Functional Expression Of Ion Channels Research Articles

Kv2 channels contribute to neuronal activity within the vagal afferent-nTS reflex arc.

Diversity in the functional expression of ion channels contributes to the unique patterns of activity generated in visceral sensory A-type myelinated neurons versus C-type unmyelinated neurons in response to their natural stimuli. In the present study, Kv2 channels were identified as underlying a previously uncharacterized delayed rectifying potassium current expressed in both A- and C-type nodose ganglion neurons. Kv2.1 and 2.2 appear confined to the soma and initial segment of these sensory neurons; however, neither was identified in their central presynaptic terminals projecting onto relay neurons in the nucleus of the solitary tract (nTS). Kv2.1 and Kv2.2 were also not detected in the peripheral axons and sensory terminals in the aortic arch. Functionally, in nodose neuron somas, Kv2 currents exhibited frequency-dependent current inactivation and contributed to action potential repolarization in C-type neurons but not A-type neurons. Within the nTS, the block of Kv2 currents does not influence afferent presynaptic calcium influx or glutamate release in response to afferent activation, supporting our immunohistochemical observations. On the other hand, Kv2 channels contribute to membrane hyperpolarization and limit action potential discharge rate in second-order neurons. Together, these data demonstrate that Kv2 channels influence neuronal discharge within the vagal afferent-nTS circuit and indicate they may play a significant role in viscerosensory reflex function.NEW & NOTEWORTHY We demonstrate the expression and function of the voltage-gated delayed rectifier potassium channel Kv2 in vagal nodose neurons. Within sensory neurons, Kv2 channels limit the width of the broader C-type but not narrow A-type action potential. Within the nucleus of the solitary tract (nTS), the location of the vagal terminal field, Kv2 does not influence glutamate release. However, Kv2 limits the action potential discharge of nTS relay neurons. These data suggest a critical role for Kv2 in the vagal-nTS reflex arc.

Electrophysiological Properties of Tetraploid Cardiomyocytes Derived from Murine Pluripotent Stem Cells Generated by Fusion of Adult Somatic Cells with Embryonic Stem Cells.

Most cardiomyocytes (CMs) in the adult mammalian heart are either binucleated or contain a single polyploid nucleus. Recent studies have shown that polyploidy in CMs plays an important role as an adaptive response to physiological demands and environmental stress and correlates with poor cardiac regenerative ability after injury. However, knowledge about the functional properties of polyploid CMs is limited. In this study, we generated tetraploid pluripotent stem cells (PSCs) by fusion of murine embryonic stem cells (ESCs) and somatic cells isolated from bone marrow or spleen and performed a comparative analysis of the electrophysiological properties of tetraploid fusion-derived PSCs and diploid ESC-derived CMs. Fusion-derived PSCs exhibited characteristics of genuine ESCs and contained a near-tetraploid genome. Ploidy features and marker expression were also retained during the differentiation of fusion-derived cells. Fusion-derived PSCs gave rise to CMs, which were similar to their diploid ESC counterparts in terms of their expression of typical cardiospecific markers, sarcomeric organization, action potential parameters, response to pharmacologic stimulation with various drugs, and expression of functional ion channels. These results suggest that the state of ploidy does not significantly affect the structural and electrophysiological properties of murine PSC-derived CMs. These results extend our knowledge of the functional properties of polyploid CMs and contribute to a better understanding of their biological role in the adult heart.

Ion channel long non-coding RNAs in neuropathic pain.

Neuropathic pain is one of the primary forms of chronic pain and is the consequence of the somatosensory system's direct injury or disease. It is a relevant public health problem that affects about 10% of the world's general population. In neuropathic pain, alteration in neurotransmission occurs at various levels, including the dorsal root ganglia, the spinal cord, and the brain, resulting from the malfunction of diverse molecules such as receptors, ion channels, and elements of specific intracellular signaling pathways. In this context, there have been exciting advances in elucidating neuropathic pain's cellular and molecular mechanisms in the last decade, including the possible role that long non-coding RNAs (lncRNAs) may play, which open up new alternatives for the development of diagnostic and therapeutic strategies for this condition. This review focuses on recent studies associated with the possible relevance of lncRNAs in the development and maintenance of neuropathic pain through their actions on the functional expression of ion channels. Recognizing the changes in the function and spatio-temporal patterns of expression of these membrane proteins is crucial to understanding the control of neuronal excitability in chronic pain syndromes.

Remodeling of Ion Channel Trafficking and Cardiac Arrhythmias

Both inherited and acquired cardiac arrhythmias are often associated with the abnormal functional expression of ion channels at the cellular level. The complex machinery that continuously traffics, anchors, organizes, and recycles ion channels at the plasma membrane of a cardiomyocyte appears to be a major source of channel dysfunction during cardiac arrhythmias. This has been well established with the discovery of mutations in the genes encoding several ion channels and ion channel partners during inherited cardiac arrhythmias. Fibrosis, altered myocyte contacts, and post-transcriptional protein changes are common factors that disorganize normal channel trafficking during acquired cardiac arrhythmias. Channel availability, described notably for hERG and KV1.5 channels, could be another potent arrhythmogenic mechanism. From this molecular knowledge on cardiac arrhythmias will emerge novel antiarrhythmic strategies.

Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons.

Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons (NGN2iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.

Balance of Na+, K+, and Cl- Unidirectional Fluxes in Normal and Apoptotic U937 Cells Computed With All Main Types of Cotransporters.

Fluxes of monovalent ions through the multiple pathways of the plasma membrane are highly interdependent, and their assessment by direct measurement is difficult or even impossible. Computation of the entire flux balance helps to identify partial flows and study the functional expression of individual transporters. Our previous computation of unidirectional fluxes in real cells ignored the ubiquitous cotransporters NKCC and KCC. Here, we present an analysis of the entire balance of unidirectional Na+, K+, and Cl– fluxes through the plasma membrane in human lymphoid U937 cells, taking into account not only the Na/K pump and electroconductive channels but all major types of cotransporters NC, NKCC, and KCC. Our calculations use flux equations based on the fundamental principles of macroscopic electroneutrality of the system, water balance, and the generally accepted thermodynamic dependence of ion fluxes on the driving force, and they do not depend on hypotheses about the molecular structure of the channel and transporters. A complete list of the major inward and outward Na+, K+, and Cl– fluxes is obtained for human lymphoid U937 cells at rest and during changes in the ion and water balance for the first 4 h of staurosporine-induced apoptosis. It is shown how the problem of the inevitable multiplicity of solutions to the flux equations, which arises with an increase in the number of ion pathways, can be solved in real cases by analyzing the ratio of ouabain-sensitive and ouabain-resistant parts of K+ (Rb+) influx (OSOR) and using additional experimental data on the effects of specific inhibitors. It is found that dynamics of changes in the membrane channels and transporters underlying apoptotic changes in the content of ions and water in cells, calculated without taking into account the KCC and NKCC cotransporters, differs only in details from that calculated for cells with KCC and NKCC. The developed approach to the assessment of unidirectional fluxes may be useful for understanding functional expression of ion channels and transporters in other cells under various conditions. Attached software allows reproduction of all calculated data under presented conditions and to study the effects of the condition variation.

Identification of TREK-2 K+ channels in human mesenchymal stromal cells

The maintenance of pluripotency of mesenchymal stromal cells (MSCs), their proliferation and initiation of differentiation may critically depend on functional expression of ion channels. Despite such a possibility, mechanisms of electrogenesis in MSCs remain poorly understood. In particular, little is known about a variety of ion channels active in resting MSCs or activated upon MSC stimulation. Here we aimed at uncovering ion channels operating in MSCs, including those being active at rest, using the patch clamp technique and inhibitory analysis. In trying to evaluate a contribution of anion channels in MSC resting potential, we employed a number of diverse inhibitors of anion channels and transporters, including niflumic acid (NFA). Basically, NFA caused hyperpolarization of MSCs that was accompanied by a marked increase in ion conductance of their plasma membranes. The blockage of Cl− channels could not underlie such a NFA effect, given that cells dialyzed with a CsCl solution were weakly or negligibly sensitive to this blocker. This and other findings indicated that NFA affected the MSC ion permeability not by targeting Cl− channels but by stimulating K+ channels. NFA-activated K+ current was TEA and diltiazem blockable, and K+ channels involved were potentiated from outside by solution acidification and Cu2+ ions. Taken together, the data obtained implicated two-pore domain K+ channels of the TREK-2 subtype in mediating stimulatory effects of NFA on MSCs. The notable inference from our work is that TREK-2 channels should be expressed and functional virtually in every MSC, given that all cells examined by us (n > 100) similarly responded to NFA by increasing their TREK-2-like K+ conductance.

Presympathetic neuron dysfunction--time to reconsider increased intrinsic activity as the cause of neurogenic hypertension.

Presympathetic neuron dysfunction--time to reconsider increased intrinsic activity as the cause of neurogenic hypertension.

Disruption of Ion Homeostasis in the Neurogliovascular Unit Underlies the Pathogenesis of Ischemic Cerebral Edema

Cerebral edema is a major cause of morbidity and mortality following ischemic stroke, but its underlying molecular pathophysiology is incompletely understood. Recent data have revealed the importance of ion flux via channels and transporters expressed in the neurogliovascular unit in the development of ischemia-triggered cytotoxic edema, vasogenic edema, and hemorrhagic conversion. Disruption of homeostatic mechanisms governing cell volume regulation and epithelial/endothelial ion transport due to ischemia-associated energy failure results in the thermodynamically driven re-equilibration of solutes and water across the CSF-blood and blood-brain barriers that ultimately increases the brain's extravascular volume. Additionally, hypoxia, inflammation, and other stress-triggered increases in the functional expression of ion channels and transporters normally expressed at low levels in the neurogliovascular unit cause disruptions in ion homeostasis that contribute to ischemic cerebral edema. Here, we review the pathophysiological significance of several molecular mediators of ion transport expressed in the neurogliovascular unit, including targets of existing FDA-approved drugs, which might be potential nodes for therapeutic intervention.

Functional expression of smooth muscle-specific ion channels in TGF-β1-treated human adipose-derived mesenchymal stem cells

Human adipose tissue-derived mesenchymal stem cells (hASCs) have the power to differentiate into various cell types including chondrocytes, osteocytes, adipocytes, neurons, cardiomyocytes, and smooth muscle cells. We characterized the functional expression of ion channels after transforming growth factor-β1 (TGF-β1)-induced differentiation of hASCs, providing insights into the differentiation of vascular smooth muscle cells. The treatment of hASCs with TGF-β1 dramatically increased the contraction of a collagen-gel lattice and the expression levels of specific genes for smooth muscle including α-smooth muscle actin, calponin, smooth mucle-myosin heavy chain, smoothelin-B, myocardin, and h-caldesmon. We observed Ca(2+), big-conductance Ca(2+)-activated K(+) (BKCa), and voltage-dependent K(+) (Kv) currents in TGF-β1-induced, differentiated hASCs and not in undifferentiated hASCs. The currents share the characteristics of vascular smooth muscle cells (SMCs). RT-PCR and Western blotting revealed that the L-type (Cav1.2) and T-type (Cav3.1, 3.2, and 3.3), known to be expressed in vascular SMCs, dramatically increased along with the Cavβ1 and Cavβ3 subtypes in TGF-β1-induced, differentiated hASCs. Although the expression-level changes of the β-subtype BKCa channels varied, the major α-subtype BKCa channel (KCa1.1) clearly increased in the TGF-β1-induced, differentiated hASCs. Most of the Kv subtypes, also known to be expressed in vascular SMCs, dramatically increased in the TGF-β1-induced, differentiated hASCs. Our results suggest that TGF-β1 induces the increased expression of vascular SMC-like ion channels and the differentiation of hASCs into contractile vascular SMCs.

Ovarian cancer: Ion channel and aquaporin expression as novel targets of clinical potential

Ovarian cancer is associated with limited overall survival, due to problems in early detection and therapy. Membrane ion channels have been proposed to play a significant, concerted role in the cancer process, from initial proliferation to metastasis, and promise to be early, functional biomarkers. We review the evidence for ion channel and aquaporin expression and functioning in human ovarian cancer cells and tissues. In vitro, K+ channels, mainly voltage-gated, including Ca2+-activated channels, have been found to control the cell cycle, as in other cancers. Voltage-gated, volume-regulated and intracellular Cl− channels have been detected in vitro and in vivo and shown to be involved in proliferation, adhesion and invasion. Evidence for ‘transient receptor potential’, voltage-gated sodium and calcium channels, which have been shown to contribute to pathogenesis of other carcinomas, is also emerging in ovarian cancer. Aquaporins may be involved in cell growth, migration and formation of ascites via increased water permeability of micro-vessels. It is concluded that functional expression of ion channels and their regulation by steroid hormones and growth factors are an integral part of ovarian cancer development and progression. Furthermore, ion channels may be involved in multidrug resistance, commonly associated with treatment of ovarian cancer. We propose that ion channel studies can facilitate our understanding of the pathobiology of ovarian cancer and, ultimately, can serve as viable novel targets for its clinical management.

Involvement of cationic channels in proliferation and migration of human mesenchymal stem cells

Human mesenchymal stem cells (HMSCs) have been applied in various clinic settings. Ion channels play an important role in cellular physiology. However, the potential role of cationic channels in regulating the proliferation and migration properties of hMSCs remains to be determined. In the present study, the functional expression of ion channels in hMSCs was investigated by patch clamp. MTT assay and BrdU stainings were used to assess the proliferation of hMSCs. hMSC migration was evaluated by Transwell migration assays. The results show that sodium-, L-type calcium, potassium currents have been identified in hMSCs. TEA (K+ channel blocker), nifedipine (Ca2+ channel blocker) can inhibit both proliferation and migration of hMSCs. The increase of extracellular Ca2+ concentration promoted both proliferation and migration of hMSCs. TTX, a Na+ channel blocker, promoted cell proliferation but inhibited cell migration. Our data suggest that cationic channels (sodium, L-type calcium, potassium channels) play important roles in regulating proliferation and migration of hMSCs.

Cardiogenic Regulation of Stem-Cell Electrical Properties in a Laser-Patterned Biochip

Normal cardiomyocytes are highly dependent on the functional expression of ion channels to form action potentials and electrical coupling with other cells. To fully determine the scientific and therapeutic potential of stem cells for cardiovascular-disease treatment, it is necessary to assess comprehensively the regulation of stem-cell electrical properties during stem cell-cardiomyocyte interaction. It has been reported in the literature that contact with native cardiomyocytes induced and regulated stem-cell cardiogenic differentiation. However, in conventional cell-culture models, the importance of cell-cell contact for stem-cell functional coupling with cardiomyocytes has not been elucidated due to insufficient control of the cell-contact mode of individual cells. Using microfabrication and laser-guided cell micropatterning techniques, we created two biochips with contact-promotive and -preventive microenvironments to systematically study the effect of contact on cardiogenic regulation of stem-cell electrical properties. In contact-promotive biochips, connexin 43 expression was upregulated and relocated to the junction area between one stem cell and one cardiomyocyte. Only stem cells in contact with cardiomyocytes were induced by adjacent cardiomyocytes to acquire electrophysiological properties for action-potential formation similar to that of a cardiomyocyte.

Robust L‐type calcium current expression following heterozygous knockout of the Cav1.2 gene in adult mouse heart

Mechanisms that contribute to maintaining expression of functional ion channels at relatively constant levels following perturbations of channel biosynthesis are likely to contribute significantly to the stability of electrophysiological systems in some pathological conditions. In order to examine the robustness of L-type calcium current expression, the response to changes in Ca²⁺ channel Cav1.2 gene dosage was studied in adult mice. Using a cardiac-specific inducible Cre recombinase system, Cav1.2 mRNA was reduced to 11 ± 1% of control values in homozygous floxed mice and the mice died rapidly (11.9 ± 3 days) after induction of gene deletion. In these homozygous knockout mice, echocardiographic analysis showed that myocardial contractility was reduced to 14 ± 1% of control values shortly before death. For these mice, no effective compensatory changes in ion channel gene expression were triggered following deletion of both Cav1.2 alleles, despite the dramatic decay in cardiac function. In contrast to the homozygote knockout mice, following knockout of only one Cav1.2 allele, cardiac function remained unchanged, as did survival.Cav1.2mRNAexpression in the left ventricle of heterozygous knockout mice was reduced to 58 ± 3% of control values and there was a 21 ± 2% reduction in Cav1.2 protein expression. There was no significant reduction in L-type Ca²⁺ current density in these mice. The results are consistent with a model of L-type calcium channel biosynthesis in which there are one or more saturated steps, which act to buffer changes in both total Cav1.2 protein and L-type current expression.

Review: Placental syncytiotrophoblast membranes – domains, subdomains and microdomains

Human placental syncytiotrophoblast (STB) is an epithelium responsible for materno-fetal exchange. Ions play multiple roles in STB, as in other transport epithelia. We have been interested in the character and functional expression of ion channels in STB membrane fractions. Characterization of ion channels and their relationship with different domains, subdomains and microdomains of STB membranes is important to explain the intracellular mechanisms operating in the placental barrier. The aim of this paper is to summarize our work on this subject. We isolated and purified basal membrane (BM) and two fractions from the apical membrane, a classical fraction (MVM) and a light fraction (LMVM). They were used either for reconstitution into giant liposomes or for transplantation into Xenopus oocyte membranes followed by electrophysiological recordings to characterize chloride and cationic channels in STB from term human placenta. In addition, Western blot analysis, using ion channel antibodies, was performed on purified apical and basal membrane fractions. We also reported the presence of two functional microdomains (lipid rafts) in LMVM and MVM, using detergent resistant membranes (DRMs) and cholesterol-sensitive depletion. Moreover we found evidence of cytoskeletal participation in lipid rafts of different composition. Our results contribute to knowledge of the ion channels present in STB membranes and their participation in the physiology of this epithelium in normal and pathological pregnancies.

Functional ion channels in stem cells

Bioelectrical signals generated by ion channels play crucial roles in excitation genesis and impulse conduction in excitable cells as well as in cell proliferation, migration and apoptosis in proliferative cells. Recent studies have demonstrated that multiple ion channels are heterogeneously present in different stem cells; however, patterns and phenotypes of ion channels are species- and/or origin-dependent. This editorial review focuses on the recent findings related to the expression of functional ion channels and the roles of these channels in regulation of cell proliferation in stem cells. Additional effort is required in the future to clarify the ion channel expression in different types of stem cells; special attention should be paid to the relationship between ion channels and stem cell proliferation, migration and differentiation.

Functional Expression of Ion Channels in Mesenchymal Stem Cells Derived from Umbilical Cord Vein

Mesenchymal stem cells have the ability to renew and differentiate into various lineages of mesenchymal tissues. We used undifferentiated human mesenchymal-like stem cells from human umbilical cord vein (hUC-MSCs), a cell line which contains several mesenchymal cell markers. We characterized functional ion channels in cultured hUC-MSCs with whole-cell patch clamp and reverse transcription-polymerase chain reaction (RT-PCR). Three types of outward current were found in these cells: the Ca(2+)-activated K(+) channel (IK(Ca)), a transient outward K(+) current (I(to)), and a delayed rectifier K(+) current (IK(DR)). IK(Ca) and IK(DR) were totally suppressed by tetraethylammonium, and IK(Ca) was sensitive to a specific blocker, iberiotoxin. I(to) was inhibited by 4-aminopyridine. Another type of inward rectifier K(+) current (K(ir)) was also detected in approximately 5% of hUC-MSCs. Elevation of external potassium ion concentration increased the K(ir) current amplitude and positively shifted its reversal potential. In addition, inward Na(+) current (I(Na)) was found in these cells ( approximately 30%); the current was blocked by tetrodotoxin and verapamil. In the RT-PCR analysis, Kv1.1, Kv4.2, Kv1.4, Kir2.1, heag1, MaxiK, hNE-Na, and TWIK-1 were detected. These results suggested that multiple functional ion channel currents, IK(Ca), IK(DR), I(to), I(Na), and K(ir), are expressed in hUC-MSCs. Disclosure of potential conflicts of interest is found at the end of this article.

Serotonin-induced ion channel modulations in mesenteric artery myocytes from normotensive and DOCA-salt hypertensive rats

Although serotonin (5-hydroxytryptamine, 5-HT) has been found to be a potent vasoconstrictor, a pivotal role of 5-HT in the control of appetite and mood control by the modulation of neuronal synapse has also been proposed. Selective 5-HT reuptake inhibitors (SSRIs) are frequently used to suppress appetite and treat depressive disorder, and the target protein of SSRIs is the 5-HT transporter (5-HTT) in the neuronal synapse. However, SSRIs may increase the free 5-HT concentration in circulating blood because platelets and vascular smooth muscles express functional 5-HTT. In addition, enhanced vasoactive action of 5-HT and alterations in 5-HT receptor subtypes have been reported in some types of hypertension. Therefore, we can infer that the use of drugs such as SSRIs in some hypertensive patients is potentially risky. Altered functional expression of ion channels in vascular smooth muscle is suggested to be a mechanism for the enhanced vasoconstriction by vasoactive agonists, including 5-HT. In this brief review, we compared the electrophysiological properties of mesenteric artery myocytes and their modulation by 5-HT between sham-operated control and deoxycorticosterone acetate (DOCA)-salt hypertensive rats.

Electrophysiological characterization of murine HL-5 atrial cardiomyocytes

HL-5 cells are cultured murine atrial cardiomyocytes and have been used in studies to address important cellular and molecular questions. However, electrophysiological features of HL-5 cells have not been characterized. In this study, we examined such properties using whole cell patch-clamp techniques. Membrane capacitance of the HL-5 cells was from 8 to 62 pF. The resting membrane potential was -57.8 +/- 1.4 mV (n = 51). Intracellular injection of depolarizing currents evoked action potentials (APs) with variable morphologies in 71% of the patched cells. Interestingly, the incidence of successful, current-induced APs positively correlated with the hyperpolarizing degrees of resting membrane potentials (r = 0.99, P < 0.001). Only a few of the patched cells (4 of 51, 7.8%) exhibited spontaneous APs. The muscarinic agonist carbachol activated the acetylcholine-activated K(+) current and significantly shortened the duration of APs. Immunostaining confirmed the presence of the muscarinic receptor type 2 in HL-5 cells. The hyperpolarization-activated cation current (I(f)) was detected in 39% of the patched cells. The voltage to activate 50% of I(f) channels was -73.4 +/- 1.2 mV (n = 12). Voltage-gated Na(+), Ca(2+), and K(+) currents were observed in the HL-5 cells with variable incidences. Compared with the adult mouse cardiomyocytes, the HL-5 cells had prolonged APs and small outward K(+) currents. Our data indicate that HL-5 cells display significant electrophysiological heterogeneity of morphological appearance of APs and expression of functional ion channels. Compared with adult murine cardiomyocytes, HL-5 cells show an immature phenotype of cardiac AP morphology.

Alterations in glutamatergic and gabaergic ion channel activity in hippocampal neurons following exposure to the abused inhalant toluene

Toluene, a representative member of the large class of abused inhalants, decreases neuronal activity and depresses behavior in both animals and humans. The sites of action of toluene are not completely known but recent studies suggest that ion channels that regulate neuronal excitability may be particularly sensitive. Previous studies with recombinant receptors showed that toluene decreases currents carried by N-methyl- d-aspartate (NMDA)-glutamate receptors without affecting those gated by non-NMDA receptors. In addition, toluene increases currents generated by GABA and glycine receptors. In the present study, primary cultures of rat hippocampal neurons were used to investigate the effects of acute and chronic toluene exposure on native excitatory and inhibitory ligand-gated ion channels. Toluene dose-dependently inhibited NMDA-mediated currents (IC 50 1.5 mM) but had no effect on responses evoked by the non-NMDA agonist kainic acid. Prolonged treatment of neurons with toluene (1 mM; 4 days) increased whole-cell responses to exogenously applied NMDA, reduced those evoked by GABA but did not alter responses generated by kainic acid. Immunoblot analysis revealed that prolonged toluene exposure increased levels of NR2A and NR2B NMDA receptor subunits with no change in NR1. Immunohistochemical analysis with confocal imaging showed that toluene-treated neurons had significant increases in the density of NR1 subunits as compared with control neurons. Toluene exposure increased the amplitude of synaptic NMDA currents and decreased those activated by GABA. The results from this study suggest that toluene induces compensatory responses in the functional expression of ion channels that regulate neuronal excitability.