Hypoxia suppresses glutamate transport in astrocytes

Glutamate uptake by astrocytes is fundamentally important in the regulation of CNS function. Disruption of uptake can lead to excitotoxicity and is implicated in various neurodegenerative processes as well as a consequence of hypoxic/ischemic events. Here, we investigate the effect of hypoxia on activity and expression of the key glutamate transporters excitatory amino acid transporter 1 (EAAT1) [GLAST (glutamate-aspartate transporter)] and EAAT2 [GLT-1 (glutamate transporter 1)]. Electrogenic, Na+-dependent glutamate uptake was monitored via whole-cell patch-clamp recordings from cortical astrocytes. Under hypoxic conditions (2.5 and 1% O2 exposure for 24 h), glutamate uptake was significantly reduced, and pharmacological separation of uptake transporter subtypes suggested that the EAAT2 subtype was preferentially reduced relative to the EAAT1. This suppression was confirmed at the level of EAAT protein expression (via Western blots) and mRNA levels (via real-time PCR). These effects of hypoxia to inhibit glutamate uptake current and EAAT protein levels were not replicated by desferrioxamine, cobalt, FG0041, or FG4496, agents known to mimic effects of hypoxia mediated via the transcriptional regulator, hypoxia-inducible factor (HIF). Furthermore, the effects of hypoxia were not prevented by topotecan, which prevents HIF accumulation. In stark contrast, inhibition of nuclear factor-kappaB (NF-kappaB) with SN50 fully prevented the effects of hypoxia on glutamate uptake and EAAT expression. Our results indicate that prolonged hypoxia can suppress glutamate uptake in astrocytes and that this effect requires activation of NF-kappaB but not of HIF. Suppression of glutamate uptake via this mechanism may be an important contributory factor in hypoxic/ischemic triggered glutamate excitotoxicity.

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

Potassium Current in Mature Bovine Spermatozoa

Ion channels have been assigned a pivotal importance in various sperm functions and are therefore promising targets for contraceptive development. The lack of data on channel functionality and pharmacology has hampered this goal. This is a consequence of technical problems of applying electrophysiological techniques to spermatozoa due to their small size and form. By using a laminin coating to increase adherence of spermatozoa and nystatin in the patch pipette for pore formation, we have adapted the whole-cell recording technique to study currents in mature uncapacitated bovine spermatozoa. Employing these conditions, in the head region, patched spermatozoa could be transferred into the whole-cell configuration. For the first time we document an outward rectifying current in mature bovine spermatozoa was blocked by tetraethyl ammonium (TEA) chloride. The observation of a shift in the reversal potential as a response to changes in the extracellular concentration of K+ ions allowed us to identify this current as K+ selective. This result shows that K+ channels in the head region of mature uncapacitated bovine spermatozoa can be suitably investigated using the whole-cell recording patch-clamp technique.

Elektrophysiologische, molekularbiologische und verhaltensbiologische Untersuchungen zur Funktion von N-Methyl-D-Aspartat-Rezeptoren bei Gehirnentwicklung und Lernvorgängen

Im Zentralnervensystem der Säuger steuern N-Methyl-D-Aspartat-(NMDA)-Rezeptoren viele neuronale Prozesse, insbesondere während der Ontogenese sowie bei Lern- und Gedächtnisvorgängen. In der vorliegenden Arbeit wurde die Bedeutung dieser Rezeptoren während der Kortexentwicklung und bei Lernvorgängen mittels elektrophysiologischer, molekularbiologischer, pharmakologischer, histologischer, genetischer und verhaltensbiologischer Methoden an der Maus untersucht. Oszillatorische Netzwerkaktivität ist für die gesunde Entwicklung des Kortex essentiell. Mittels gepaarter patch-clamp Experimente an neonatalen Subplattenzellen wurde festgestellt, dass diese Neurone elektrisch gekoppelt sind. Damit könnten sie einen wichtigen Beitrag zur Entstehung bzw. Verstärkung von Netzwerkoszillationen leisten. Subplattenzellen erhalten afferenten Eingang aus dem Thalamus sowie von benachbarten Subplattenzellen. Die funktionellen und molekularen Eigenschaften dieser Synapsen differierten in eingangsspezifischer Weise. Subplatteninterne Verbindungen besaßen Integrations- und Summationsfähigkeiten, wenig synaptische Ermüdung, Paarpulsfazilitierung und einen erhöhten NR2D-Anteil in ihren NMDA-Rezeptoren. CA1-Pyramidenzellen des adulten Hippocampus zeigten eine den Subplattenzellen vergleichbare eingangsspezifische Verteilung der NMDA-Rezeptor-Untereinheiten. Synapsen von Schaffer-Kollateralen besaßen einen höheren NR2B-Anteil als temporo-ammonische Verbindungen. Die Aktivierung von Dopamin-Rezeptoren potenzierte NR2B-vermittelte synaptische Ströme in CA1-Neuronen. Bei komplexen Lernvorgängen, wie der Extinktion einer traumatischen Erinnerung, spielten NMDA-Rezeptoren von hippocampalen CA1-Zellen eine entscheidende Rolle. CA1-NMDA-Rezeptor-ko-Mäuse zeigten erhebliche Extinktionsdefizite nach Angstkonditionierung. Zudem entwickelten diese Mäuse erhöhte Ängstlichkeit und Hyperaktivität. Das sind beim Menschen Symptome für psychiatrische Angststörungen. Daher könnten CA1-NMDA-Rezeptor-ko-Mäuse als neues Tiermodell für solche Störungen dienen, die durch ein traumatisches Erlebnis ausgelöst werden, wie beim Posttraumatischen Stresssyndrom (PTSD).

Resonance properties of different neuronal populations in the immature mouse neocortex

Subthreshold resonance is a characteristic membrane property of different neuronal classes, is critically involved in the generation of network oscillations, and tunes the integration of synaptic inputs to particular frequency ranges. In order to investigate whether resonance properties of distinct neuronal populations in the immature neocortex contribute to these network oscillations, I performed whole-cell patch-clamp recordings from visually identified neurons in tangential and coronal neocortical slices from postnatal day (P) P0-P7 C57Bl/6 and P6-P13 GAD67-GFP knock-in mice. Subthreshold resonance was analyzed by sinusoidal current injection of varying frequency. All Cajal-Retzius cells showed subthreshold resonance with an average frequency of 2.6 ± 0.1 Hz (n=60), which was massively reduced by ZD7288, a blocker of hyperpolarization-activated cation currents. About 65.6% (n=61) of the supragranular pyramidal neurons showed subthreshold resonance with an average frequency of 1.4 ± 0.1 Hz (n=40). Application of 1 mM Ni2+ suppressed subthreshold resonance, suggesting that low-threshold Ca2+ currents contribute to resonance in these neurons. About 63.6% (n=77) of the layer V pyramidal neurons showed subthreshold resonance with an average frequency of 1.4 ± 0.2 Hz (n=49), which was abolished by ZD7288. Only 44.1% (n=59) of the subplate neurons showed subthreshold resonance with an average frequency of 1.3 ± 0.2 Hz (n=26) and a small resonance strength. Finally, 50% of the investigated GABAergic interneurons showed subthreshold resonance with an average frequency of 2.0 ± 0.2 Hz (n=42). Membrane hyperpolarization to –86 mV attenuated the frequency and strength of subthreshold resonance. Subthreshold resonance was virtually abolished in the presence of 1 mM Ni2+, suggesting that t-type Ca2+ currents are critically involved in the generation of resonance, while ZD7288 had no effect. Application of 0.4 µM TTX suppressed subthreshold resonance at depolarized, but not hyperpolarized membrane potential, suggesting that persistent Na+ current contribute to the amplification of membrane resonance. rnIn summary, these results demonstrate that all investigated neuronal subpopulations reveal resonance behavior, with either hyperpolarization-activated cation or low-threshold Ca2+ currents contributing to the subthreshold resonance. GABAergic interneurons also express subthreshold resonance at low frequencies, with t-type Ca2+ and persistent Na+ currents underlying the generation of membrane resonance. The membrane resonance of immature neurons may contribute to the generation of slow oscillatory activity pattern in the immature neocortex and enhance the temporal precision of synaptic integration in developing cortical neurons.rn

Development of a fluorescent-based single liposome assay for studying calcium transporter LMCA1

The morphological and functional unit of all the living organisms is the cell. The transmembrane proteins, localized in the plasma membrane of cells, play a key role in the survival of the cells themselves. These proteins perform a variety of different tasks, for example the control of the homeostasis. In order to control the homeostasis, these proteins have to regulate the concentration of chemical elements, like ions, inside and outside the cell. These regulations are fundamental for the survival of the cell and to understand them we need to understand how transmembrane proteins work. Two of the most important categories of transmembrane proteins are ion channels and transporter proteins. The ion channels have been depth studied at the single molecule level since late 1970s with the development of patch-clamp technique. It is not possible to apply this technique to study the transporter proteins so a new technique is under development in order to investigate the behavior of transporter proteins at the single molecule level. This thesis describes the development of a nanoscale single liposome assay for functional studies of transporter proteins based on quantitative fluorescence microscopy in a highly-parallel manner and in real time. The transporter of interest is the prokaryotic transporter Listeria Monocytogenes Ca2+-ATPase1 (LMCA1), a structural analogue of the eukaryotic calcium pumps SERCA and PMCA. This technique will allow the characterization of LMCA1 functionality at the single molecule level. Three systematically characterized fluorescent sensors were tested at the single liposome scale in order to investigate if their properties are suitable to study the function of the transporter of interest. Further studies will be needed in order to characterize the selected calcium sensor and pH sensor both implemented together in single liposomes and in presence of the reconstituted protein LMCA1.

Dendritic spikes in apical dendrites of neocortical layer 2/3 pyramidal neurons

Layer 2/3 (L2/3) pyramidal neurons are the most abundant cells of the neocortex. Despite their key position in the cortical microcircuit, synaptic integration in dendrites of L2/3 neurons is far less understood than in L5 pyramidal cell dendrites, mainly because of the difficulties in obtaining electrical recordings from thin dendrites. Here we directly measured passive and active properties of the apical dendrites of L2/3 neurons in rat brain slices using dual dendritic-somatic patch-clamp recordings and calcium imaging. Unlike L5 cells, L2/3 dendrites displayed little sag in response to long current pulses, which suggests a low density of I(h) in the dendrites and soma. This was also consistent with a slight increase in input resistance with distance from the soma. Brief current injections into the apical dendrite evoked relatively short (half-width 2-4 ms) dendritic spikes that were isolated from the soma for near-threshold currents at sites beyond the middle of the apical dendrite. Regenerative dendritic potentials and large concomitant calcium transients were also elicited by trains of somatic action potentials (APs) above a critical frequency (130 Hz), which was slightly higher than in L5 neurons. Initiation of dendritic spikes was facilitated by backpropagating somatic APs and could cause an additional AP at the soma. As in L5 neurons, we found that distal dendritic calcium transients are sensitive to a long-lasting block by GABAergic inhibition. We conclude that L2/3 pyramidal neurons can generate dendritic spikes, sharing with L5 pyramidal neurons fundamental properties of dendritic excitability and control by inhibition.

Basolateral membrane targeting of a renal-epithelial inwardly rectifying potassium channel from the cortical collecting duct, CCD-IRK3, in MDCK cells

We recently cloned an inward-rectifying K channel (Kir) cDNA, CCD-IRK3 (mKir 2.3), from a cortical collecting duct (CCD) cell line. Although this recombinant channel shares many functional properties with the “small-conductance” basolateral membrane Kir channel in the CCD, its precise subcellular localization has been difficult to elucidate by conventional immunocytochemistry. To circumvent this problem, we studied the targeting of several different epitope-tagged CCD-IRK3 in a polarized renal epithelial cell line. Either the 11-amino acid span of the vesicular stomatitis virus (VSV) G glycoprotein (P5D4 epitope) or a 6-amino acid epitope of the bovine papilloma virus capsid protein (AU1) was genetically engineered on the extreme N terminus of CCD-IRK3. As determined by patch-clamp and two-microelectrode voltage-clamp analyses in Xenopus oocytes, neither tag affected channel function; no differences in cation selectivity, barium block, single channel conductance, or open probability could be distinguished between the wild-type and the tagged constructs. MDCK cells were transfected with tagged CCD-IRK3, and several stable clonal cell lines were generated by neomycin-resistance selection. Immunoprecipitation studies with anti-P5D4 or anti-AU1 antibodies readily detected the predicted-size 50-kDa protein in the transfected cells lines but not in wild-type or vector-only (PcB6) transfected MDCK cells. As visualized by indirect immunofluorescence and confocal microscopy, both the tagged CCD-IRK3 forms were exclusively detected on the basolateral membrane. To assure that the VSV G tag was not responsible for the targeting, the P5D4 epitope modified by a site-directed mutagenesis (Y2F) to remove a potential basolateral targeting signal contained in this tag. VSV(Y2F) was also detected exclusively on the basolateral membrane, confirming bona fide IRK3 basolateral expression. These observations, with our functional studies, suggest that CCD-IRK3 may encode the small-conductance CCD basolateral K channel.

KATP channel inhibition by ATP requires distinct functional domains of the cytoplasmic C terminus of the pore-forming subunit

ATP-sensitive potassium (“KATP”) channels are rapidly inhibited by intracellular ATP. This inhibition plays a crucial role in the coupling of electrical activity to energy metabolism in a variety of cells. The KATP channel is formed from four each of a sulfonylurea receptor (SUR) regulatory subunit and an inwardly rectifying potassium (Kir6.2) pore-forming subunit. We used systematic chimeric and point mutagenesis, combined with patch-clamp recording, to investigate the molecular basis of ATP-dependent inhibition gating of mouse pancreatic β cell KATP channels expressed in Xenopus oocytes. We identified distinct functional domains of the presumed cytoplasmic C-terminal segment of the Kir6.2 subunit that play an important role in this inhibition. Our results suggest that one domain is associated with inhibitory ATP binding and another with gate closure.

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP3) binding to its receptors (IP3R) in the endoplasmic reticulum (ER) activates Ca2+ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca2+ concentration signals including temporal oscillations and propagating waves. IP3-mediated Ca2+ release is also controlled by cytoplasmic Ca2+ concentration with both positive and negative feedback. Single-channel properties of the IP3R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP3R on cytoplasmic Ca2+ and IP3 concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca2+ concentration ([Ca2+]i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP3, even as [Ca2+]i was raised to high concentrations, displaying two distinct types of functional Ca2+ binding sites: activating sites with half-maximal activating [Ca2+]i (Kact) of 210 nM and Hill coefficient (Hact) ≈2; and inhibitory sites with half-maximal inhibitory [Ca2+]i (Kinh) of 54 μM and Hill coefficient (Hinh) ≈4. Lowering IP3 concentration was without effect on Ca2+ activation parameters or Hinh, but decreased Kinh with a functional half-maximal activating IP3 concentration (KIP3) of 50 nM and Hill coefficient (HIP3) of 4 for IP3. These results demonstrate that Ca2+ is a true receptor agonist, whereas the sole function of IP3 is to relieve Ca2+ inhibition of IP3R. Allosteric tuning of Ca2+ inhibition by IP3 enables the individual IP3R Ca2+ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca2+ concentration signaling and quantal Ca2+ release.

Starburst amacrine cells change from spiking to nonspiking neurons during retinal development.

The membrane excitability of cholinergic (starburst) amacrine cells was studied in the rabbit retina during postnatal development. Whole-cell patch-clamp recordings were made from 110 displaced starburst cells in a thin retina] slice preparation of rabbits between postnatal days P1 and P56 old. We report that displaced starburst cells undergo a dramatic transition from spiking to nonspiking, caused by a loss of voltage-gated Na currents. This change in membrane excitability occurred just after eye opening (P10), such that all of the starburst cells tested before eye opening had conspicuous tetrodotoxin-sensitive Na currents and action potentials, but none tested after the first 3 postnatal weeks had detectable Na currents or spikes. Our results suggest that starburst cells use action potentials transiently during development and probably play a functional role in visual development. These cells then cease to spike as the retina matures, presumably consistent with their role in visual processing in the mature retina.

Receptor stimulation causes slow inhibition of IRK1 inwardly rectifying K+ channels by direct protein kinase A-mediated phosphorylation.

Strongly rectifying IRK-type inwardly rectifying K+ channels are involved in the control of neuronal excitability in the mammalian brain. Whole-cell patch-clamp experiments show that cloned rat IRK1 (Kir 2.1) channels, when heterologously expressed in mammalian COS-7 cells, are inhibited following the activation of coexpressed serotonin (5-hydroxytryptamine) type 1A receptors by receptor agonists. Inhibition is mimicked by internal perfusion with GTP[gamma-S] and elevation of internal cAMP concentrations. Addition of the catalytic subunits of protein kinase A (PKA) to the internal recording solution causes complete inhibition of wild-type IRK1 channels, but not of mutant IRK1(S425N) channels in which a C-terminal PKA phosphorylation site has been removed. Our data suggest that in the nervous system serotonin may negatively control IRK1 channel activity by direct PKA-mediated phosphorylation.

Antisense oligodeoxynucleotide inhibition of a swelling-activated cation channel in osteoblast-like osteosarcoma cells.

By patch-clamp analysis, we have shown that chronic, intermittent mechanical strain (CMS) increases the activity of stretch-activated cation channels of osteoblast-like UMR-106.01 cells. CMS also produces a swelling-activated whole-cell conductance (Gm) regulated by varying strain levels. We questioned whether the swelling-activated conductance was produced by stretch-activated cation channel activity. We have identified a gene involved in the increase in conductance by using antisense oligodeoxynucleotides (ODN) derived from the alpha 1-subunit genes of calcium channels found in UMR-106.01 cells (alpha1S, alpha1C, and alpha1D). We demonstrate that alpha 1C antisense ODNs abolish the increase in Gm in response to hypotonic swelling following CMS. Antisense ODNs to alpha1S and alpha1D, sense ODNs to alpha1C, and sham permeabilization had no effect on the conductance increase. In addition, during cell-attached patch-clamp studies, antisense ODNs to alpha1c completely blocked the swelling-activated and stretch-activated nonselective cation channel response to strain. Antisense ODNs to alpha1S treatment produced no effect on either swelling-activated or stretch-activated cation channel activity. There were differences in the stretch-activated and swelling-activated cation channel activity, but whether they represent different channels could not be determined from our data. Our data indicate that the alpha1C gene product is involved in the Gm and the activation of the swelling-activated cation channels induced by CMS. The possibility that swelling-activated cation channel genes are members of the calcium channel superfamily exists, but if alpha1c is not the swelling-activated cation channel itself, then its expression is required for induction of swelling-activated cation channel activity by CMS.

Presynaptic modulation of cortical synaptic activity by calcineurin.

Synaptic plasticity is modulated by Ca(2+)-induced alterations in the balance between phosphorylation and dephosphorylation. Recent evidence suggests that calcineurin, the Ca(2+)-calmodulin-dependent phosphatase (2B), modulates the activity of postsynaptic glutamate receptors. However, in rat cortex, calcineurin is enriched mainly in presynaptic, not postsynaptic, fractions. To determine if calcineurin modulates glutamatergic neurotransmission through a presynaptic mechanism, we used whole-cell patch clamp experiments to test effects of two specific calcineurin inhibitors, cyclosporin A (CsA) and FK506, on synaptic activity in fetal rat cortical neurons. The rate of spontaneous action-potential firing was markedly increased by either CsA or FK506 but was unaffected by rapamycin, a structural analog of FK506 which has no effect on calcineurin. In voltage-clamp experiments, CsA increased the rate but not the amplitude of glutamate receptor-mediated, excitatory postsynaptic currents, suggesting an increased rate of glutamate release. CsA had no effect on the amplitude of currents evoked by brief bath application of selective glutamate receptor agonists, providing further evidence for a pre- rather than postsynaptic site of action. In conclusion, these data indicate that calcineurin modulates glutamatergic neurotransmission in rat cortical neurons through a presynaptic mechanism.

Plasmodium induces swelling-activated ClC-2 anion channels in the host erythrocyte

Intraerythrocytic growth of the human malaria parasite Plasmodium falciparum depends on delivery of nutrients. Moreover, infection challenges cell volume constancy of the host erythrocyte requiring enhanced activity of cell volume regulatory mechanisms. Patch clamp recording demonstrated inwardly and outwardly rectifying anion channels in infected but not in control erythrocytes. The molecular identity of those channels remained elusive. We show here for one channel type that voltage dependence, cell volume sensitivity, and activation by oxidation are identical to ClC-2. Moreover, Western blots and FACS analysis showed protein and functional ClC-2 expression in human erythrocytes and erythrocytes from wild type (Clcn2(+/+)) but not from Clcn2(-/-) mice. Finally, patch clamp recording revealed activation of volume-sensitive inwardly rectifying channels in Plasmodium berghei-infected Clcn2(+/+) but not Clcn2(-/-) erythrocytes. Erythrocytes from infected mice of both genotypes differed in cell volume and inhibition of ClC-2 by ZnCl(2) (1 mm) induced an increase of cell volume only in parasitized Clcn2(+/+) erythrocytes. Lack of ClC-2 did not inhibit P. berghei development in vivo nor substantially affect the mortality of infected mice. In conclusion, activation of host ClC-2 channels participates in the altered permeability of Plasmodium-infected erythrocytes but is not required for intraerythrocytic parasite survival.