Gated Cation Channel Research Articles

The Anti-Human P2X7 Monoclonal Antibody (Clone L4) Can Mediate Complement-Dependent Cytotoxicity of Human Leukocytes.

P2X7 is an extracellular adenosine 5'-triphosphate (ATP)-gated cation channel that plays various roles in inflammation and immunity. P2X7 is present on peripheral blood monocytes, dendritic cells (DCs), and innate and adaptive lymphocytes. The anti-human P2X7 monoclonal antibody (mAb; clone L4), used for immunolabelling P2X7 or blocking P2X7 activity, is a murine IgG2b antibody, but its ability to mediate complement-dependent cytotoxicity (CDC) is unknown. In this study the functionality of this mAb was confirmed by inhibition of ATP-induced Ca2+ responses in HEK-293 cells expressing P2X7 (HEK-P2X7). Spectrophotometric measurements of lactate dehydrogenase release demonstrated that the anti-P2X7 mAb mediated CDC in HEK-P2X7 but not HEK-293 cells. Further, flow cytometric measurements of the viability dye, 7-aminoactinomycin D, showed that this mAb mediated CDC in human RPMI 8226 but not mouse J774 cells. Immunolabelling with this mAb and flow cytometry revealed that relative amounts of cell surface P2X7 varied between human peripheral blood leukocytes. As such, the anti-P2X7 mAb preferentially mediated CDC of leukocytes that displayed relatively high cell surface P2X7, namely monocytes, DCs, natural killer T cells, myeloid-derived suppressor cells, and T helper 17 cells. Together, this data highlights a novel approach to target cellular P2X7 and to limit unwanted P2X7 functions.

Extracellular ATP inhibits excitatory synaptic input on parvalbumin positive interneurons and attenuates gamma oscillations via P2X4 receptors.

P2X4 receptors (P2X4R) are ligand gated cation channels that are activated by extracellular ATP released by neurons and glia. The receptors are widely expressed in the brain and have fractional calcium currents comparable with NMDA receptors. Although P2X4Rs have been reported to modulate synaptic transmission and plasticity, their involvement in shaping neuronal network activity remains to be elucidated. We investigated the effects of P2X receptors at network and synaptic level using local field potential electrophysiology, whole cell patch clamp recordings and calcium imaging in fast spiking parvalbumin positive interneurons (PVINs) in rat and mouse hippocampal slices. The stable ATP analogue ATPγS, selective antagonists and P2X4R knockout mice were used. The P2XR agonist ATPγS reversibly decreased the power of gamma oscillations. This inhibition could be antagonized by the selective P2X4R antagonist PSB-12062 and was not observed in P2X4-/- mice. The phasic excitatory inputs of CA3 PVINs were one of the main regulators of the gamma power. Associational fibre compound excitatory postsynaptic currents (cEPSCs) in CA3 PVINs were inhibited by P2X4R activation. This effect was reversible, dependent on intracellular calcium and dynamin-dependent internalization of AMPA receptors. The results indicate that P2X4Rs are an important source of dendritic calcium in CA3 PVINs, thereby regulating excitatory synaptic inputs onto the cells and presumably the state of gamma oscillations in the hippocampus. P2X4Rs represent an effective target to modulate hippocampal network activity in pathophysiological conditions such as Alzheimer's disease and schizophrenia.

Pre-clinical validation of a pan-cancer CAR-T cell immunotherapy targeting nfP2X7

Chimeric antigen receptor (CAR)-T cell immunotherapy is a novel treatment that genetically modifies the patients’ own T cells to target and kill malignant cells. However, identification of tumour-specific antigens expressed on multiple solid cancer types, remains a major challenge. P2X purinoceptor 7 (P2X7) is a cell surface expressed ATP gated cation channel, and a dysfunctional version of P2X7, named nfP2X7, has been identified on cancer cells from multiple tissues, while being undetectable on healthy cells. We present a prototype -human CAR-T construct targeting nfP2X7 showing potential antigen-specific cytotoxicity against twelve solid cancer types (breast, prostate, lung, colorectal, brain and skin). In xenograft mouse models of breast and prostate cancer, CAR-T cells targeting nfP2X7 exhibit robust anti-tumour efficacy. These data indicate that nfP2X7 is a suitable immunotherapy target because of its broad expression on human tumours. CAR-T cells targeting nfP2X7 have potential as a wide-spectrum cancer immunotherapy for solid tumours in humans.

The role of P21-activated kinase (Pak1) in sinus node function

Sinoatrial node (SAN) dysfunction (SND) and atrial arrhythmia frequently occur simultaneously with a hazard ratio of 4.2 for new onset atrial fibrillation (AF) in SND patients. In the atrial muscle attenuated activity of p21-activated kinase 1 (Pak1) increases the risk for AF by enhancing NADPH oxidase 2 dependent production of reactive oxygen species (ROS). However, the role of Pak1 dependent ROS regulation in SAN function has not yet been determined. We hypothesize that Pak1 activity maintains SAN activity by regulating the expression of the hyperpolarization activated cyclic nucleotide gated cation channel (HCN).To determine Pak1 dependent changes in heart rate (HR) regulation we quantified the intrinsic sinus rhythm in wild type (WT) and Pak1 deficient (Pak1−/−) mice of both sexes in vivo and in isolated Langendorff perfused hearts. Pak1−/− hearts displayed an attenuated HR in vivo after autonomic blockage and in isolated hearts. The contribution of the Ca2+ clock to pacemaker activity remained unchanged, but Ivabradine (3 μM), a blocker of HCN channels that are a membrane clock component, eliminated the differences in SAN activity between WT and Pak1−/− hearts. Reduced HCN4 expression was confirmed in Pak1−/− right atria. The reduced HCN activity in Pak1−/− could be rescued by class II HDAC inhibition (LMK235), ROS scavenging (TEMPOL) or attenuation of Extracellular Signal-Regulated Kinase (ERK) 1/2 activity (SCH772984). No sex specific differences in Pak1 dependent SAN regulation were determined.Our results establish Pak1 as a class II HDAC regulator and a potential therapeutic target to attenuate SAN bradycardia and AF susceptibility.

A point mutation turns an inactivated potassium channel into a hyperpolarization activated cation channel.

In potassium channels, the slow inactivation (C-type) process decreases the ionic conduction by affecting the selectivity filter. The W434F mutation in Shaker potassium channel stabilizes it in the inactivated conformation. The recent determination of the structure of the Shaker W434F channel shows a dilated selectivity filter, compared to Shaker WT structure, reminiscent to the hyperpolarization activated cyclic nucleotide gated channel (HCN). This suggests a common link between inactivation and hyperpolarization gating, which is explored in this work. In Shaker the P475D (Sh-P475D) is a mutation in the bundle crossing region that stabilizes the activation gate in the open conformation over a wide voltage range (−140 mV to +100). Sh-P475D was expressed in Xenopus oocytes and inactivation was favored by removing external potassium. Under these conditions, the channel displays robust hyperpolarization activated currents reminiscent of HCN. (Similar currents are observed when the W434F mutation is added in Sh-P475D background.) The currents elicited by hyperpolarizing voltages in the Sh-P475D mutant are non-selective (PK/PNa∼1) and even N-methyl-D-glucamine and calcium are permeable in this mutant. We analyzed the role of residues known to affect pore inactivation, the contribution of the selectivity filter, and the influence of the voltage sensor movement on the kinetics and steady state characteristics of the P475D-induced hyperpolarization activated currents. Our results suggest a mechanism of hyperpolarization gating and inactivation at the selectivity filter that seems to be common among voltage gated cation channels. Supported by NIH GM03076.

The role of mechanical channel in the proliferation and migration of colon cancer

Piezo channel is the first family of mechanically gated cation channels found in mammals and has been shown correlated with the proliferation and metastasis of tumors in recent years. In a variety of cancer tissues, colon cancer tissues are particularly affected by mechanical stimulation. In this paper, we reported that the role of piezo1 in the proliferation and migration of colon cancer. Firstly, bioinformatics indicated that high levels of piezo1 expression existed in CRC tissues and were associated with poor prognosis. In vitro experiments, SW1116 cells were cultured in DMSO solutions, and CCK-8 assay and transwell assay were separately performed. It turned out that the inhibition of PIEZO1 by Dooku1 resulted in diminished cell proliferation and metastasis, while the activation of PIEZO1 by Yoda1 led to enhanced proliferation and migration of colon cells. Collectively, these findings demonstrated the facilitation role of piezo1 in colon cancer proliferation and migration, suggesting that piezo1 has the potential as a novel therapeutic target for drug design and treatment of colon cancer.

Recent Advances in the Development of Antidepressants Targeting the Purinergic P2X7 Receptor.

The purinergic P2X7 receptor (P2X7R) is an adenosine triphosphate (ATP)- gated cation channel protein. Although extracellular ATP (eATP) is maintained at the nanomolar concentration range under normal conditions, it is elevated to micromolar levels in response to cell stress or damage, resulting in activation of P2X7R in the brain. The binding of eATP to P2X7R in glial cells in the brain activates the NLRP3 inflammasome and releases pro-inflammatory cytokines, such as IL-1β, IL-6, IL-18, and TNFα. Depression has been demonstrated to be strongly associated with neuroinflammation activated by P2X7R. Therefore, P2X7R is an attractive therapeutic target for depression. Multinational pharmaceutical companies, including AstraZeneca, GlaxoSmithKline, Janssen, Lundbeck, and Pfizer, have developed CNS-penetrating P2RX7 antagonists. Several of these have been evaluated in clinical trials. This review summarizes the recent development of P2X7R antagonists as novel antidepressant agents in terms of structural optimization, as well as in vitro/in vivo evaluation and physicochemical properties of representative compounds.

Mechanisms of Action of the Peptide Toxins Targeting Human and Rodent Acid-Sensing Ion Channels and Relevance to Their In Vivo Analgesic Effects.

Acid-sensing ion channels (ASICs) are voltage-independent H+-gated cation channels largely expressed in the nervous system of rodents and humans. At least six isoforms (ASIC1a, 1b, 2a, 2b, 3 and 4) associate into homotrimers or heterotrimers to form functional channels with highly pH-dependent gating properties. This review provides an update on the pharmacological profiles of animal peptide toxins targeting ASICs, including PcTx1 from tarantula and related spider toxins, APETx2 and APETx-like peptides from sea anemone, and mambalgin from snake, as well as the dimeric protein snake toxin MitTx that have all been instrumental to understanding the structure and the pH-dependent gating of rodent and human cloned ASICs and to study the physiological and pathological roles of native ASICs in vitro and in vivo. ASICs are expressed all along the pain pathways and the pharmacological data clearly support a role for these channels in pain. ASIC-targeting peptide toxins interfere with ASIC gating by complex and pH-dependent mechanisms sometimes leading to opposite effects. However, these dual pH-dependent effects of ASIC-inhibiting toxins (PcTx1, mambalgin and APETx2) are fully compatible with, and even support, their analgesic effects in vivo, both in the central and the peripheral nervous system, as well as potential effects in humans.

6-Furopyridine Hexamethylene Amiloride Is a Non-Selective P2X7 Receptor Antagonist.

P2X7 is an extracellular adenosine 5′-triphopshate (ATP)-gated cation channel present on leukocytes, where its activation induces pro-inflammatory cytokine release and ectodomain shedding of cell surface molecules. Human P2X7 can be partially inhibited by amiloride and its derivatives at micromolar concentrations. This study aimed to screen a library of compounds derived from amiloride or its derivative 5-(N,N-hexamethylene) amiloride (HMA) to identify a potential P2X7 antagonist. 6-Furopyridine HMA (6-FPHMA) was identified as a novel P2X7 antagonist and was characterized further. 6-FPHMA impaired ATP-induced dye uptake into human RPMI8226 multiple myeloma cells and human P2X7-HEK293 cells, in a concentration-dependent, non-competitive manner. Likewise, 6-FPHMA blocked ATP-induced Ca2+ fluxes in human P2X7-HEK293 cells in a concentration-dependent, non-competitive manner. 6-FPHMA inhibited ATP-induced dye uptake into human T cells, and interleukin-1β release within human blood and CD23 shedding from RPMI8226 cells. 6-FPHMA also impaired ATP-induced dye uptake into murine P2X7- and canine P2X7-HEK293 cells. However, 6-FPHMA impaired ATP-induced Ca2+ fluxes in human P2X4-HEK293 cells and non-transfected HEK293 cells, which express native P2Y1, P2Y2 and P2Y4. In conclusion, 6-FPHMA inhibits P2X7 from multiple species. Its poor selectivity excludes its use as a specific P2X7 antagonist, but further study of amiloride derivatives as P2 receptor antagonists is warranted.

Discovery of 5-methyl-1H-benzo[d]imidazole derivatives as novel P2X3 Receptor antagonists

Drug discovery programs targeting P2X3 receptors (P2X3R), an extracellular adenosine 5′-triphosphate (ATP) gated cation channel family, have been actively investigated for several CNS-related diseases. The current unmet need in the field of P2X3R targeted drugs is to avoid a side effect, the loss of taste, that could be reduced by increase of the P2X3R selectivity vs P2X2/3R. In this study, 5-methyl-1H-benzo[d]imidazole derivatives were designed and synthesized from the analysis of key pharmacophores of current antagonists. In the structure–activity relationship study, the most potent compounds 17a-b was discovered as potent P2X3R antagonists with IC50 values of 145 and 206 nM, and selectivity index of 60 and 41, respectively. In addition, 17a-b showed the not-competitive antagonism, but poor binding score in the docking study at the known allosteric binding site of Gefapixant binding site, indicating that another allosteric binding site might be existing for the novel P2X3R antagonists.

P2X4 Inhibition reduces microglia inflammation and apoptosis by NLRP3 and improves nervous system defects in rat brain trauma model

The purinergic receptor P2X4 is an adenosine triphosphate (ATP)-gated cation channel, which plays an essential role in regulating various biological activities in the organism. This study was designed to investigate the potential role and mechanism of P2X4 in the traumatic brain injury (TBI) rat model. Real-time PCR, Western blot, immunofluorescence, apoptosis, brain water content and neurological score analysis were evaluated. We found that the expression level of P2X4 surrounding the injured area of the brain in the TBI rat model increased significantly after 48 h. Following the P2X4 selective antagonist 5-BDBD treatment, the neurological damage after TBI was significantly improved and brain edema was reduced. The inhibition of P2X4 effectively reduced the inflammation and apoptosis of microglia, and NLRP3 may be involved in this process. Our results indicate that inhibition of P2X4 may be a potential therapeutic approach for TBI by reducing the occurrence of inflammation and apoptosis of microglia, alleviating brain edema, and improving neurological deficits.

Delta glutamate receptors are functional glycine- and ᴅ-serine-gated cation channels in situ.

Delta receptors are members of the ionotropic glutamate receptor superfamily and form trans-synaptic connections by interacting with the extracellular scaffolding protein cerebellin-1 and presynaptic transmembrane protein neurexin-1β. Unlike other family members, however, direct agonist-gated ion channel activity has not been recorded in delta receptors. Here, we show that the GluD2 subtype of delta receptor forms cation-selective channels when bound to cerebellin-1 and neurexin-1β. Using fluorescence lifetime measurements and chemical cross-linking, we reveal that tight packing of the amino-terminal domains of GluD2 permits glycine- and d-serine–induced channel openings. Thus, cerebellin-1 and neurexin-1β act as biological cross-linkers to stabilize the extracellular domains of GluD2 receptors, allowing them to function as ionotropic excitatory neurotransmitter receptors in synapses.

Analgesic effect of ivabradine against inflammatory pain mediated by hyperpolarization-activated cyclic nucleotide-gated cation channels expressed on primary afferent terminals in the spinal dorsal horn.

Ivabradine, a hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel blocker and clinically approved bradycardic agent, has analgesic effects against neuropathic pain. Although the expression of HCN channels in the spinal dorsal horn (SDH) is augmented under inflammatory pain, spinal responses to centrally and peripherally applied ivabradine remain poorly understood. We investigated the spinal action and cellular mechanisms underlying the drug's analgesic effects against inflammatory pain using inflammatory pain model rats. Intraperitoneal and intrathecal injections of ivabradine inhibited mechanical allodynia (6 rats/dose; P < 0.05), and immunohistochemical staining showed that ivabradine suppresses the phosphorylated extracellular signal-regulated kinase activation in the SDH (6 rats/group, P < 0.01). In vitro whole-cell patch-clamp and in vivo extracellular recordings showed that direct application of ivabradine to the spinal cord decreases the mean miniature excitatory postsynaptic currents' frequency (13 rats; P < 0.01), and direct and peripheral application of ivabradine suppresses the spinal response to mechanical stimulation-evoked firing (8 rats/group, P < 0.01). Moreover, ivabradine reduces the amplitudes of monosynaptic excitatory postsynaptic currents evoked by Aδ-fiber and C-fiber stimulation (6 rats; P < 0.01) and induces a stronger inhibition of those evoked by C-fiber stimulation. These phenomena were inhibited by forskolin, an activator of HCN channels. In conclusion, spinal responses mediated by HCN channels on primary afferent terminals are suppressed by central and peripheral administration of ivabradine; the drug also exhibits analgesic effects against inflammatory pain. In addition, ivabradine preferentially acts on C-fiber terminals of SDH neurons and induces a stronger inhibition of neuronal excitability in inflammatory pain.

Advances in Implantable Optogenetic Technology for Cardiovascular Research and Medicine.

Optogenetic technology provides researchers with spatiotemporally precise tools for stimulation, sensing, and analysis of function in cells, tissues, and organs. These tools can offer low-energy and localized approaches due to the use of the transgenically expressed light gated cation channel Channelrhodopsin-2 (ChR2). While the field began with many neurobiological accomplishments it has also evolved exceptionally well in animal cardiac research, both in vitro and in vivo. Implantable optical devices are being extensively developed to study particular electrophysiological phenomena with the precise control that optogenetics provides. In this review, we highlight recent advances in novel implantable optogenetic devices and their feasibility in cardiac research. Furthermore, we also emphasize the difficulties in translating this technology toward clinical applications and discuss potential solutions for successful clinical translation.

Regulation of P2X1 receptors by modulators of the cAMP effectors PKA and EPAC

P2X1 receptors are adenosine triphosphate (ATP)-gated cation channels that are functionally important for male fertility, bladder contraction, and platelet aggregation. The activity of P2X1 receptors is modulated by lipids and intracellular messengers such as cAMP, which can stimulate protein kinase A (PKA). Exchange protein activated by cAMP (EPAC) is another cAMP effector; however, its effect on P2X1 receptors has not yet been determined. Here, we demonstrate that P2X1 currents, recorded from human embryonic kidney (HEK) cells transiently transfected with P2X1 cDNA, were inhibited by the highly selective EPAC activator 007-AM. In contrast, EPAC activation enhanced P2X2 current amplitude. The PKA activator 6-MB-cAMP did not affect P2X1 currents, but inhibited P2X2 currents. The inhibitory effects of EPAC on P2X1 were prevented by triple mutation of residues 21 to 23 on the amino terminus of P2X1 subunits to the equivalent amino acids on P2X2 receptors. Double mutation of residues 21 and 22 and single mutation of residue 23 also protected P2X1 receptors from inhibition by EPAC activation. Finally, the inhibitory effects of EPAC on P2X1 were also prevented by NSC23766, an inhibitor of Rac1, a member of the Rho family of small GTPases. These data suggest that EPAC is an important regulator of P2X1 and P2X2 receptors.

Electrophysiological and Biochemical Characterization of the Human P2X7B Receptor Inxenopus Laevisoocytes

P2X receptors (P2XR) are trimeric ATP-gated ion channels consisting of seven isoforms, P2X1-P2X7, which are permeable to cations. A hallmark of the 595 residues long P2X7R subunit is that its 2nd transmembrane domain (TM2) is linked with a cytoplasmic domain that is significantly larger than that of any other P2XR. From the partial or complete loss of the ATP4--activated ion channel activity due to various C-terminal deletions, we know that this endodomain plays an important functional role. Interestingly, several human P2X7 (hP2X7) splice variants were identified, including four C-terminally truncated variants named hP2X7B, hP2XE, hP2XG and hP2X7J. The amino acid sequences of P2X7A and P2X7B are identical up to Leu346, which lies approximately in the middle of TM2. The subsequent hP2X7B-unique part comprises the C-terminal portion of TM2 (347VRDSLFHALG356), followed by a very short cytoplasmic domain of only eight residues in total (357KWFGEGSD364). Here, we compared the P2X7AR and hP2X7BR as ATP4--gated cation channels and their oligomeric structure and expression on the cell surface. Shortening the hP2X7B variant C-terminal to Leu346 to study only the hP2X7A-identical portion reduced surface expression and homotrimeric assembly. On the other hand, if the truncation mutant contained all TM2 residues including Gly356, but without the eight residues of the cytoplasmic tail, homotrimeric assembly and surface expression was as efficient as with the full-length hP2X7B. ATP4--induced currents mediated by hP2X7BR desensitized relatively quickly as compared to the hP2X7AR, which has a non-desensitizing phenotype. The desensitizing behavior of hP2X7BR supports the view that the long C-terminal endodomain of the non-desensitizing hP2X7AR serves as a gating module that by forming a cytoplasmic cap, stabilizes the ATP4--induced open state of the P2X7AR.However, C-terminal endodomain is not absolutely needed for the channel function.

The cGMP-Dependent Protein Kinase 2 Contributes to Cone Photoreceptor Degeneration in the Cnga3-Deficient Mouse Model of Achromatopsia.

Mutations in the CNGA3 gene, which encodes the A subunit of the cyclic guanosine monophosphate (cGMP)-gated cation channel in cone photoreceptor outer segments, cause total colour blindness, also referred to as achromatopsia. Cones lacking this channel protein are non-functional, accumulate high levels of the second messenger cGMP and degenerate over time after induction of ER stress. The cell death mechanisms that lead to loss of affected cones are only partially understood. Here, we explored the disease mechanisms in the Cnga3 knockout (KO) mouse model of achromatopsia. We found that another important effector of cGMP, the cGMP-dependent protein kinase 2 (Prkg2) is crucially involved in cGMP cytotoxicity of cones in Cnga3 KO mice. Virus-mediated knockdown or genetic ablation of Prkg2 in Cnga3 KO mice counteracted degeneration and preserved the number of cones. Analysis of markers of endoplasmic reticulum stress and unfolded protein response confirmed that induction of these processes in Cnga3 KO cones also depends on Prkg2. In conclusion, we identified Prkg2 as a novel key mediator of cone photoreceptor degeneration in achromatopsia. Our data suggest that this cGMP mediator could be a novel pharmacological target for future neuroprotective therapies.

Manipulations of the olfactory circuit highlight the role of sensory stimulation in regulating sleep amount.

While wake duration is a major sleep driver, an important question is if wake quality also contributes to controlling sleep. In particular, we sought to determine whether changes in sensory stimulation affect sleep in Drosophila. As Drosophila rely heavily on their sense of smell, we focused on manipulating olfactory input and the olfactory sensory pathway. Sensory deprivation was first performed by removing antennae or applying glue to antennae. We then measured sleep in response to neural activation, via expression of the thermally gated cation channel TRPA1, or inhibition, via expression of the inward rectifying potassium channel KIR2.1, of subpopulations of neurons in the olfactory pathway. Genetically restricting manipulations to adult animals prevented developmental effects. We find that olfactory deprivation reduces sleep, largely independently of mushroom bodies that integrate olfactory signals for memory consolidation and have previously been implicated in sleep. However, specific neurons in the lateral horn, the other third-order target of olfactory input, affect sleep. Also, activation of inhibitory second-order projection neurons increases sleep. No single neuronal population in the olfactory processing pathway was found to bidirectionally regulate sleep, and reduced sleep in response to olfactory deprivation may be masked by temperature changes. These findings demonstrate that Drosophila sleep is sensitive to sensory stimulation, and identify novel sleep-regulating neurons in the olfactory circuit. Scaling of signals across the circuit may explain the lack of bidirectional effects when neuronal activity is manipulated. We propose that olfactory inputs act through specific circuit components to modulate sleep in flies.

Upregulation of ASIC1a channels in an in vitro model of Fabry disease

Neuropathic pain is one of the key features of the classical phenotype of Fabry disease (FD). Acid sensing ion channels (ASICs) are H+-gated cation channels, which belong to the epithelial sodium channel/DeGenerin superfamily, sensitive to the diuretic drug Amiloride. Molecular cloning has identified several distinct ASIC subunits. In particular the ASIC1a subunit has been associated to pain and its upregulation has been documented in animal models of pain. We analyzed the expression of ASIC1a channels in cellular models that mimic the accumulation of glycosphingolipids in FD (FD-GLs) like Gb3, and LysoGb3. We used mouse primary neurons from brain cortex and hippocampus -supraspinal structures that accumulate FD-GLs-, as well as HEK293 cells. Incubation with Gb3, lysoGb3 and the inhibitor (1-deoxy-galactonojirymicin, DJG) of the enzyme α-galactosidase A (Gla) lead to the upregulation of ASIC1a channels. In addition, activation of ASIC1a results in the activation of the MAPK ERK pathway, a signaling pathway associated with pain. Moreover, accumulation of glycosphingolipids results in activation of ERK, an effect that was prevented by blocking ASIC1a channels with the specific blocker Psalmotoxin. Our results suggest that FD-GLs accumulation and triggering of the ERK pathway via ASIC channels might be involved in the mechanism responsible for pain in FD, thus providing a new therapeutic target for pain relief treatment.

The C-terminal domains of the NMDA receptor: How intrinsically disordered tails affect signalling, plasticity and disease.

NMDA receptors are part of the ionotropic glutamate receptor family, and are crucial for neurotransmission and memory. At the cellular level, the effects of activating these receptors include long-term potentiation (LTP) or depression (LTD). The NMDA receptor is a stringently gated cation channel permeable to Ca2+ , and it shares the molecular architecture of a tetrameric ligand-gated ion channel with the other family members. Its subunits, however, have uniquely long cytoplasmic C-terminal domains (CTDs). While the molecular gymnastics of the extracellular domains have been described in exquisite detail, much less is known about the structure and function of these CTDs. The CTDs vary dramatically in length and sequence between receptor subunits, but they all have a composition characteristic of intrinsically disordered proteins. The CTDs affect channel properties, trafficking and downstream signalling output from the receptor, and these functions are regulated by alternative splicing, protein-protein interactions, and post-translational modifications such as phosphorylation and palmitoylation. Here, we review the roles of the CTDs in synaptic plasticity with a focus on biochemical mechanisms. In total, the CTDs play a multifaceted role as a modifier of channel function, a regulator of cellular location and abundance, and signalling scaffold control the downstream signalling output.