Interactive cloning with the SH3 domain of N-src identifies a new brain specific ion channel protein, with homology to Eag and cyclic nucleotide-gated channels

We have isolated a novel cDNA, that appears to represent a new class of ion channels, by using the yeast two-hybrid system and the SH3 domain of the neural form of Src (N-src) as a bait. The encoded polypeptide, BCNG-1, is distantly related to cyclic nucleotide-gated channels and the voltage-gated channels, Eag and H-erg. BCNG-1 is expressed exclusively in the brain, as a glycosylated protein of ≈132 kDa. Immunohistochemical analysis indicates that BCNG-1 is preferentially expressed in specific subsets of neurons in the neocortex, hippocampus, and cerebellum, in particular pyramidal neurons and basket cells. Within individual neurons, the BCNG-1 protein is localized to either the dendrites or the axon terminals depending on the cell type. Southern blot analysis shows that several other BCNG-related sequences are present in the mouse genome, indicating the emergence of an entire subfamily of ion channel coding genes. These findings suggest the existence of a new type of ion channel, which is potentially able to modulate membrane excitability in the brain and could respond to regulation by cyclic nucleotides.

Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through dephosphorylation of a specific tyrosine residue

Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca2+, which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the α-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.

Rat hippocampal neurons express genes for both rod retinal and olfactory cyclic nucleotide-gated channels: novel targets for cAMP/cGMP function.

Cyclic nucleotide-gated (CNG) channels are Ca(2+)-permeable, nonspecific cation channels that can be activated through direct interaction with cAMP and/or cGMP. Recent electrophysiological evidence for these channels in cultured hippocampal neurons prompted us to investigate the expression of CNG channel genes in hippocampus. PCR amplification detected the expression of transcripts for subunit 1 of both the rod photoreceptor (RCNGC1) and the olfactory receptor cell (OCNGC1) subtype of CNG channel in adult rat hippocampus. In situ hybridization detected expression of both channel subtypes in most principal neurons, including pyramidal cells of the CA1 through CA3 regions and granule cells of the dentate gyrus. From the hybridization patterns, we conclude that the two genes are colocalized in individual neurons. Comparison of the patterns of expression of type 1 cGMP-dependent protein kinase and the CNG channels suggests that hippocampal neurons can respond to changes in cGMP levels with both rapid changes in CNG channel activity and slower changes induced by phosphorylation. Future models of hippocampal function should include CNG channels and their effects on both electrical responses and intracellular Ca2+ levels.

Subunit interactions in coordination of Ni2+ in cyclic nucleotide-gated channels.

Cyclic nucleotide-gated (CNG) channels present a unique model for studying the molecular mechanisms of channel gating. We have studied the mechanism of potentiation of expressed rod CNG channels by Ni2+ as a first step toward understanding the channel gating process. Here we report that coordination of Ni2+ between histidine residues (H420) on adjacent channel subunits occurs when the channels are open. Mutation of H420 to lysine completely eliminated the potentiation by Ni2+ but did not markedly alter the apparent cGMP affinity of the channel, indicating that the introduction of positive charge at the Ni(2+)-binding site was not sufficient to produce potentiation. Deletion or mutation of most of the other histidines present in the channel did not diminish potentiation by Ni2+. We studied the role of subunit interactions in Ni2+ potentiation by generating heteromultimeric channels using tandem dimers of the rod CNG channel sequence. Injection of single heterodimers in which one subunit contained H420 and the other did not (wt/H420Q or H420Q/wt) resulted in channels that were not potentiated by Ni2+. However, coinjection of both heterodimers into Xenopus oocytes resulted in channels that exhibited potentiation. The H420 residues probably occurred predominantly in nonadjacent subunits when each heterodimer was injected individually, but, when the two heterodimers were coinjected, the H420 residues could occur in adjacent subunits as well. These results suggest that the mechanism of Ni2+ potentiation involves intersubunit coordination of Ni2+ by H420. Based on the preferential binding of Ni2+ to open channels, we suggest that alignment of H420 residues of neighboring subunits into the Ni(2+)-coordinating position may be associated with channel opening.

Probing the transmembrane topology of cyclic nucleotide-gated ion channels with a gene fusion approach.

Cyclic nucleotide-gated (CNG) cation channels contain two short sequence motifs--a residual voltage-sensor (S4) and a pore-forming (P) segment--that are reminiscent of similar segments in voltage-activated Shaker-type K+ channels. It has been tacitly assumed that CNG channels and this K+ channel subfamily share a common overall topology, characterized by a hydrophobic domain comprising six membrane-spanning segments. We have systematically investigated the topology of CNG channels from bovine rod photoreceptor and Drosophila melanogaster by a gene fusion approach using the bacterial reporter enzymes alkaline phosphatase and beta-galactosidase, which are active only in the periplasm and only in the cytoplasm, respectively. Enzymatic activity was determined after expression of fusion constructs in Escherichia coli. CNG channels were found to have six membrane-spanning segments, suggesting that CNG and Shaker-type K+ channels, albeit distant relatives within a gene superfamily of ion channels, share a common topology.

A pore-lining glutamic acid in the olfactory cyclic nucleotide-gated channel controls external spermine block

Spermine is a potent, voltage-dependent blocker of the olfactory cyclic nucleotide-gated channel from both the intracellular and extracellular sides. However, its sites of action are unknown. This study investigated the external spermine binding site in the rat CNC alpha3 subunit. Neutralization of a glutamic acid residue (E342Q) in the P-loop region eliminated voltage-dependence of block by externally applied spermine. The charge-conservative E342D mutation had little effect on spermine block. Thus, E342 forms the binding site for externally applied spermine. However, spermine remained a potent voltage-independent blocker of the E342Q mutant channel, suggesting that the mutation either created a novel binding site outside the membrane electrical field or that it dramatically changed the properties of the existing pore site. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.

Depolarisation and suppression of burst firing activity in the mouse subthalamic nucleus by dopamine D1/D5 receptor activation of a cyclic-nucleotide gated non-specific cation conductance

Neuronal burst firing in the subthalamic nucleus (STN) is one of the hallmarks of dopamine depletion in Parkinson's disease. Here, we have determined the postsynaptic effects of dopamine in the STN and the functional consequences of dopamine receptor modulation on burst firing in vitro. STN cells displayed regular spiking activity at a rate of 7.9 +/- 0.5 Hz. Application of dopamine (30 mu M) induced membrane depolarisations accompanied by an increase in firing rate of mean 12.0 +/- 0.6 Hz in all 69 cells. The dopamine effect was mimicked by the dopamine D1/D5 receptor agonist SKF38393 (10 mu M, 17 cells) and the dopamine D2-like receptor agonist quinpirole (10 mu M, 35 cells), partly reduced by D1/D5 antagonist SCH23390 (2 mu M, seven cells), but unaffected by the D2 antagonists sulpiride (10 mu M, seven cells) or eticlopride (10 mu M, six cells). Using voltage ramps, dopamine induced an inward current of 69 +/- 9.4 pA at a holding potential of -60 mV (n = 17). This current was accompanied by an increase in input conductance of 1.55 +/- 0.35 nS which reversed at -30.6 +/- 2.3 mV, an effect mimicked by SKF38393 (10 AM, nine cells). Similar responses were observed when measuring instantaneous current evoked by voltage steps and in the presence of the I-h blocker, ZD7288, indicating effects independent of I-h. The increase in conductance was blocked by SCH23390 (2 mu M, n = 4), mimicked by the activator of adenylyl cyclase forskolin (10 mu M, n = 7) and blocked by H-89, an inhibitor of cyclic AMP dependent protein kinase A (10 PM, n = 6). These results indicate that the dopamine depolarisation is in part mediated by D1/D5 receptor mediated activation of a cyclic-nucleotide gated (CNG) non-specific cation conductance. This conductance contributes to the membrane depolarisation that changes STN neuronal bursting to more regular activity by significantly increasing burst duration and number of spikes per burst.

Remyelination in vitro following protein kinase C activator-induced demyelination.

In previous work we found that mezerein, a C kinase activator, as well as basic fibroblast growth factor (FGF-2) induce demyelination and partial oligodendrocyte dedifferentiation in highly differentiated aggregating brain cell cultures. Here we show that following protein kinase C activator-induced demyelination, effective remyelination occurs. We found that mezerein or FGF-2 caused a transient increase in DNA synthesis following a pronounced decrease of the myelin markers myelin basic protein and 2',3'-cyclic nucleotide 3'-phosphohydrolase. Both oligodendrocytes and astrocytes were involved in this mitogenic response. Within 17 days after demyelination, myelin was restored to the level of the untreated controls. Transient mitotic activity was indispensable for remyelination. The present results suggest that myelinating oligodendrocytes retain the capacity to reenter the cell cycle, and that this plasticity is important for the regeneration of the oligodendrocyte lineage and remyelination. Although it cannot be excluded that a quiescent population of oligodendrocyte precursor cells was present in the aggregates and able to proliferate, differentiate and remyelinate, we could not find evidence supporting this view.

Phosphodiesterase-5 inhibition mimics intermittent reoxygenation and improves cardioprotection in the hypoxic myocardium.

Although chronic hypoxia is a claimed myocardial risk factor reducing tolerance to ischemia/reperfusion (I/R), intermittent reoxygenation has beneficial effects and enhances heart tolerance to I/R. AIM OF THE STUDY: To test the hypothesis that, by mimicking intermittent reoxygenation, selective inhibition of phosphodiesterase-5 activity improves ischemia tolerance during hypoxia. Adult male Sprague-Dawley rats were exposed to hypoxia for 15 days (10% O₂) and treated with placebo, sildenafil (1.4 mg/kg/day, i. p.), intermittent reoxygenation (1 h/day exposure to room air) or both. Controls were normoxic hearts. To assess tolerance to I/R all hearts were subjected to 30-min regional ischemia by left anterior descending coronary artery ligation followed by 3 h-reperfusion. Whereas hypoxia depressed tolerance to I/R, both sildenafil and intermittent reoxygenation reduced the infarct size without exhibiting cumulative effects. The changes in myocardial cGMP, apoptosis (DNA fragmentation), caspase-3 activity (alternative marker for cardiomyocyte apoptosis), eNOS phosphorylation and Akt activity paralleled the changes in cardioprotection. However, the level of plasma nitrates and nitrites was higher in the sildenafil+intermittent reoxygenation than sildenafil and intermittent reoxygenation groups, whereas total eNOS and Akt proteins were unchanged throughout. CONCLUSIONS: Sildenafil administration has the potential to mimic the cardioprotective effects led by intermittent reoxygenation, thereby opening the possibility to treat patients unable to be reoxygenated through a pharmacological modulation of NO-dependent mechanisms.

Stable transmission of targeted gene modification using single-stranded oligonucleotides with flanking LNAs.

Targeted mutagenesis directed by oligonucleotides (ONs) is a promising method for manipulating the genome in higher eukaryotes. In this study, we have compared gene editing by different ONs on two new target sequences, the eBFP and the rd1 mutant photoreceptor betaPDE cDNAs, which were integrated as single copy transgenes at the same genomic site in 293T cells. Interestingly, antisense ONs were superior to sense ONs for one target only, showing that target sequence can by itself impart strand-bias in gene editing. The most efficient ONs were short 25 nt ONs with flanking locked nucleic acids (LNAs), a chemistry that had only been tested for targeted nucleotide mutagenesis in yeast, and 25 nt ONs with phosphorothioate linkages. We showed that LNA-modified ONs mediate dose-dependent target modification and analyzed the importance of LNA position and content. Importantly, when using ONs with flanking LNAs, targeted gene modification was stably transmitted during cell division, which allowed reliable cloning of modified cells, a feature essential for further applications in functional genomics and gene therapy. Finally, we showed that ONs with flanking LNAs aimed at correcting the rd1 stop mutation could promote survival of photoreceptors in retinas of rd1 mutant mice, suggesting that they are also active in vivo.

Myelin gene expression during demyelination and remyelination in aggregating brain cell cultures.

Remyelination can be studied in aggregating rat brain cell cultures after limited demyelination. Demyelination was induced using a monoclonal antibody against myelin/oligodendrocyte glycoprotein (MOG mAb), in the presence of complement. De- and remyelination were assessed by measuring myelin basic protein (MBP). Two days after removing the MOG mAb, MBP levels reached 50% of controls and after 7 days 93%. During this period, cell proliferation determined by [14C]thymidine incorporation was similar in remyelinating and control cultures. Hormones and growth factors were tested for possible stimulatory effect on remyelinating cultures. Bovine growth hormone (bGH), triiodothyronine (T3), basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) did not improve remyelination. Only epidermal growth factor (EGF) increased the level of remyelination. PDGF increased the rate of cell proliferation in both control and remyelinating cultures. A significant proportion of oligodendrocytes entered the cell division cycle and were not available for remyelination. The results obtained with PDGF and FGF (inhibition) support the idea that a pool of progenitor cells was still present and able to proliferate and differentiate into myelinating oligodendrocytes. The levels of myelin protein mRNAs were investigated during de- and remyelination. During demyelination, myelin protein mRNA levels decreased to approximately 50% of control cultures and returned to normal during remyelination. These preliminary results indicate that normal levels of gene transcription are sufficient to meet the increased need for newly synthesized myelin proteins during remyelination.

Single-stranded oligonucleotide-mediated in vivo gene repair in the rd1 retina.

PURPOSE: The aim of this study was to test whether oligonucleotide-targeted gene repair can correct the point mutation in genomic DNA of PDE6b(rd1) (rd1) mouse retinas in vivo. METHODS: Oligonucleotides (ODNs) of 25 nucleotide length and complementary to genomic sequence subsuming the rd1 point mutation in the gene encoding the beta-subunit of rod photoreceptor cGMP-phosphodiesterase (beta-PDE), were synthesized with a wild type nucleotide base at the rd1 point mutation position. Control ODNs contained the same nucleotide bases as the wild type ODNs but with varying degrees of sequence mismatch. We previously developed a repeatable and relatively non-invasive technique to enhance ODN delivery to photoreceptor nuclei using transpalpebral iontophoresis prior to intravitreal ODN injection. Three such treatments were performed on C3H/henJ (rd1) mouse pups before postnatal day (PN) 9. Treatment outcomes were evaluated at PN28 or PN33, when retinal degeneration was nearly complete in the untreated rd1 mice. The effect of treatment on photoreceptor survival was evaluated by counting the number of nuclei of photoreceptor cells and by assessing rhodopsin immunohistochemistry on flat-mount retinas and sections. Gene repair in the retina was quantified by allele-specific real time PCR and by detection of beta-PDE-immunoreactive photoreceptors. Confirmatory experiments were conducted using independent rd1 colonies in separate laboratories. These experiments had an additional negative control ODN that contained the rd1 mutant nucleotide base at the rd1 point mutation site such that the sole difference between treatment with wild type and control ODN was the single base at the rd1 point mutation site. RESULTS: Iontophoresis enhanced the penetration of intravitreally injected ODNs in all retinal layers. Using this delivery technique, significant survival of photoreceptors was observed in retinas from eyes treated with wild type ODNs but not control ODNs as demonstrated by cell counting and rhodopsin immunoreactivity at PN28. Beta-PDE immunoreactivity was present in retinas from eyes treated with wild type ODN but not from those treated with control ODNs. Gene correction demonstrated by allele-specific real time PCR and by counts of beta-PDE-immunoreactive cells was estimated at 0.2%. Independent confirmatory experiments showed that retinas from eyes treated with wild type ODN contained many more rhodopsin immunoreactive cells compared to retinas treated with control (rd1 sequence) ODN, even when harvested at PN33. CONCLUSIONS: Short ODNs can be delivered with repeatable efficiency to mouse photoreceptor cells in vivo using a combination of intravitreal injection and iontophoresis. Delivery of therapeutic ODNs to rd1 mouse eyes resulted in genomic DNA conversion from mutant to wild type sequence, low but observable beta-PDE immunoreactivity, and preservation of rhodopsin immunopositive cells in the outer nuclear layer, suggesting that ODN-directed gene repair occurred and preserved rod photoreceptor cells. Effects were not seen in eyes treated with buffer or with ODNs having the rd1 mutant sequence, a definitive control for this therapeutic approach. Importantly, critical experiments were confirmed in two laboratories by several different researchers using independent mouse colonies and ODN preparations from separate sources. These findings suggest that targeted gene repair can be achieved in the retina following enhanced ODN delivery.

Developmental effects of basic fibroblast growth factor and platelet-derived growth factor on glial cells in a three-dimensional cell culture system.

In order to study peptide growth factor action in a three-dimensional cellular environment, aggregating cell cultures prepared from 15-day fetal rat telencephalon were grown in a chemically defined medium and treated during an early developmental stage with either bovine fibroblast growth factor (bFGF) or platelet-derived growth factor (PDGF homodimers AA and BB). A single dose (5-50 ng/ml) of either growth factor given to the cultures on day 3 greatly enhanced the developmental increase of the two glia-specific enzyme activities, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and glutamine synthetase (GS), whereas it had relatively little effect on total protein and DNA content. Distinct patterns of dose-dependency were found for CNP and GS stimulation. At low concentrations of bFGF (0.5-5 ng/ml) and at all PDGF concentrations applied, the oligodendroglial marker enzyme CNP was the most affected. A relatively small but significant mitogenic effect was observed after treatment with PDGF, particularly at higher concentrations or after repetitive stimulation. The two PDGF homodimers AA and BB were similar in their biological effects and potency. The present results show that under histotypic conditions both growth factors, bFGF and PDGF, promote the maturation rather than the proliferation of immature oligodendrocytes and astrocytes.

Inflammatory responses in aggregating rat brain cell cultures subjected to different demyelinating conditions.

To study inflammatory reactions occurring in relation to demyelination, aggregating rat brain cell cultures were subjected to three different demyelinating insults, i.e., (i) lysophosphatidylcholine (LPC), (ii) interferon-gamma combined with lipopolysaccharide (IFN-gamma+LPS), and (iii) anti-MOG antibodies plus complement (alpha-MOG+C). Demyelination was assessed by measuring the expression of myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), and the activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP). The accompanying inflammatory reactions were examined by the quantification of microglia-specific staining, by immunostaining for glial fibrillary acidic protein (GFAP), and by measuring the mRNA expression of a panel of inflammation-related genes. It was found that all three demyelinating insults decreased the expression of MBP and MOG, and induced microglial reactivity. LPC and alpha-MOG+C, but not IFN-gamma+LPS, decreased CNP activity; they also caused the appearance of macrophagic microglia, and increased GFAP staining indicating astrogliosis. LPC affected also the integrity of neurons and astrocytes. LPC and IFN-gamma+LPS upregulated the expression of the inflammation-related genes IL-6, TNF-alpha, Ccl5, Cxcl1, and iNOS, although to different degrees. Other inflammatory markers were upregulated by only one of the three insults, e.g., Cxcl2 by LPC; IL-1beta and IL-15 by IFN-gamma+LPS; and IFN-gamma by alpha-MOG+C. These findings indicate that each of the three demyelinating insults caused distinct patterns of demyelination and inflammatory reactivity, and that of the demyelinating agents tested only LPC exhibited general toxicity.

Epidermal growth factor (EGF) stimulation of cultured brain cells. I. Enhancement of the developmental increase in glial enzymatic activity.

Serum-free aggregating cell cultures of fetal rat telencephalon grown in the presence of 3 ng/ml (5 X 10(-10) M) epidermal growth factor (EGF) until day 12 showed 2- to 3-fold increased activities in the two glial enzymes, glutamine synthetase (GLU-S) and 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase). This effect was concentration-dependent, with maximal stimulation in cultures treated daily with 3 ng/ml EGF. Addition of EGF during the first 10 culture days was sufficient to produce a maximal stimulation of both GLU-S and CNPase on day 19, whereas treatments starting on day 12 were ineffective. The stimulation of GLU-S preceded that of CNPase. The EGF-induced increase in GLU-S activity was not directly dependent on the presence of insulin, triiodothyronine, or hydrocortisone in the medium, whereas insulin was required for the stimulation of CNPase. A single dose of 5 ng/ml EGF on day 2 caused a slight but significant decrease in DNA synthesis after day 6. The present results indicate that in serum-free aggregating cell cultures of fetal rat telencephalon EGF partially inhibits DNA synthesis, and stimulates an early step in glial differentiation.