Influence of epidermal growth factor on long-term potentiation in the hippocampal slice.

Rat hippocampal slices were perfused with epidermal growth factor (EGF) at a concentration of 10−8 M. EGF is a well known mitogen which exhibits neurotrophic action on neurons of the CNS. During extra- and intracellular recordings in the pyramidal cell body layer of the CA1-region no influence of EGF on evoked potentials was seen if single-pulse or paired-pulse stimulation was used. Furthermore EGF has no influence on the resting membrane potential of the cells investigated. However after tetanic stimulation a significant increase in the magnitude of long-term potentiation was observed. Therefore it is concluded, that EGF might be involved in modulation of neuronal plasticity

Characterization of the Conus bullatus genome and its venom-duct transcriptome

<p>Abstract</p> <p>Background</p> <p>The venomous marine gastropods, cone snails (genus <it>Conus</it>), inject prey with a lethal cocktail of conopeptides, small cysteine-rich peptides, each with a high affinity for its molecular target, generally an ion channel, receptor or transporter. Over the last decade, conopeptides have proven indispensable reagents for the study of vertebrate neurotransmission. <it>Conus bullatus </it>belongs to a clade of <it>Conus </it>species called <it>Textilia</it>, whose pharmacology is still poorly characterized. Thus the genomics analyses presented here provide the first step toward a better understanding the enigmatic <it>Textilia </it>clade.</p> <p>Results</p> <p>We have carried out a sequencing survey of the <it>Conus bullatus </it>genome and venom-duct transcriptome. We find that conopeptides are highly expressed within the venom-duct, and describe an <it>in silico </it>pipeline for their discovery and characterization using RNA-seq data. We have also carried out low-coverage shotgun sequencing of the genome, and have used these data to determine its size, genome-wide base composition, simple repeat, and mobile element densities.</p> <p>Conclusions</p> <p>Our results provide the first global view of venom-duct transcription in any cone snail. A notable feature of <it>Conus bullatus </it>venoms is the breadth of A-superfamily peptides expressed in the venom duct, which are unprecedented in their structural diversity. We also find SNP rates within conopeptides are higher compared to the remainder of <it>C. bullatus </it>transcriptome, consistent with the hypothesis that conopeptides are under diversifying selection.</p

Calmodulin Interaction with hEAG1 Visualized by FRET Microscopy

BACKGROUND: Ca(2+)-mediated regulation of ion channels provides a link between intracellular signaling pathways and membrane electrical activity. Intracellular Ca(2+) inhibits the voltage-gated potassium channel EAG1 through the direct binding of calmodulin (CaM). Three CaM binding sites (BD-C1: 674-683, BD-C2: 711-721, BD-N: 151-165) have been identified in a peptide screen and were proposed to mediate binding. The participation of the three sites in CaM binding to the native channel, however, remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: Here we studied the binding of Ca(2+)/CaM to the EAG channel by visualizing the interaction between YFP-labeled CaM and Cerulean-labeled hEAG1 in mammalian cells by FRET. The results of our cellular approach substantiate that two CaM binding sites are predominantly involved; the high-affinity 1-8-14 based CaM binding domain in the N-terminus and the second C-terminal binding domain BD-C2. Mutations at these sites completely abolished CaM binding to hEAG1. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the BD-N and BD-C2 binding domains are sufficient for CaM binding to the native channel, and, therefore, that BD-C1 is unable to bind CaM independently

Rapid Internalization of the Oncogenic K+ Channel KV10.1

KV10.1 is a mammalian brain voltage-gated potassium channel whose ectopic expression outside of the brain has been proven relevant for tumor biology. Promotion of cancer cell proliferation by KV10.1 depends largely on ion flow, but some oncogenic properties remain in the absence of ion permeation. Additionally, KV10.1 surface populations are small compared to large intracellular pools. Control of protein turnover within cells is key to both cellular plasticity and homeostasis, and therefore we set out to analyze how endocytic trafficking participates in controlling KV10.1 intracellular distribution and life cycle. To follow plasma membrane KV10.1 selectively, we generated a modified channel of displaying an extracellular affinity tag for surface labeling by α-bungarotoxin. This modification only minimally affected KV10.1 electrophysiological properties. Using a combination of microscopy and biochemistry techniques, we show that KV10.1 is constitutively internalized involving at least two distinct pathways of endocytosis and mainly sorted to lysosomes. This occurs at a relatively fast rate. Simultaneously, recycling seems to contribute to maintain basal KV10.1 surface levels. Brief KV10.1 surface half-life and rapid lysosomal targeting is a relevant factor to be taken into account for potential drug delivery and targeting strategies directed against KV10.1 on tumor cells

The Terebridae and teretoxins: Combining phylogeny and anatomy for concerted discovery of bioactive compounds

The Conoidea superfamily, comprised of cone snails, terebrids, and turrids, is an exceptionally promising group for the discovery of natural peptide toxins. The potential of conoidean toxins has been realized with the distribution of the first Conus (cone snail) drug, Prialt (ziconotide), an analgesic used to alleviate chronic pain in HIV and cancer patients. Cone snail toxins (conotoxins) are highly variable, a consequence of a high mutation rate associated to duplication events and positive selection. As Conus and terebrids diverged in the early Paleocene, the toxins from terebrids (teretoxins) may demonstrate highly divergent and unique functionalities. Recent analyses of the Terebridae, a largely distributed family with more than 300 described species, indicate they have evolutionary and pharmacological potential. Based on a three gene (COI, 12S and 16S) molecular phylogeny, including ~50 species from the West-Pacific, five main terebrid lineages were discriminated: two of these lineages independently lost their venom apparatus, and one venomous lineage was previously unknown. Knowing the phylogenetic relationships within the Terebridae aids in effectively targeting divergent lineages with novel peptide toxins. Preliminary results indicate that teretoxins are similar in structure and composition to conotoxins, suggesting teretoxins are an attractive line of research to discover and develop new therapeutics that target ion channels and receptors. Using conotoxins as a guideline, and innovative natural products discovery strategies, such as the Concerted Discovery Strategy, the potential of the Terebridae and their toxins are explored as a pioneering pharmacological resource