as voltage-dependent calcium channels (VDCCs), N-methyl-D-aspartate receptors (NMDA), and amino-3- hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPA-Rs) are also permeable to Zn(2+). Thus, the importance of Zn(2+) in physiological and pathophysiological processes is now more widely appreciated. In this review, we describe Zn(2+)- permeable membrane molecules, especially Zn(2+)-permeable ion channels, in intracellular Zn(2+)dynamics and Zn(2+) mediated physiology/pathophysiology.

C-Fiber Recovery Cycle Supernormality Depends on Ion Concentration and Ion Channel Permeability

Following each action potential, C-fiber nociceptors undergo cyclical changes in excitability, including a period of superexcitability, before recovering their basal excitability state. The increase in superexcitability during this recovery cycle depends upon their immediate firing history of the axon, but also determines the instantaneous firing frequency that encodes pain intensity. To explore the mechanistic underpinnings of the recovery cycle phenomenon a biophysical model of a C-fiber has been developed. The model represents the spatial extent of the axon including its passive properties as well as ion channels and the Na/K-ATPase ion pump. Ionic concentrations were represented inside and outside the membrane. The model was able to replicate the typical transitions in excitability from subnormal to supernormal observed empirically following a conducted action potential. In the model, supernormality depended on the degree of conduction slowing which in turn depends upon the frequency of stimulation, in accordance with experimental findings. In particular, we show that activity-dependent conduction slowing is produced by the accumulation of intraaxonal sodium. We further show that the supernormal phase results from a reduced potassium current Kdr as a result of accumulation of periaxonal potassium in concert with a reduced influx of sodium through Nav1.7 relative to Nav1.8 current. This theoretical prediction was supported by data from an in vitro preparation of small rat dorsal root ganglion somata showing a reduction in the magnitude of tetrodotoxin-sensitive relative to tetrodotoxin -resistant whole cell current. Furthermore, our studies provide support for the role of depolarization in supernormality, as previously suggested, but we suggest that the basic mechanism depends on changes in ionic concentrations inside and outside the axon. The understanding of the mechanisms underlying repetitive discharges in recovery cycles may provide insight into mechanisms of spontaneous activity, which recently has been shown to correlate to a perceived level of pain.

TRPM3 expression in mouse retina.

Transient receptor potential (TRP) channels constitute a large family of cation permeable ion channels that serve crucial functions in sensory systems by transducing environmental changes into cellular voltage and calcium signals. Within the retina, two closely related members of the melastatin TRP family, TRPM1 and TRPM3, are highly expressed. TRPM1 has been shown to be required for the depolarizing response to light of ON-bipolar cells, but the role of TRPM3 in the retina is unknown. Immunohistochemical staining of mouse retina with an antibody directed against the C-terminus of TRPM3 labeled the inner plexiform layer (IPL) and a subset of cells in the ganglion cell layer. Within the IPL, TRPM3 immunofluorescence was markedly stronger in the OFF sublamina than in the ON sublamina. Electroretinogram recordings showed that the scotopic and photopic a- and b-waves of TRPM3-/- mice are normal indicating that TRPM3 does not play a major role in visual processing in the outer retina. TRPM3 activity was measured by calcium imaging and patch-clamp recording of immunopurified retinal ganglion cells. Application of the TRPM3 agonist, pregnenolone sulfate (PS), stimulated increases in intracellular calcium in ~40% of cells from wild type and TRPM1‑/‑ mice, and the PS-stimulated increases in calcium were blocked by co-application of mefenamic acid, a TRPM3 antagonist. No PS-stimulated changes in fluorescence were observed in ganglion cells from TRPM3-/- mice. Similarly, PS-stimulated currents that could be blocked by mefenamic acid were recorded from wild type retinal ganglion cells but were absent in ganglion cells from TRPM3-/- mice.

A role for calcium in the regulation of ATP-binding cassette, sub-family C, member 3 (ABCC3) gene expression in a model of epidermal growth factor-mediated breast cancer epithelial–mesenchymal transition

Epithelial–mesenchymal transition (EMT), a process implicated in cancer metastasis, is associated with the transcriptional regulation of members of the ATP-binding cassette superfamily of efflux pumps, and drug resistance in breast cancer cells. Epidermal growth factor (EGF)-induced EMT in MDA-MB-468 breast cancer cells is calcium signal dependent. In this study induction of EMT was shown to result in the transcriptional up-regulation of ATP-binding cassette, subfamily C, member 3 (ABCC3), a member of the ABC transporter superfamily, which has a recognized role in multidrug resistance. Buffering of cytosolic free calcium inhibited EGF-mediated ABCC3 increases, indicating a calcium-dependent mode of regulation. Silencing of TRPM7 (an ion channel involved in EMT associated vimentin induction) did not inhibit ABCC3 up-regulation. Silencing of the store operated calcium entry (SOCE) pathway components ORAI1 and STIM1 also did not alter ABCC3 induction by EGF. However, the calcium permeable ion channel transient receptor potential cation channel, subfamily C, member 1 (TRPC1) appears to contribute to the regulation of both basal and EGF-induced ABCC3 mRNA. Improved understanding of the relationship between calcium signaling, EMT and the regulation of genes important in therapeutic resistance may help identify novel therapeutic targets for breast cancer.

Cognitive Function and Calcium. Structures and functions of Ca2+-permeable channels

Calcium is essential for living organisms where the increase in intracellular Ca2+ concentration functions as a second messenger for many cellular processes including synaptic transmission and neural plasticity. The cytosolic concentration of Ca2+ is finely controlled by many Ca2+-permeable ion channels and transporters. The comprehensive view of their expression, function, and regulation will advance our understanding of neural and cognitive functions of Ca2+, which leads to the future drug discovery.

Inhibition of antigen receptor-dependent Ca2+ signals and NF-AT activation by P2X7 receptors in human B lymphocytes

One of the first intracellular signals after antigen binding by the antigen receptor of B lymphocytes is the increased intracellular Ca2+ concentration ([Ca2+]i), which is followed by several intracellular signaling events like the nuclear translocation of the transcription factor NF-AT controlling the fate of B lymphocytes after their activation. Extracellular ATP, which is released from cells under several pathological conditions, is considered a danger-associated signal serving as an immunomodulator. We investigated the interaction of antigen receptor (BCR) and P2X7 receptor (P2X7R) activation on [Ca2+]i signaling and on nuclear translocation of the transcription factor NF-AT in human B lymphocytes. Although the P2X7R is an ATP-gated Ca2+-permeable ion channel, P2X7R activation inhibits the BCR-mediated [Ca2+]i responses. This effect is mimicked by cell membrane depolarization induced by an increase in the extracellular K+ concentration or by application of the Na+ ionophore gramicidin, but is abolished by stabilization of the membrane potential using the K+ ionophore valinomycin, by extracellular Mg2+, which is known to inhibit P2X7R-dependent effects, or by replacing Na+ by the less P2X7R-permeable Tris+ ion. Furthermore, P2X7R activation by ATP inhibits the BCR-dependent translocation of the transcription factor NF-ATc1 to the nucleus. We therefore conclude that extracellular ATP via the P2X7R mediates inhibitory effects on B cell activation. This may be of relevance for understanding of the activation of the BCR under pathological conditions and for the development of therapeutic strategies targeting human B lymphocytes or P2X7 receptors.

Evolutionary Diversity of Protein Nanodomains within Mammalian Sperm

Mammalian sperm motility and fertility potential are regulated by ion homeostasis which is controlled by a suite of ion permeable channels and transporters working in concert. While all mammalian sperm share in their goal to navigate the female reproductive tract in search of an egg, the molecular regulation of this process is extremely diverse and differs dramatically among species. Here we report that electrophysiological recordings from sperm cells of different species illustrate marked differences between rodent, human and ruminant sperm on the basis of their ion channel make-up and regulation. Furthermore, localization patterning of ion channels along the length of sperm flagella show species specific configuration. It is from these observed differences that we can now begin to explain the disparity in motility parameters among species as well as determine the effect proper spatiotemporal regulation of ion concentrations have on proper sperm motility.

Effect of the CALHM1 G330D and R154H human variants on the control of cytosolic Ca2+ and Aβ levels.

CALHM1 is a plasma membrane voltage-gated Ca2+-permeable ion channel that controls amyloid-β (Aβ) metabolism and is potentially involved in the onset of Alzheimer's disease (AD). Recently, Rubio-Moscardo et al. (PLoS One (2013) 8: e74203) reported the identification of two CALHM1 variants, G330D and R154H, in early-onset AD (EOAD) patients. The authors provided evidence that these two human variants were rare and resulted in a complete loss of CALHM1 function. Recent publicly available large-scale exome sequencing data confirmed that R154H is a rare CALHM1 variant (minor allele frequency (MAF) = 0.015%), but that G330D is not (MAF = 3.5% in an African American cohort). Here, we show that both CALHM1 variants exhibited gating and permeation properties indistinguishable from wild-type CALHM1 when expressed in Xenopus oocytes. While there was also no effect of the G330D mutation on Ca2+ uptake by CALHM1 in transfected mammalian cells, the R154H mutation was associated with defects in the control by CALHM1 of both Ca2+ uptake and Aβ levels in this cell system. Together, our data show that the frequent CALHM1 G330D variant has no obvious functional consequences and is therefore unlikely to contribute to EOAD. Our data also demonstrate that the rare R154H variant interferes with CALHM1 control of cytosolic Ca2+ and Aβ accumulation. While these results strengthen the notion that CALHM1 influences Aβ metabolism, further investigation will be required to determine whether CALHM1 R154H, or other natural variants in CALHM1, is/are associated with EOAD.

Abstract 4067: TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways

Abstract A small fraction of cancer stem cells (CSC) initiate and maintain tumors thus driving glioma tumorigenesis and are responsible for resistance to classical chemo- and radio-therapies. It is desirable to identify signaling pathways related to CSC to develop novel therapies to selectively target them. TRPM7 is a ubiquitous Ca2+ and Mg2+ permeable ion channel which contains a serine/threonine kinase. Recent studies have demonstrated that TRPM7 plays an important role in the growth and proliferation of various tumor cells. To determine the possibility of an interaction among TRPM7, Notch, STAT3 and the ALDH1 signaling in the context of brain tumors, we investigated the effects of TRPM7 pathway activation on glioma cells and stem-like cells derived from them. Methods: 1) We analyzed human stem cell signaling pathways using real time RT2 Profile PCR arrays in the A172 glioma cells upon TRPM7 gene silencing. 2) We then examined Notch and STAT3 pathway activation, Aldehyde dehydrogenase 2 family, mitochondrial (ALDH1) and CD133 expressions in parental and glioma stem cell (GSC) populations using quantitative real time-PCR, Western blot, and immunofluorence images. 3) We conducted a ChIP analysis to examine the effects of STAT3 on ALDH1 promoter by introducing constitutive STAT3 (caSTAT3) and dominant STAT3 (dnSTAT3) into A172 cells. 4) Finally, we examined the role of TRPM7 in glioma cell migration and invasion using transwell invasion assay and wound healing assay. Results: 1) RT2 Profile PCR arrays showed that TRPM7 gene silencing down-regulated genes (≥2 fold-change) involved in Notch pathway [Notch1, Jagged 1(JAG1) and Delta-like 1, Drosophila (DLL1)], Wnt pathway, Wingless-type MMTV integration site family, member 1(WNT1) and ALDH1. 2) STAT3 activation and Notch1 expression were decreased in response to TRPM7 silencing. 3) Notch-regulated genes including Notch1, Hey2, JAG1, and Survivin as well as ALDH1 were significantly expressed higher in spheroid glioma CSCs when compared with monolayer cultures. 4) ALDH1 promoter activity was activated by STAT3 in A172 cells. 5) Knockdown of TRPM7 inhibited migration and invasion of A172 cells. Conclusions: TRPM7 activates JAK2/STAT3 and/or Notch signaling pathways and leads to increased cell proliferation and migration. These findings for the first time demonstrate that TRPM7 (1) activates a previously unrecognized STAT3→ALDH1 pathway, and (2) promotes the induction of ALDH1 activity in glioma cells. (This work was supported by the National Institutes of Health grant numbers NIH R01NS047506, R01NS066027, UL1 RR025008, U54 RR026137, AHA 0840132N, and ALZ IIRG-10-173350.) Citation Format: Mingli Liu, Koichi Inoue, Tiandong Leng, Shanchun Guo, Zhigang Xiong. TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2014-4067

TRPM7 channels regulate glioma stem cell through STAT3 and Notch signaling pathways

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults with median survival time of 14.6months. A small fraction of cancer stem cells (CSC) initiate and maintain tumors thus driving glioma tumorigenesis and being responsible for resistance to classical chemo- and radio-therapies. It is desirable to identify signaling pathways related to CSC to develop novel therapies to selectively target them. Transient receptor potential cation channel, subfamily M, member 7, also known as TRPM7 is a ubiquitous, Ca2+ and Mg2+ permeable ion channels that are special in being both an ion channel and a serine/threonine kinase. In studies of glioma cells silenced for TRPM7, we demonstrated that Notch (Notch1, JAG1, Hey2, and Survivin) and STAT3 pathways are down regulated in glioma cells grown in monolayer. Furthermore, phospho-STAT3, Notch target genes and CSC markers (ALDH1 and CD133) were significantly higher in spheroid glioma CSCs when compared with monolayer cultures. The results further show that tyrosine-phosphorylated STAT3 binds and activates the ALDH1 promoters in glioma cells. We found that TRMP7-induced upregulation of ALDH1 expression is associated with increases in ALDH1 activity and is detectable in stem-like cells when expanded as spheroid CSCs. Finally, TRPM7 promotes proliferation, migration and invasion of glioma cells. These demonstrate that TRPM7 activates JAK2/STAT3 and/or Notch signaling pathways and leads to increased cell proliferation and migration. These findings for the first time demonstrates that TRPM7 (1) activates a previously unrecognized STAT3→ALDH1 pathway, and (2) promotes the induction of ALDH1 activity in glioma cells.

Involvement of Ectodomain Leu 214 in ATP Binding and Channel Desensitization of the P2X4 Receptor

P2X receptors are trimeric ATP-gated cation permeable ion channels. When ATP binds, the extracellular head and dorsal fin domains are predicted to move closer to each other. However, there are scant functional data corroborating the role of the dorsal fin in ligand binding. Here using site-directed mutagenesis and electrophysiology, we show that a dorsal fin leucine, L214, contributes to ATP binding. Mutant receptors containing a single substitution of alanine, serine, glutamic acid, or phenylalanine at L214 of the rat P2X4 receptor exhibited markedly reduced sensitivities to ATP. Mutation of other dorsal fin side chains, S216, T223, and D224, did not significantly alter ATP sensitivity. Exposure of L214C to sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES(-)) or (2-aminoethyl) methanethiosulfonate hydrobromide in the absence of ATP blocked responses evoked by subsequent ATP application. In contrast, when MTSES(-) was applied in the presence of ATP, no current inhibition was observed. Furthermore, L214A also slightly reduced the inhibitory effect of the antagonist 2',3'-O-(2,4,6-trinitrophenyl)-ATP, and the blockade was more rapidly reversible after washout. Certain L214 mutants also showed effects on current desensitization in the continued presence of ATP. L214I exhibited an accelerated current decline, whereas L214M exhibited a slower rate. Taken together, these data reveal that position L214 participates in both ATP binding and conformational changes accompanying channel opening and desensitization, providing compelling evidence that the dorsal fin domain indeed has functional properties that are similar to those previously reported for the body domains.