Synnöve Beckh

Oncogenic potential of EAG K+ channels

We have investigated the possible implication of the cell cycle‐regulated K+ channel ether à go‐go (EAG) in cell proliferation and transformation. We show that transfection of EAG into mammalian cells confers a transformed phenotype. In addition, human EAG mRNA is detected in several somatic cancer cell lines, despite being preferentially expressed in brain among normal tissues. Inhibition of EAG expression in several of these cancer cell lines causes a significant reduction of cell proliferation. Moreover, the expression of EAG favours tumour progression when transfected cells are injected into immune‐depressed mice. These data provide evidence for the oncogenic potential of EAG.

Cloning and expression of a human voltage-gated potassium channel. A novel member of the RCK potassium channel family.

We have isolated and characterized a human cDNA (HBK2) that is homologous to novel member (RCK2) of the K+ channel RCK gene family expressed in rat brain. RCK2 mRNA was detected predominantly in midbrain areas and brainstem. The primary sequences of the HBK2/RCK2 K+ channel proteins exhibit major differences to other members of the RCK gene family. The bend region between segments S1 and S2 is unusually long and does not contain the N‐glycosylation site commonly found in this region. They might be O‐glycosylated instead. Functional characterization of the HBK2/RCK2 K+ channels in Xenopus laevis oocytes following micro‐injection in in vitro transcribed HBK2 or RCK2 cRNA showed that the HBK2/RCK2 proteins form voltage‐gated K+ channels with novel functional and pharmacological properties. These channels are different to RCK1, RCK3, RCK4 and RCK5 K+ channels.

Differential expression of sodium channel mRNAs in rat peripheral nervous system and innervated tissues

RNA blot hybridization analyses using probes specific for sodium channels I, II and III revealed high levels of sodium channel I mRNA and low levels of sodium channel II and III mRNAs in peripheral nervous system (PNS) tissues. The developmental expression patterns of these mRNAs were generally similar to those described for the central nervous system. The small amounts of sodium channel I and III mRNAs present in tongue muscle were greatly reduced after partial denervation. Expression of the three sodium channels thus appears to be restricted to the nervous system. Putative novel additional mRNAs, specifically expressed in the PNS, were detected with a probe that recognizes nucleotide sequences common to sodium channels I, II and III.

A method of in situ hybridization combined with immunocytochemistry, histochemistry, and tract tracing to characterize the mRNA expressing cell types in heterogeneous neuronal populations

A rapid, sensitive, non-isotopic in situ hybridization histochemistry protocol is presented to study the expression of mRNA at the single cell level in anatomically complex structures of the mammalian central nervous system. The protocol uses digoxigenin-UTP-labeled riboprobes, freefloating sections, and alkaline phosphatase and horseradish peroxidase detection. Modifications have been introduced which preserve the integrity of marker molecules, and as such enable the simultaneous identification and characterization of CNS cell types by tract tracing, histochemical, and immunocytochemical detection of intra- and extracellular markers. All pretreatments that enhance probe penetration have been omitted without substantial loss in sensitivity. The protocol has been successfully extended to vibratome sections with subsequent plastic-embedding and semithin sectioning, which considerably broadens the general applicability of this fast and easy ISHH method.

Neurotrophic and Neurite Promoting Activities in Astroglial Conditioned Medium

The pre-eminent hypothesis in the field of trophic factors is still that put foreward in 1928 by Ramon y Cajal who proposed that nonneuronal cells play an important physiological role in the trophic support of neurons. Since then many investigators have found that peripheral and central glial cells provide factors needed for the survival and differentiation of neurons and for the guidance and growth of neurites (58, 40, 36). Significant progress has been made in studying such factors in the peripheral nervous system (PNS) since the first neurotrophic agent, nerve growth factor (NGF), was discovered in a mouse sarcoma (26). While the action of NGF and its correlation to structurally similar peptides has been extensively explored (17; for reviews see 19,11), little is known about neurotrophic factors acting on neurons specific for cells in the central nervous system (CNS). This despite the progress made studying sensory neurons located within the CNS which respond in addition to NGF to skeletal muscle extract and a specific central neurotrophic protein, the so called brain-derived neurotrophic factor (BDNF), purified by Thoenen and coworkers (4,12).

Molecular properties of the glutamate receptor mediating synaptic excitation in rat hypothalamic neurons.

AMPA-type glutamate receptors (GluRs) mediate synaptic excitation in networks of cultured rat hypothalamic neurons [18, 25]. Under voltage clamp the agonists quisqualate and AMPA induce current responses which consist of a maintained and/or transient component depending on the concentrations applied. The current-voltage relationship for both components is linear. The biphasic response patterns are due to receptor desensitization which is fast and does not require intracellular second messengers for its activation. Several GluR-subtype-encoding transcripts were found in these neurons using polymerase chain reaction (PCR) methods. While mRNAs encoding the GluR2 and 3 flip forms are expressed early, mRNAs encoding the GluR1, 2 and 3 flop forms and the GluR4 flip form appear only in cultures older than 3 weeks. By comparison to recombinant receptors, the properties of the native receptor can be accommodated by a heteromeric receptor containing GluR2 as one of the subunits.

Functional and Molecular Characteristics of the Glutamate Receptor Involved in Synaptic Transmission in the Hypothalamusa

Non-N-methyl-D-aspartate (NMDA) receptors mediate fast glutamatergic neurotransmission in most parts of the mammalian central nervous system (for a review see ref. 5). The existence of multiple GluR subtypes that are differentially expressed in the brain”Is has been demonstrated. Specific subtype combinations expressed in vitro exhibit current-voltage relationships found with native neuronal receptors.*J0J6 Functional characteristics of the native receptors, however, cannot be easily correlated with the receptor subtypes expressed in vitro. Recently, we have shown that excitatory-synaptic transmission in cultured neurons from embryonic rat hypothalamus is mediated by a non-NMDA receptor of the a-amino-3-hydroxy-5-methyl-isoxazole-propionic acid (AMPA) In the present study, we have attempted to gain more insight into the functional characteristics of this receptor, using rapid agonist application to neurons under whole-cell voltage clamp and microfluorometric measurements with fura-2. In addition, the expression of GluR subunits in the cultured neurons during network formation was analyzed.