Distinct ClC-6 and ClC-7 Cl- sensitivities provide insight into ClC-7's role in lysosomal Cl- homeostasis.

ClC-6 and ClC-7 are closely related, intracellular Cl- /H+ antiporters belonging to the CLC family of channels and transporters. They localize to acidic late endosomes and lysosomes and probably function in ionic homeostasis of these contiguous compartments. ClC-7 transport function requires association with the accessory protein Ostm1, whereas ClC-6 transport does not. To elucidate their roles in endo-lysosomes, we measured Cl- - and pH-dependences of over-expressed wild-type ClC-6 and ClC-7, as well as disease-associated mutants, using high-resolution recording protocols. Lowering extracellular Cl- (corresponding to luminal Cl- in endo-lysosomes) reduced ClC-6 currents, whereas it increased transport activity of ClC-7/Ostm1. Low extracellular Cl- activated ClC-7/Ostm 1 under acidic extracellular conditions, as well as under conditions of low intracellular chloride. Activation is conserved in ClC-7Y713C , a variant displaying disrupted PI(3,5)P2 inhibition. Detailed biophysical analysis of disease-associated ClC-6 and ClC-7 gain-of-function (GoF) variants, ClC-6Y553C and ClC-7Y713C , and the ClC-7Y577C and ClC-6Y781C correlates, identified additional functional nuances distinguishing ClC-6 and ClC-7. ClC-7Y577C recapitulated GoF produced by ClC-6Y553C . ClC-6Y781C displayed transport activation qualitatively similar to ClC-7Y713C , although current density did not differ from that of wild-type ClC-6. Finally, rClC-7R760Q , homologous to hClC-7R762Q , an osteopetrosis variant with fast gating kinetics, appeared indifferent to extracellular Cl- , identifying altered Cl- sensitivity as a plausible mechanism underlying disease. Collectively, the present studies underscore the distinct roles of ClC-6 and ClC-7 within the context of their respective localization to late endosomes and lysosomes. In particular, we suggest the atypical inhibition of ClC-7 by luminal Cl- serves to limit excessive intraluminal Cl- accumulation. KEY POINTS: ClC-6 and ClC-7 are late endosomal and lysosomal 2 Cl- /1 H+ exchangers, respectively. When targeted to the plasma membrane, both activate slowly at positive voltages. ClC-6 activity is decreased in low extracellular (i.e. luminal) chloride, whereas ClC-7 is activated by low luminal chloride, even at acidic pH. The functional gain-of-function phenotypes of the ClC-6 and ClC-7 disease mutations ClC-6Y553C and ClC-7Y715C are maintained when introduced in their respective homologues, ClC-7Y577C and ClC-6Y781C , with all mutations retaining chloride dependence of the respective wild type (WT). An osteopetrosis mutation of ClC-7 displaying fast gating kinetics (R762Q) was less sensitive to extracellular chloride compared to WT. The opposing substrate dependences of ClC-6 and ClC-7 Cl- / H+ exchangers point to non-overlapping physiological functions, leading us to propose that inhibition of ClC-7 by luminal chloride and protons serves to prevent osmotic stress imposed by hyper-accumulation of chloride.

IK Channel-Independent Effects of Clotrimazole and Senicapoc on Cancer Cells Viability and Migration.

Many studies highlighted the importance of the IK channel for the proliferation and the migration of different types of cancer cells, showing how IK blockers could slow down cancer growth. Based on these data, we wanted to characterize the effects of IK blockers on melanoma metastatic cells and to understand if such effects were exclusively IK-dependent. For this purpose, we employed two different blockers, namely clotrimazole and senicapoc, and two cell lines: metastatic melanoma WM266-4 and pancreatic cancer Panc-1, which is reported to have little or no IK expression. Clotrimazole and senicapoc induced a decrease in viability and the migration of both WM266-4 and Panc-1 cells irrespective of IK expression levels. Patch-clamp experiments on WM266-4 cells revealed Ca2+-dependent, IK-like, clotrimazole- and senicapoc-sensitive currents, which could not be detected in Panc-1 cells. Neither clotrimazole nor senicapoc altered the intracellular Ca2+ concentration. These results suggest that the effects of IK blockers on cancer cells are not strictly dependent on a robust presence of the channel in the plasma membrane, but they might be due to off-target effects on other cellular targets or to the blockade of IK channels localized in intracellular organelles.

Molecular determinants underlying volume-regulated anion channel subunit-dependent oxidation sensitivity.

The volume-regulated anion channel (VRAC) is formed by LRRC8 subunits. Besides their role in the maintenance of cell homeostasis, VRACs are critically involved in oxidative stress mechanisms: reactive oxygen species directly modulate VRACs in a subunit-dependent manner. It was reported that LRRC8A-LRRC8E heteromeric channels are activated by oxidation, whereas LRRC8A-LRRC8C heteromers are inhibited. Here we adopted chimeric- as well as concatemeric-based strategies to identify residues responsible for the divergent effect of oxidants. We identified two cysteines in the first two leucine rich repeats of LRRC8E, C424 and C448, as the targets of oxidation. Oxidation likely results in the formation of a disulfide bond between the two cysteines, which in turn induces a conformational change leading to channel activation. Additionally, we found that LRRC8C inhibition is caused by oxidation of the first methionine. We thus identified crucial molecular elements involved in channel activation, which are conceivably relevant in determining physiological ROS effects. KEY POINTS: Volume-regulated anion channels (VRACs) are heterohexameric complexes composed of an essential LRRC8A subunit and a variable number of LRRC8B-E subunits. VRACs are directly regulated by oxidation, with LRRC8A-LRRC8E heteromers being potentiated and LRRC8A-LRRC8C heteromers being inhibited by oxidation. We identified two LRRC8E specific intracellular cysteines that form a disulfide bond upon oxidation leading to LRRC8A-LRRC8E potentiation. Inhibition of LRRC8A-LRRC8C heteromers is mediated by the oxidation of the start methionine, being additionally dependent on the identity of the LRR domain. Besides providing physiological insights concerning the outcome of reactive oxygen species modulation, the results point to key structural elements involved in VRAC activation.

Mechanisms of Activation of LRRC8 Volume Regulated Anion Channels.

Volume regulated anion channels (VRACs) are ubiquitously expressed in all vertebrate cells. Despite many years of research, the fundamental mechanisms underlying VRAC activation are not understood. The recent molecular identification of the LRRC8 genes underlying VRAC revealed that VRACs are formed by a hexameric assembly of members of the LRRC8 gene family. Knowing the genes underlying VRACs allowed the discovery of novel VRAC functions into cell volume regulation, and first structure function studies revealed important insight in channel activation mechanisms. The determination of cryo-EM structures of homomeric LRRC8A and LRRC8D complexes provide a framework for a rational approach to investigate biophysical mechanisms. We discuss several recent advances within the structural framework, and we critically review the literature on the main mechanisms proposed to be involved in VRAC activation, including low intracellular ionic strength, membrane unfolding, oxidation, phosphorylation and G-protein coupling.

Biophysical phenotyping of mesenchymal stem cells along the osteogenic differentiation pathway.

Mesenchymal stem cells represent an important resource, for bone regenerative medicine and therapeutic applications. This review focuses on new advancements and biophysical tools which exploit different physical and chemical markers of mesenchymal stem cell populations, to finely characterize phenotype changes along their osteogenic differentiation process. Special attention is paid to recently developed label-free methods, which allow monitoring cell populations with minimal invasiveness. Among them, quantitative phase imaging, suitable for single-cell morphometric analysis, and nanoindentation, functional to cellular biomechanics investigation. Moreover, the pool of ion channels expressed in cells during differentiation is discussed, with particular interest for calcium homoeostasis.Altogether, a biophysical perspective of osteogenesis is proposed, offering a valuable tool for the assessment of the cell stage, but also suggesting potential physiological links between apparently independent phenomena.

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase.

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline-a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc-a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole-two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

Gain of function of sporadic/familial hemiplegic migraine-causing SCN1A mutations: Use of an optimized cDNA.

INTRODUCTION: Familial hemiplegic migraine 3 is an autosomal dominant headache disorder associated with aura and transient hemiparesis, caused by mutations of the neuronal voltage-gated sodium channel Nav1.1. While a gain-of function phenotype is generally assumed to underlie familial hemiplegic migraine, this has not been fully explored. Indeed, a major obstacle in studying in vitro neuronal sodium channels is the difficulty in propagating and mutagenizing expression plasmids containing their cDNAs. The aim of this work was to study the functional effect of two previously uncharacterized hemiplegic migraine causing mutations, Leu1670Trp (L1670W) and Phe1774Ser (F1774S). METHODS: A novel SCN1A containing-plasmid was designed in silico and synthesized, and migraine mutations were inserted in this background. Whole-cell patch clamp was performed to investigate the functional properties of mutant Nav1.1 transiently expressed in Human Embryonic Kidney 293 cells. RESULTS AND CONCLUSIONS: We generated an optimized Nav1.1 expression plasmid that was extremely simple to handle and used the novel plasmid to study the functional effects of two migraine mutations. We observed that L1670W, but not F1774S, reduced current density and that both mutations led to a dramatic increase in persistent sodium currents, a depolarizing shift of the steady state-inactivation voltage-dependence, and a faster recovery from inactivation. The results are consistent with a major gain-of function effect underlying familial hemiplegic migraine 3. Our optimization strategy will help to characterize in an efficient manner the effect in vitro of mutations of neuronal voltage-gated sodium channels.

Subunit-dependent oxidative stress sensitivity of LRRC8 volume-regulated anion channels.

KEY POINTS: Swelling-activated anion currents are modulated by oxidative conditions, but it is unknown if oxidation acts directly on the LRRC8 channel-forming proteins or on regulatory factors. We found that LRRC8A-LRRC8E heteromeric channels are dramatically activated by oxidation of intracellular cysteines, whereas LRRC8A-LRRC8C and LRRC8A-LRRC8D heteromers are inhibited by oxidation. Volume-regulated anion currents in Jurkat T lymphocytes were inhibited by oxidation, in agreement with a low expression of the LRRC8E subunit in these cells. Our results show that LRRC8 channel proteins are directly modulated by oxidation in a subunit-specific manner. ABSTRACT: The volume-regulated anion channel (VRAC) is formed by heteromers of LRRC8 proteins containing the essential LRRC8A subunit and at least one among the LRRC8B-E subunits. Reactive oxygen species (ROS) play physiological and pathophysiological roles and VRAC channels are highly ROS sensitive. However, it is unclear if ROS act directly on the channels or on molecules involved in the activation pathway. We used fluorescently tagged LRRC8 proteins that yield large constitutive currents to test direct effects of oxidation. We found that 8A/8E heteromers are dramatically potentiated (more than 10-fold) by oxidation of intracellular cysteine residues by chloramine-T or tert-butyl hydroperoxide. Oxidation was, however, not necessary for hypotonicity-induced activation. In contrast, 8A/8C and 8A/8D heteromers were strongly inhibited by oxidation. Endogenous VRAC currents in Jurkat T lymphocytes were similarly inhibited by oxidation, in agreement with the finding that LRRC8C and LRRC8D subunits were more abundantly expressed than LRRC8E in Jurkat cells. Our results show that LRRC8 channels are directly modulated by oxidation in a subunit-dependent manner.

In vitro effect of temperature on the conformational structure and collagen binding of SdrF, a Staphylococcus epidermidis adhesin.

Staphylococcus epidermidis is the leading etiologic agent of device-related infections. S. epidermidis is able to bind, by means of the adhesins of its cell wall, the host matrix proteins filming the artificial surfaces. Thence, bacteria cling to biomaterials and infection develops. The effect of temperature on integrity, structure, and biological activity of the collagen-binding adhesin (SdrF) of S. epidermidis has been here investigated. By cloning in E. coli XL1-Blue, a recombinant of the SdrF binding domain B (rSdrFB), carrying an N-terminal polyhistidine, was obtained. Purification was by HiTrap(TM) Chelating HP columns. Assessment of purity, molecular weight, and integrity was by SDS-PAGE. The rSdrFB-collagen binding was investigated by ELISA. A full three-dimensional reconstruction of rSdrFB was achieved by small-angle X-ray scattering (SAXS). At 25 °C, rSdrFB bound to type I collagen in a dose-dependent, saturable manner, with a Kd of 2.48 × 10(-7) M. When temperature increased from 25 to 37 °C, a strong conformational change occurred, together with the abolition of the rSdrFB-collagen binding. The rSdrFB integrity was not affected by temperature variation. SdrFB-collagen binding is switched on/off depending on the temperature. Implications with the infection pathogenesis are enlightened.

The P2X7 receptor as a route for non-exocytotic glutamate release: dependence on the carboxyl tail.

P2X7 receptors trigger Ca(2+) -dependent exocytotic glutamate release, but also function as a route for non-exocytotic glutamate release from neurons or astrocytes. To gain an insight into the mechanisms involving the P2X7 receptor as a direct pathway for glutamate release, we compared the behavior of a full-length rat P2X7 receptor, a truncated rat P2X7 receptor in which the carboxyl tail had been deleted, a rat P2X7 receptor with the 18-amino acid cysteine-rich motif of the carboxyl tail deleted, and a rat P2X2 receptor, all of which are expressed in HEK293 cells. We found that the P2X7 receptor function as a route for glutamate release was antagonized in a non-competitive way by extracellular Mg(2+) , did not require the recruitment of pore-forming molecules, and was dependent on the carboxyl tail. Indeed, the truncated P2X7 receptor and the P2X7 receptor with the deleted cysteine-rich motif both lost their function as a pathway for glutamate release, while still evoking intracellular Ca(2+) elevation. No glutamate efflux was observed through the P2X2 receptor. Notably, HEK293 cells (lacking the machinery for Ca(2+) -dependent exocytosis), when transfected with P2X7 receptors, appear to be a suitable model for investigating the P2X7 receptor as a route for non-exocytotic glutamate efflux.

Voltage-Gated Sodium Channel Dysfunctions in Neurological Disorders.

The pore-forming subunits (α subunits) of voltage-gated sodium channels (VGSC) are encoded in humans by a family of nine highly conserved genes. Among them, SCN1A, SCN2A, SCN3A, and SCN8A are primarily expressed in the central nervous system. The encoded proteins Nav1.1, Nav1.2, Nav1.3, and Nav1.6, respectively, are important players in the initiation and propagation of action potentials and in turn of the neural network activity. In the context of neurological diseases, mutations in the genes encoding Nav1.1, 1.2, 1.3 and 1.6 are responsible for many forms of genetic epilepsy and for Nav1.1 also of hemiplegic migraine. Several pharmacological therapeutic approaches targeting these channels are used or are under study. Mutations of genes encoding VGSCs are also involved in autism and in different types of even severe intellectual disability (ID). It is conceivable that in these conditions their dysfunction could indirectly cause a certain level of neurodegenerative processes; however, so far, these mechanisms have not been deeply investigated. Conversely, VGSCs seem to have a modulatory role in the most common neurodegenerative diseases such as Alzheimer's, where SCN8A expression has been shown to be negatively correlated with disease severity.

Biophysical Aspects of Neurodegenerative and Neurodevelopmental Disorders Involving Endo-/Lysosomal CLC Cl-/H+ Antiporters.

Endosomes and lysosomes are intracellular vesicular organelles with important roles in cell functions such as protein homeostasis, clearance of extracellular material, and autophagy. Endolysosomes are characterized by an acidic luminal pH that is critical for proper function. Five members of the gene family of voltage-gated ChLoride Channels (CLC proteins) are localized to endolysosomal membranes, carrying out anion/proton exchange activity and thereby regulating pH and chloride concentration. Mutations in these vesicular CLCs cause global developmental delay, intellectual disability, various psychiatric conditions, lysosomal storage diseases, and neurodegeneration, resulting in severe pathologies or even death. Currently, there is no cure for any of these diseases. Here, we review the various diseases in which these proteins are involved and discuss the peculiar biophysical properties of the WT transporter and how these properties are altered in specific neurodegenerative and neurodevelopmental disorders.

Melatonin protects Kir2.1 function in an oxidative stress-related model of aging neuroglia.

Melatonin is a pleiotropic biofactor and an effective antioxidant and free radical scavenger and, as such, can be protective in oxidative stress-related brain conditions including epilepsy and aging. To test the potential protective effect of melatonin on brain homeostasis and identify the corresponding molecular targets, we established a new model of oxidative stress-related aging neuroglia represented by U-87 MG cells exposed to D-galactose (D-Gal). This model was characterized by a substantial elevation of markers of oxidative stress, lipid peroxidation, and protein oxidation. The function of the inward rectifying K+ channel Kir2.1, which was identified as the main Kir channel endogenously expressed in these cells, was dramatically impaired. Kir2.1 was unlikely a direct target of oxidative stress, but the loss of function resulted from a reduction of protein abundance, with no alterations in transcript levels and trafficking to the cell surface. Importantly, melatonin reverted these changes. All findings, including the melatonin antioxidant effect, were reproduced in heterologous expression systems. We conclude that the glial Kir2.1 can be a target of oxidative stress and further suggest that inhibition of its function might alter the extracellular K+ buffering in the brain, therefore contributing to neuronal hyperexcitability and epileptogenesis during aging. Melatonin can play a protective role in this context.

Probing cytoskeleton organisation of neuroblastoma cells with single-cell force spectroscopy.

Single-cell force spectroscopy is an emerging technique in the field of biomedicine because it has proved to be a unique tool to obtain mechanical and functional information on living cells, with force resolution up to single molecular bonds. This technique was applied to the study of the cytoskeleton organisation of neuroblastoma cells, a life-threatening cancer typically developing during childhood, and the results were interpreted on the basis of reference experiments on human embryonic kidney cell line. An intimate connection emerges among cellular state, cytoskeleton organisation and experimental outcome that can be potentially exploited towards a new method for cancer stadiation of neuroblastoma cells.

The Murine PSE/TATA-dependent transcriptome: evidence of functional homologies with its human counterpart.

A series of recent studies demonstrated an unexpectedly high frequency of intronic RNA polymerase (pol) III transcription units spread throughout the human genome. The investigation of a subset of these transcripts revealed their tissue/cell-specific transcription together with the involvement in relevant physiopathological pathways. Despite this evidence, these transcripts did not seem to have murine orthologs, based on their nucleotide sequence, resulting in a limitation of the experimental approaches aimed to study their function. In this work, we have extended our investigation to the murine genome identifying 121 pairs of mouse/human transcripts displaying syntenic subchromosomal localization. The analysis in silico of this set of putative noncoding (nc)RNAs suggest their association with alternative splicing as suggested by recent experimental evidence. The investigation of one of these pairs taken as experimental model in mouse hippocampal neurons provided evidence of a human/mouse functional homology that does not depend on underlying sequence conservation. In this light, the collection of transcriptional units here reported can be considered as a novel source for the identification and the study of novel regulatory elements involved in relevant biological processes.

Is Neuronal Fatigue the Cause of Migraine?

The pathological basis of migraine is not fully understood. Familial hemiplegic migraines (FHM) are monogenic forms of severe migraine, caused by mutations in genes encoding various neuronal and/or astrocytic ion transporting proteins. The leading hypothesis regarding the mechanism underlying migraine in FHM is that enhanced electrical excitability leads to increased extracellular potassium levels with subsequent cortical spreading depression. In this short commentary we would like to propose an additional mechanism distinct from enhanced electrical excitability per se. Rather, we propose that FHM mutations cause substantially increased energy expenditure of neurons for re-establishing ion gradients and/or for increased synaptic activity, a mechanism we call neuronal fatigue. Such a metabolic mechanism had been proposed earlier for common migraine and has received recent experimental evidence in particular for the case of FHM3. The hypothesis could be tested in future studies of FHM related models that would need to take metabolic parameters into account.

BK Channel in the Physiology and in the Cancer of Pancreatic Duct: Impact and Reliability of BK Openers.

BK (KCa 1.1, Slo-1) is a K+ channel characterized by an allosteric regulation of the gating mechanism by Ca2+ binding and voltage, and a high unitary conductance. The channel is expressed in many different tissues, where it is involved in the regulation or the fine-tuning of many physiological processes. Among other organs, BK is expressed in the pancreatic duct, a part of the gland important for the correct ionic composition of the pancreatic juice. Unfortunately, the pancreatic duct is also the site where one of the deadliest cancer types, the pancreatic duct adenocarcinoma (PDAC), develops. In the past years, it has been reported that continuous exposure of cancer cells to BK openers can have a significant impact on cell viability as well as on the ability to proliferate and migrate. Here, we first summarize the main BK channel properties and its roles in pancreatic duct physiology. Then we focus on the potential role of BK as a pharmacological target in PDAC. Moreover, we discuss how results obtained when employing BK activators on cancer cells can, in some cases, be misleading.

Ion Channel Involvement in Tumor Drug Resistance.

Over 90% of deaths in cancer patients are attributed to tumor drug resistance. Resistance to therapeutic agents can be due to an innate property of cancer cells or can be acquired during chemotherapy. In recent years, it has become increasingly clear that regulation of membrane ion channels is an important mechanism in the development of chemoresistance. Here, we review the contribution of ion channels in drug resistance of various types of cancers, evaluating their potential in clinical management. Several molecular mechanisms have been proposed, including evasion of apoptosis, cell cycle arrest, decreased drug accumulation in cancer cells, and activation of alternative escape pathways such as autophagy. Each of these mechanisms leads to a reduction of the therapeutic efficacy of administered drugs, causing more difficulty in cancer treatment. Thus, targeting ion channels might represent a good option for adjuvant therapies in order to counteract chemoresistance development.

The VRAC blocker DCPIB directly gates the BK channels and increases intracellular Ca2+ in melanoma and pancreatic duct adenocarcinoma cell lines.

BACKGROUND AND PURPOSE: The volume regulated anion channel (VRAC) is known to be involved in different aspects of cancer cell behaviour and response to therapies. For this reason, we investigated the effect of DCPIB, a presumably specific blocker of VRAC, in two types of cancer: pancreatic duct adenocarcinoma (PDAC) and melanoma. EXPERIMENTAL APPROACH: We used patch-clamp electrophysiology, supported by Ca2+ imaging, gene expression analysis, docking simulation and mutagenesis. We employed two PDAC lines (Panc-1 and MiaPaCa-2), as well as a primary (IGR39) and a metastatic (IGR37) melanoma line. KEY RESULTS: DCPIB markedly increased whole-cell currents in Panc-1, MiaPaca2 and IGR39, but not in IGR37 cells. The currents were mostly mediated by KCa 1.1 channels, commonly known as BK channels. We confirmed DCPIB activation of BK channels also in HEK293 cells transfected with α subunits of this channel. Further experiments showed that in IGR39, and to a smaller degree also in Panc-1 cells, DCPIB induced a rapid Ca2+ influx. This, in turn, indirectly potentiated BK channels and, in IGR39 cells, additionally activated other Ca2+ -dependent channels. However, Ca2+ influx was not required for activation of BK channels by DCPIB, as such activation involved the extracellular part of the protein and we have identified a residue crucial for binding. CONCLUSION AND IMPLICATIONS: DCPIB directly targeted BK channels and, also, acutely increased intracellular Ca2+ . Our findings extend the list of DCPIB effects that should be taken into consideration for future development of DCPIB-based modulators of ion channels and other membrane proteins.

Acquisition of neuron-like electrophysiological properties in neuroblastoma cells by controlled expression of NDM29 ncRNA.

Neuroblastoma is a pediatric cancer characterized by high malignancy and remarkable cell heterogeneity within the tumor nodules. It has been previously shown that the over-expression of a specific non-coding RNA, NDM29, reduces neuroblastoma development promoting cell differentiation. We have used neuroblastoma cells expressing NDM29 at its basal level (Mock cells) or at 5.4-fold higher levels (S1 cells) to investigate whether a functional differentiation correlates with morphological and biochemical development induced by NDM29 expression. First, analyzing the expression of specific markers we demonstrated that NDM29 expression is accompanied by a well coordinated differentiation process toward a neuron-like, rather than toward a glial-like, phenotype. Next, we defined the neuron-like traits of S1 in terms of secretion of cytokines involved in axon guidance, synapse formation and neurite outgrowth. Finally, we characterized the ionic channel apparatus of S1 cells by patch-clamp technique and compared with the Mock counterpart. S1 cells showed much higher levels of fast inactivating Na(+) current and were able to generate mature action potentials. Moreover, they developed expression of functional GABA(A) receptors on their membrane. In contrast, the two cell lines shared very similar pools of functional K(+) channels, although slight quantitative differences can be described. Our results suggest that a maturation occurs in neuroblastoma as a consequence of NDM29 expression, inducing the appearance of neuronal-like properties. In this context, S1 cells may represent a novel in vitro tool for electrophysiological and pharmacological studies of human cells of the neural lineage.