Shift In Reversal Potential Research Articles

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.

Commentary: “Nitric oxide releases Cl− from acidic organelles in retinal amacrine cells”

In their recent article (Krishnan and Gleason, 2015) Vijai Krishnan and Evanna Gleason investigate the cellular mechanisms underlying the shift in the GABA reversal potential upon application of nitric oxide (NO). Functional alteration in GABAergic signaling by alterations in the GABA reversal potential has been identified as an important mechanism of plasticity (Raimondo et al., 2012) and NO is clearly one key substance involved in plasticity (Prast and Philippu, 2001). Therefore, the investigation of the mechanisms behind the NO induced shift in GABAergic effects is an important issue. However, in my opinion the authors neglected a possible explanation of their observations in the discussion section of their recent article. The detailed investigation in the present article was based on the seminal observation by the same group that moderate concentrations of NO donors induced a slight increase in GABAA receptor mediated responses without affecting the reversal potential, while the application of a saturated NO solution (resulting in “hundreds of nanomolar to low micromolar” NO concentrations) led to a significant shift in the GABA reversal potential (Hoffpauir et al., 2006). The latter effect was independent of the soluble guanylate cyclase, which is elementary for the classical NO signaling pathway, and persisted in nominally Cl−-free extracellular solution, suggesting that NO may trigger a Cl− release from intracellular stores (Hoffpauir et al., 2006). Further experiments by the authors suggest that a NO-induced intracellular acidification triggers the release of Cl− from intracellular compartments (McMains and Gleason, 2011). In the present study they used nominally Cl−-free extracellular and intracellular (pipette-) solutions to “wash out both cytosolic and protein-bound Cl−” and thereby isolate Cl− stores “contained within an intracellular membrane-bound compartment” (Krishnan and Gleason, 2015). These experiments demonstrated that application of NO or NO-donors re-established GABAergic currents under nominally Cl−-free conditions, and that this effect was attenuated in the presence of the V-type ATPase inhibitor bafilomycin and was abolished by the uncoupling agent FCCP. From these experimental findings they came to the well supported conclusion, that acidic compartments are a possible source for the NO-induced increase in intracellular Cl− anions. However, when such high NO concentrations are used, it should be considered that the mitochondrial cytochrome c oxidase can reduce NO to nitrite (NO2-), although no information about the maximal rates for this process is available (Sarti et al., 2012). The permeability of NO2- through GABAA receptors is higher than their Cl− permeability (Bormann et al., 1987). Thus the intracellular generation of NO2- might contribute to the re-occurrence of inward currents after NO application under nominally Cl−-free solutions. Notably, the oxidation of NO to NO2- relies on functional oxidative phosphorylation (Sarti et al., 2012). Thus, the reduction of NO-induced GABA currents by bafilomycin and its abolishment by FCCP (Krishnan and Gleason, 2015) may be caused by the fact, that both substances induce mitochondrial dysfunction via uncoupling (Zhdanov et al., 2012). Therefore, I suggest that the authors should consider the formation of NO2- as an additional hypothesis to explain their observations. To differentiate between both hypotheses, a conclusive experiment would be to test whether the NO-induced GABA currents in nominally Cl−-free intra- and extra-cellular solutions are depletable (indicating a limited intracellular reservoir of Cl− ions) or whether repetitive NO applications induced sustained GABA currents (indicating a NO-dependent generation of anions permeable via the GABAA receptor).

Prenatal Hypoxia-Ischemia Induces Abnormalities in CA3 Microstructure, Potassium Chloride Co-Transporter 2 Expression and Inhibitory Tone.

Infants who suffer perinatal brain injury, including those with encephalopathy of prematurity, are prone to chronic neurological deficits, including epilepsy, cognitive impairment, and behavioral problems, such as anxiety, inattention, and poor social interaction. These deficits, especially in combination, pose the greatest hindrance to these children becoming independent adults. Cerebral function depends on adequate development of essential inhibitory neural circuits and the appropriate amount of excitation and inhibition at specific stages of maturation. Early neuronal synaptic responses to γ-amino butyric acid (GABA) are initially excitatory. During the early postnatal period, GABAAR responses switch to inhibitory with the upregulation of potassium-chloride co-transporter KCC2. With extrusion of chloride by KCC2, the Cl− reversal potential shifts and GABA and glycine responses become inhibitory. We hypothesized that prenatal hypoxic–ischemic brain injury chronically impairs the developmental upregulation of KCC2 that is essential for cerebral circuit formation. Following late gestation hypoxia–ischemia (HI), diffusion tensor imaging in juvenile rats shows poor microstructural integrity in the hippocampal CA3 subfield, with reduced fractional anisotropy and elevated radial diffusivity. The loss of microstructure correlates with early reduced KCC2 expression on NeuN-positive pyramidal neurons, and decreased monomeric and oligomeric KCC2 protein expression in the CA3 subfield. Together with decreased inhibitory post-synaptic currents during a critical window of development, we document for the first time that prenatal transient systemic HI in rats impairs hippocampal CA3 inhibitory tone. Failure of timely development of inhibitory tone likely contributes to a lower seizure threshold and impaired cognitive function in children who suffer perinatal brain injury.

Human SLC4A11 Is a Novel NH3/H+ Co-transporter

SLC4A11 has been proposed to be an electrogenic membrane transporter, permeable to Na(+), H(+) (OH(-)), bicarbonate, borate, and NH4 (+). Recent studies indicate, however, that neither bicarbonate or borate is a substrate. Here, we examined potential NH4 (+), Na(+), and H(+) contributions to electrogenic ion transport through SLC4A11 stably expressed in Na(+)/H(+) exchanger-deficient PS120 fibroblasts. Inward currents observed during exposure to NH4Cl were determined by the [NH3]o, not [NH4 (+)]o, and current amplitudes varied with the [H(+)] gradient. These currents were relatively unaffected by removal of Na(+), K(+), or Cl(-) from the bath but could be reduced by inclusion of NH4Cl in the pipette solution. Bath pH changes alone did not generate significant currents through SLC4A11, except immediately following exposure to NH4Cl. Reversal potential shifts in response to changing [NH3]o and pHo suggested an NH3/H(+)-coupled transport mode for SLC4A11. Proton flux through SLC4A11 in the absence of ammonia was relatively small, suggesting that ammonia transport is of more physiological relevance. Methylammonia produced currents similar to NH3 but with reduced amplitude. Estimated stoichiometry of SLC4A11 transport was 1:2 (NH3/H(+)). NH3-dependent currents were insensitive to 10 μM ethyl-isopropyl amiloride or 100 μM 4,4'- diisothiocyanatostilbene-2,2'-disulfonic acid. We propose that SLC4A11 is an NH3/2H(+) co-transporter exhibiting unique characteristics.

The contribution of raised intraneuronal chloride to epileptic network activity.

Altered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory if intraneuronal chloride rises. However, it has proved difficult to separate the role of raised chloride from other contributory factors in complex network phenomena, such as epileptic pathology. Therefore, we asked what patterns of activity are associated with chloride dysregulation by making novel use of Halorhodopsin to load clusters of mouse pyramidal cells artificially with Cl(-). Brief (1-10 s) activation of Halorhodopsin caused substantial positive shifts in the GABAergic reversal potential that were proportional to the charge transfer during the illumination and in adult neocortical pyramidal neurons decayed with a time constant of τ = 8.0 ± 2.8s. At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high-frequency oscillations with evidence of out-of-phase firing (Ibarz et al., 2010). We show how such firing patterns can arise from quite small shifts in the mean intracellular Cl(-) level, within heterogeneous neuronal populations. Notably, however, chloride loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, subepileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that chloride loading has at most an adjunctive role in ictogenesis. Our simulations also show how chloride loading can affect the jitter of action potential timing associated with imminent recruitment to an ictal event (Netoff and Schiff, 2002).

Sevoflurane Potentiates Kv Channels by Inhibiting a Late Non-Conducting State: A Plausible Mechanism of General Anesthetic Action Implicating the Selectivity Filter

Previous work has shown that sevoflurane potentiates gating of Kv channels, including mammalian Kv1.2, Kv1.5 and Drosophila K-Shaw2 (J Biol Chem, 287: 40425-0432, 2012). Kinetic modeling additionally showed that this potentiation could result from the elimination of non-conducting states connected to the open state but outside the main activation pathway. To test this hypothesis, we examined the effect of sevoflurane on K-Shaw2 inactivation. Consistent with the presence of a classical P/C-type mechanism, K-Shaw2 inactivation increases Na+ selectivity and is inhibited by elevated external K+. To ask whether sevoflurane inhibits K-Shaw2 P/C-type inactivation, we investigated the positive reversal potential shift that occurs as inactivation develops. As expected for an increase in relative Na+ selectivity induced by P/C-type inactivation, initial results showed that the magnitude of the inward tail current increases as the outward current slowly decays, and that 1 mM sevoflurane does not affect this relationship (albeit the outward peak current at strongly depolarized voltages increases as shown previously). Thus, K-Shaw2 potentiation by sevoflurane does not result from inhibition of P/C-type inactivation. Alternatively, inhibition of a flicker non-conducting state outside the main activation pathway might explain the potentiation by sevoflurane. Preliminary unitary current recordings of the K-Shaw2 channel indicate the presence of a short-lived flicker state. We will present additional K-Shaw2 single channel results and Kv1.2 molecular dynamics simulations, which more directly implicate the selectivity filter region as a possible site of sevoflurane action. Supported by NIH Grant P01 GM055876-14.

Modification of glial response in hibernation: a patch-clamp study on glial cells acutely isolated from hibernating land snail.

Hibernation is a dormant state of some animal species that enables them to survive harsh environmental conditions during the winter seasons. In the hibernating state, preservation of neuronal rhythmic activity at a low level is necessary for maintenance of suspended forms of behavior. As glial cells support rhythmic activity of neurons, preservation of brain function in the hibernating state implies accompanying modification of glial activity. A supportive role of glia in regulating neuronal activity is reflected through the activity of inwardly rectifying K+ channels (Kir). Therefore, we examined electrophysiological response, particularly Kir current response, of glial cells in mixture with neurons acutely isolated from active and hibernating land snail Helix pomatia. Our data show that hibernated glia have significantly lower inward current density, specific membrane conductance, and conductance density compared with active glia. The observed reduction could be attributed to the Kir currents, since the Ba2+-sensitive Kir current density was significantly lower in hibernated glia. Accordingly, a significant positive shift of the current reversal potential indicated a more depolarized state of hibernated glia. Data obtained show that modification of glial current response could be regulated by serotonin (5-HT) through an increase of cGMP as a secondary messenger, since extracellular addition of 5-HT or intracellular administration of cGMP to active glia induced a significant reduction of inward current density and thus mimicked the reduced response of hibernated glia. Lower Kir current density of hibernated glia accompanied the lower electrical activity of hibernated neurons, as revealed by a decrease in neuronal fast inward Na+ current density. Our findings reveal that glial response is reduced in the hibernating state and suggest seasonal modulation of glial activity. Maintenance of low glial activity in hibernation could be important for preservation of brain rhythmic activity and survival of the animal.

Increased Spinal Cord Na+-K+-2Cl− Cotransporter-1 (NKCC1) Activity Contributes to Impairment of Synaptic Inhibition in Paclitaxel-induced Neuropathic Pain

Microtubule-stabilizing agents, such as paclitaxel (Taxol), are effective chemotherapy drugs for treating many cancers, and painful neuropathy is a major dose-limiting adverse effect. Cation-chloride cotransporters, such as Na(+)-K(+)-2Cl(-) cotransporter-1 (NKCC1) and K(+)-Cl(-) cotransporter-2 (KCC2), critically influence spinal synaptic inhibition by regulating intracellular chloride concentrations. Here we show that paclitaxel treatment in rats significantly reduced GABA-induced membrane hyperpolarization and caused a depolarizing shift in GABA reversal potential of dorsal horn neurons. However, paclitaxel had no significant effect on AMPA or NMDA receptor-mediated glutamatergic input from primary afferents to dorsal horn neurons. Paclitaxel treatment significantly increased protein levels, but not mRNA levels, of NKCC1 in spinal cords. Inhibition of NKCC1 with bumetanide reversed the paclitaxel effect on GABA-mediated hyperpolarization and GABA reversal potentials. Also, intrathecal bumetanide significantly attenuated hyperalgesia and allodynia induced by paclitaxel. Co-immunoprecipitation revealed that NKCC1 interacted with β-tubulin and β-actin in spinal cords. Remarkably, paclitaxel increased NKCC1 protein levels at the plasma membrane and reduced NKCC1 levels in the cytosol of spinal cords. In contrast, treatment with an actin-stabilizing agent had no significant effect on NKCC1 protein levels in the plasma membrane or cytosolic fractions of spinal cords. In addition, inhibition of the motor protein dynein blocked paclitaxel-induced subcellular redistribution of NKCC1, whereas inhibition of kinesin-5 mimicked the paclitaxel effect. Our findings suggest that increased NKCC1 activity contributes to diminished spinal synaptic inhibition and neuropathic pain caused by paclitaxel. Paclitaxel disrupts intracellular NKCC1 trafficking by interfering with microtubule dynamics and associated motor proteins.

BDNF-trkB-KCC2-GABA pathway may be related to chronic stress-induced hyperalgesia at both the spinal and supraspinal level

Chronic stresses can induce physiological and psychological changes and result in stress-related neuropsychiatry, sometimes they may also contribute to hypersensitivity to pain known as stress-induced hyperalgesia (SIH). Recently it was found that GABAergic disinhibition is closely related to morphine and chronic inflammation pain induced hyperalgesia in the PAG (periaqueductal gray)-RVM (rostral ventromedial medulla) -spinal descending facilitatory system, the changes of potassium-chloride co-transporter 2 (KCC2) function or expression modulated by brain-derived neurotrophic factor (BDNF) are involved in it. Similarly, chronic stress could induce changes of BDNF, KCC2 and a depolarizing shift in the GABA reversal potential (EGABA). Moreover, the descending facilitatory system has been suggested to be related with SIH, but whether it modulates SIH through disinhibition by the downregulation of KCC2 expression and/or transport function remains unknown. We therefore hypothesized that SIH may be owned to disinhibition caused by the activation of BDNF-trkB-KCC2-GABA pathway, which would provide a new understanding for SIH.

The origin of enhanced electrocatalytic activity of Pt–M (M = Fe, Co, Ni, Cu, and W) alloys in PEM fuel cell cathodes: A DFT computational study

The origin of enhanced electrocatalytic activity of Pt–M (M=Fe, Co, Ni, Cu, and W) alloy catalysts in PEM fuel cell cathodes have been investigated by the periodic DFT geometry optimization and electronic structure calculations. The stability of Pt–M alloy catalysts and the electrocatalytic activity have been explained, and we confirmed that the electronic structure of surface Pt is tuned after alloying Pt with non-noble transition metals, thus leading to enhancement of the electrocatalytic activity of Pt–M alloys for the ORR. Meanwhile, a positive shift in reversible potential was predicted for OHads formation from H2O oxidation on the Pt3M alloy catalysts with Pt-skin compared to the pure Pt. Therefore, this improvement of electrocatalytic activity of the ORR can be also attributed to a positive shift of the onset potential for forming OHads on the alloys relative to the Pt catalyst; thereby the overpotential for O2 reduction is reduced. In these ways we could screen new electrocatalysts for the ORR.

Nitric oxide signaling modulates synaptic inhibition in the superior paraolivary nucleus (SPN) via cGMP-dependent suppression of KCC2.

Glycinergic inhibition plays a central role in the auditory brainstem circuitries involved in sound localization and in the encoding of temporal action potential firing patterns. Modulation of this inhibition has the potential to fine-tune information processing in these networks. Here we show that nitric oxide (NO) signaling in the auditory brainstem (where activity-dependent generation of NO is documented) modulates the strength of inhibition by changing the chloride equilibrium potential. Recent evidence demonstrates that large inhibitory postsynaptic currents (IPSCs) in neurons of the superior paraolivary nucleus (SPN) are enhanced by a very low intracellular chloride concentration, generated by the neuronal potassium chloride co-transporter (KCC2) expressed in the postsynaptic neurons. Our data show that modulation by NO caused a 15 mV depolarizing shift of the IPSC reversal potential, reducing the strength of inhibition in SPN neurons, without changing the threshold for action potential firing. Regulating inhibitory strength, through cGMP-dependent changes in the efficacy of KCC2 in the target neuron provides a postsynaptic mechanism for rapidly controlling the inhibitory drive, without altering the timing or pattern of the afferent spike train. Therefore, this NO-mediated suppression of KCC2 can modulate inhibition in one target nucleus (SPN), without influencing inhibitory strength of other target nuclei (MSO, LSO) even though they are each receiving collaterals from the same afferent nucleus (a projection from the medial nucleus of the trapezoid body, MNTB).

Characterization of SLC4A11 as a novel ammonia: 2H+ transporter (893.29)

SLC4A11 mutations cause corneal endothelial dystrophies. This degenerative condition leads to corneal haze, edema, and vision loss. SLC4A11 is a recently identified membrane transporter, of which multiple substrates have been suggested. Intracellular pH measurements in SLC4A11‐expressing PS120 cells revealed that SLC4A11 is NH3/NH4+ and H+ permeable. To further characterize SLC4A11 substrates and transport stoichiometry, we performed patch‐clamp recordings in SLC4A11‐PS120 cells. We found that extracellular 10mM NH3/NH4+ induced inward current, which was inhibited by bath acidification and potentiated by bath alkalinization. Current density (pA/pF) at pHo 6.5, 7.5 and 8.5 were ‐0.22±0.19, ‐6.18±1.09, ‐18.43±3.43, respectively (mean±SE, p<0.001). Reversal potential shift in response to [NH3]o and pHo was consistent with an NH3/H+ coupled transport model. 10mM methylammonium induced smaller current (p<0.05) with similar pH sensitivity. Removal of K+, Na+, Cl‐ did not affect SLC4A11 NH3/H+ transport (K+ p=0.90, Na+ p=0.06, Cl‐ p=0.65), suggesting neither Na+, K+, nor Cl‐ is substrate. Stoichiometry of SLC4A11 NH3:H+ transport was estimated by measuring Erev at different [NH3]o. A plot of Erev against log[NH3]o was linear, and the slope was consistent with a 1:2 stoichiometry. We here identify SLC4A11 as a new member of ammonia channels/transporters, such as AQP8, Rh, MEP, and Amt.Grant Funding Source: Supported by 5R01EY008834(JAB); 5R01HL115140(AGO).

Deciphering the Kinetic and Gating Properties of Purinergic P2X7 Receptor Channels

Purinergic P2X7 receptors (P2X7Rs) are trimeric nonselective cation channels possessing three intrasubunit orthosteric binding sites and unidentified number of allosteric binding sites. In previous work, we showed that the same receptor is capable of exhibiting sensitization and pore dilation (leading to the formation of biphasic currents and cell death) and/or desensitization (leading to a decline in current amplitude and cell life signaling) during sustained application of orthosteric agonists, depending on their concentrations. This was done in part by developing a 12-state Markov model consisting of naive (not previously exposed to orthosteric agonist), desensitized, and sensitized/dilated states that reproduced whole-cell current recordings generated experimentally. In this model, we assumed that the occupancy of one or two ATP-binding sites of naive receptors favored transition from open to desensitized states, whereas the occupancy of the third binding site favored receptor sensitization. Our goal here is to extend this modeling study by examining how these model cells behave under various ionic conditions in the medium, including those that contain large organic cation N-methylD-glucamine (NMDG + ) or divalent cation such as Ca 2+ . We illustrate how the two main gating patterns behave under these ionic conditions and determine why the shift in reversal potential and the dilation of the channels are accompanied paradoxically by a decrease in the total conductance during voltage ramp protocols. The model adds more evidence to our previous hypothesis, suggesting that dilation is masking desensitization. Our results also indicate that the allosteric sites through which divalent cations inhibit P2X7R should be extracellularly located.

Non-Equilibrium Gating of CFTR Revealed by Nitrate as Charge Carriers

CFTR, a member of the ATP-binding cassette protein superfamily, is a phosphorylation-activated but ATP-gated chloride channel. Like other anion channels, CFTR's pore is permeable to a wide variety of anions, including bromide, nitrate, iodide and bicarbonate. From the shift of reversal potentials under bi-ionic conditions for chloride, bromide and nitrate, we obtained a permeability sequence: NO3- > Br- > Cl-, a result consistent with previous reports, but the macroscopic conductance sequence, NO3- > Br- > Cl-, contradicts previously published Cl- > NO3- > Br-. Nonetheless, single-channel studies reveal a conductance sequence of Cl- > NO3- > Br-, suggesting bromide and nitrate may affect CFTR gating. By analyzing single channel kinetics, we found that NO3- indeed increases the open probability (0.71±0.01 versus 0.51±0.02 with Cl-) by increasing the opening rate and prolonging the open time. Interestingly, when examining recordings from patches containing one single channel in nitrate-based bath, we observed two distinct open-channel conductance levels (the smaller O1 state and the larger O2 state), a phenomenon similar to the effect of the R352C mutation on CFTR. Furthermore, statistical analysis of the pattern of gating transitions also reveals a prevalent transition of C→O1→O2→ C (46.3%) over C→O2→O1→ C (< 5%), a telltale sign for a violation of detailed balance and hence demanding an input of free energy to drive the gating transition in a preferred direction. In addition, a considerable fraction of openings contain more than one O1→O2 transition (38%), supporting the idea that more than one ATP molecule is hydrolyzed within an opening burst. Overall our studies indicate that nitrate, as a charge carrier, can be a new tool to probe CFTR's non-equilibrium gating cycle.

High glutamate permeability and distal localization of Best1 channel in CA1 hippocampal astrocyte

BackgroundGlutamate is the major neurotransmitter that mediates a principal form of excitatory synaptic transmission in the brain. From the presynaptic terminals of neurons, glutamate is released upon exocytosis of the glutamate-packaged vesicles. In recent years, astrocytes are also known to release glutamate via various routes to modulate synaptic transmission. In particular, we have characterized a glutamate-permeable Ca2+-activated anion channel encoded by Bestrophin 1 gene (Best1) that is responsible for Ca2+-dependent, channel-mediated glutamate release in astrocyte. Best1 channel contains a large pore that is readily permeable to large molecules such as glutamate and GABA. In those studies we obtained permeability ratio of glutamate to Cl- in heterologously expressed mouse Best1 in HEK293T cells and in endogenously expressed mouse Best1 in cultured astrocytes. However, up to now, glutamate permeability of the native Best1 channel in vivo has not been reported.FindingsIn whole-cell recordings of CA1 hippocampal astrocytes, we found that opening of Best1 channel upon activation of a Gq-coupled GPCR, protease-activated receptor 1 (PAR1) generated the anion current carried by glutamate via Ca2+ increase. This Ca2+-evoked glutamate-mediated anion current was unaffected by pretreatment of the inhibitors for a gap junction hemi-channel or Ca2+-activated K+ conductance. This astrocytic anion conductance carried by glutamate was mediated by Best1 channel expression in CA1 hippocampal astrocytes, because Best1 knock-down by shRNA expression eliminated astrocytic glutamate conductance by PAR-1 activation. However, we found that these astrocytes showed a deviation in reversal potential of Best1-mediated current from the predicted value. By performing dual patch recording, we concluded that the deviation of reversal potential is due to incomplete space clamping arising from extremely leaky membrane (input resistance ranging 1–3 MΩ), very low length constant of astrocytic processes, and the localization of Best1 channel in distal microdomains near synapses. Based on the relative shift of reversal potentials by ion substitutions, we estimated the permeability ratio of glutamate and Cl- (Pglutamate/PCl) as 0.53.ConclusionsOur study shows that Best1, located at the microdomains near the synaptic junctions, has a significantly high permeability to glutamate in vivo, serving as the prominent glutamate-releasing channel in astrocytes, mediating the release of various gliotransmitters in the brain, and playing an important role in modulating synaptic transmission.

Seizure-induced alterations in fast-spiking basket cell GABA currents modulate frequency and coherence of gamma oscillation in network simulations

Gamma frequency oscillations have been proposed to contribute to memory formation and retrieval. Fast-spiking basket cells (FS-BCs) are known to underlie development of gamma oscillations. Fast, high amplitude GABA synapses and gap junctions have been suggested to contribute to gamma oscillations in FS-BC networks. Recently, we identified that, apart from GABAergic synapses, FS-BCs in the hippocampal dentate gyrus have GABAergic currents mediated by extrasynaptic receptors. Our experimental studies demonstrated two specific changes in FS-BC GABA currents following experimental seizures [Yu et al., J. Neurophysiol. 109, 1746 (2013)]: increase in the magnitude of extrasynaptic (tonic) GABA currents and a depolarizing shift in GABA reversal potential (E(GABA)). Here, we use homogeneous networks of a biophysically based model of FS-BCs to examine how the presence of extrasynaptic GABA conductance (g(GABA-extra)) and experimentally identified, seizure-induced changes in g(GABA-extra) and E(GABA) influence network activity. Networks of FS-BCs interconnected by fast GABAergic synapses developed synchronous firing in the dentate gamma frequency range (40-100 Hz). Systematic investigation revealed that the biologically realistic range of 30 to 40 connections between FS-BCs resulted in greater coherence in the gamma frequency range when networks were activated by Poisson-distributed dendritic synaptic inputs rather than by homogeneous somatic current injections, which were balanced for FS-BC firing frequency in unconnected networks. Distance-dependent conduction delay enhanced coherence in networks with 30-40 FS-BC interconnections while inclusion of gap junctional conductance had a modest effect on coherence. In networks activated by somatic current injections resulting in heterogeneous FS-BC firing, increasing g(GABA-extra) reduced the frequency and coherence of FS-BC firing when E(GABA) was shunting (-74 mV), but failed to alter average FS-BC frequency when E(GABA) was depolarizing (-54 mV). When FS-BCs were activated by biologically based dendritic synaptic inputs, enhancing g(GABA-extra) reduced the frequency and coherence of FS-BC firing when E(GABA) was shunting and increased average FS-BC firing when E(GABA) was depolarizing. Shifting E(GABA) from shunting to depolarizing potentials consistently increased network frequency to and above high gamma frequencies (>80 Hz). Since gamma oscillations may contribute to learning and memory processing [Fell et al., Nat. Neurosci. 4, 1259 (2001); Jutras et al., J. Neurosci. 29, 12521 (2009); Wang, Physiol. Rev. 90, 1195 (2010)], our demonstration that network oscillations are modulated by extrasynaptic inhibition in FS-BCs suggests that neuroactive compounds that act on extrasynaptic GABA receptors could impact memory formation by modulating hippocampal gamma oscillations. The simulation results indicate that the depolarized FS-BC GABA reversal, observed after experimental seizures, together with enhanced spillover extrasynaptic GABA currents are likely to promote generation of focal high frequency activity associated with epileptic networks.

Cl− homeodynamics in gap junction‐coupled astrocytic networks on activation of GABAergic synapses

The electrophysiological properties and functional role of GABAergic signal transmission from neurons to the gap junction-coupled astrocytic network are still unclear. GABA-induced astrocytic Cl− flux has been hypothesized to affect the driving force for GABAergic transmission by modulating [Cl−]o. Thus, revealing the properties of GABA-mediated astrocytic responses will deepen our understanding of GABAergic signal transmission. Here, we analysed the Cl− dynamics of neurons and astrocytes in CA1 hippocampal GABAergic tripartite synapses, using Cl− imaging during GABA application, and whole cell recordings from interneuron–astrocyte pairs in the stratum lacunosum-moleculare. Astrocytic [Cl−]i was adjusted to physiological conditions (40 mm). Although GABA application evoked bidirectional Cl− flux via GABAA receptors and mouse GABA transporter 4 (mGAT4) in CA1 astrocytes, a train of interneuron firing induced only GABAA receptor-mediated inward currents in an adjacent astrocyte. A GAT1 inhibitor increased the interneuron firing-induced currents and induced bicuculline-insensitive, mGAT4 inhibitor-sensitive currents, suggesting that synaptic spillover of GABA predominantly induced the astrocytic Cl− efflux because GABAA receptors are localized near the synaptic clefts. This GABA-induced Cl− efflux was accompanied by Cl− siphoning via the gap junctions of the astrocytic network because gap junction inhibitors significantly reduced the interneuron firing-induced currents. Thus, Cl− efflux from astrocytes is homeostatically maintained within astrocytic networks. A gap junction inhibitor enhanced the activity-dependent depolarizing shifts of reversal potential of neuronal IPSCs evoked by repetitive stimulation to GABAergic synapses. These results suggest that Cl− conductance within the astrocytic network may contribute to maintaining GABAergic synaptic transmission by regulating [Cl−]o.

Why do some brains seize? Molecular, cellular and network mechanisms

Why do some brains seize? Molecular, cellular and network mechanisms

Lipopolysaccharide-sensitive H+ current in dendritic cells

Dendritic cells (DCs) are the most potent antigen-presenting cells equipped to transport antigens from the periphery to lymphoid tissues and to present them to T cells. Ligation of Toll-like receptor 4 (TLR4), expressed on the DC surface, by lipopolysaccharides (LPS), elements of the Gram-negative bacteria outer wall, induces DC maturation. Initial steps of maturation include stimulation of antigen endocytosis and enhanced reactive oxygen species (ROS) production with eventual downregulation of endocytic capacity in fully matured DCs. ROS production depends on NADPH oxidase (NOX2), the activity of which requires continuous pH and charge compensation. The present study demonstrates, for the first time, the functional expression of voltage-gated proton (Hv1) channels in mouse bone marrow-derived DCs. In whole cell patch-clamp experiments, we recorded Zn(2+) (50 μM)-sensitive outwardly rectifying currents activated upon depolarization, which were highly selective for H(+), with the reversal potential shift of 38 mV per pH unit. The threshold voltage of activation (V(threshold)) was dependent on the pH gradient and was close to the empirically predicted V(threshold) for the Hv1 currents. LPS (1 μg/ml) had bimodal effects on Hv1 channels: acute LPS treatment increased Hv1 channel activity, whereas 24 h of LPS incubation significantly inhibited Hv1 currents and decreased ROS production. Activation of H(+) currents by acute application of LPS was abolished by PKC inhibitor GFX (10 nM). According to electron current measurements, acute LPS application was associated with increased NOX2 activity.

Monovalent Ion Selectivity of the Homotetrameric Bacterial Na Channel, NaChBac

NaChBac, from Bacillus halodurans, is a homotetrameric Nav channel, with each monomer having 6 putative transmembrane helices. Its selectivity filter appears to be lined by a ring of 4 glutamate residues (E191 from each monomer). We expressed NaChBac channels in HEK293 cells and studied their selectivity to monovalent organic and alkali cations. Reversal potential shifts were determined from whole-cell currents, following substitution of a test cation for extracellular Na+, and relative permeabilities (PX/PNa) were calculated using the Goldman-Hodgkin-Katz equation. Reversal potentials (Erev) were routinely measured from plots of peak INa vs V, but for ions showing small inward currents, values were checked against tail currents, measured immediately after a maximally activating prepulse. Among the alkali cations, only Na+, Li+ and K+ were measurably permeant. Of 14 organic cations, at pH 7.4, only hydrazinium (HZ+) was measurably permeant (PX/PNa, ≈ 0.8, 0.3, 0.7 respectively, for X= Li, K, HZ; <0.1 for Rb and ammonium). In contrast to eukaryotic, 4-domain Nav channels, neither ammonium nor hydroxyl-ammonium (HA+) was measurably permeant. The NaChBac mutant, E191D, showed diminished selectivity, reflected by increased values for PX/PNa. This mutation conserves the charge in the putative selectivity filter ring, but likely enlarges the filter diameter because of the shorter aspartate side chains, thereby reducing the stringent selectivity attained by the WT channel. For E191D channels, ammonium (PNH4/PNa ≈ 0.4), as well as hydroxyl-ammonium and some other small organic ions, showed measurable permeability. The recently determined crystal structure for the related channel, NavAb (Payandeh et al., 2011, Nature 475:353), opens the way, for the first time, to interpret selectivity of a voltage-dependent Na channel in the context of high-resolution structural data. Overall, our results suggest that NaChBac achieves selectivity for Na by a unique molecular strategy.