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Effect of E65 and E70 splice isoforms on electrophysiological properties of Kv10.1

by Maryna Psol (Author) Vincenzo Romaniello (Author)

Project Report 2013 16 Pages

Biology - Diseases, Health, Nutrition

Excerpt

ABSTRACT

KvlO.l, the voltage-gated non-inactivating delayed rectifier potassium channel, is overexpressed in variety of cancer cells and is involved in oncogenesis and tumor progression. Its splice variants E65 and E7O, which were discovered in melanoma cell lines, have no conducting abilities but may physically interact with KvlO.l and thereby regulate its function. Here, we investigated possible influence ofE65 and E7O on electrophysiological properties ofKvlO.l. Analysis of current-voltage relationships revealed a dose-dependentreduction ofKvlO.l currentmediatedby both splice variants. The channel demonstrated characteristic feature of activation kinetics, a Cole-Moore shift, irrespective of the isoforms presence. Both E65 and E7O were able to increase the rise time after -6O mV conditioning when expressed at l:lO ratio with full length channel. Co-expression ofE65 or E7O with Kvl.4 did not resulted in considerable changes in channel activity; therefore interactions of splice variants with KvlO.l are likely to be specific. Downregulation ofKvlO.l activity by E65 and E7O splice variants may modulate tumorigenesis and be associated with less aggressive forms of cancer.

INTRODUCTION

Cancer is one of the major causes of death, amounted to about 12.7 million cases and 7.6 million lethal outcomes worldwide in 2008 (Jemal A., 2011). Changes in the ion channel repertoire are among the hallmarks of malignant transformation (Huber S.M., 2013).

Kv10.1 (Eag1, ether á-go-go) is the first voltage-gated potassium channel whose aberrant expression was shown to be involved in oncogenesis and tumor progression (Pardo L.A., 1999; Hemmelein B., 2006). It was found in diverse cancer cell lines (SHSY-5Y, MCF-7, IGR39 etc) and solid tumors, including sarcoma, colon carcinoma, cervical and gastric cancers (Asher V., 2010), while in healthy individuals Kv10.1 is majorly restricted to brain, placenta and myoblasts of certain stage (Hemmerlein B., 2006). Activation ofKv10.1 facilitates the angiogenesis by stimulating hypoxia inducible factor-1 (HIF-1) and influences adhesion, proliferation and contact inhibition properties by re-organizing cytoskeleton (Asher V., 2010). Moreover, channel activity and surface expression are modulated depending on the stage of the cell cycle: Kv10.1 current undergoes rectification during G2-M transition and Go/G1 arrest (Pardo L.A., 1998; Kohl T., 2011).

Similar to other voltage-gated K+ channels, Kv10.1 consists of4a subunits arranged in a circle. Each subunit comprises 6 transmembrane domains (S1-S6) where S4 segment acts as a voltage sensor and S5-S6 domains take part in pore formation. The N- and C-terminal regions are intracellular and may influence kinetic properties and voltage dependence of the channel. The amino terminus includes Per-Arnt-Sim or PAS domain which activates HIF-1, providing selective advantage for tumor cells growth in hypoxic condition. Carboxyl terminus contains a cyclic nucleotide binding domain (cNBD), a calmodulin binding domain (CaM) and a tetramerizing coiled coil domain (TCC), which are necessary for correct channel subunits assembly and perinuclear localization (Ascher V., 2010; Terlau H., 1998).

Activity ofKv10.1 channel can be controlled by various mechanisms: internalization of the channel by endocytosis, control of surface expression, sorting and retention in the endoplasmic reticulum (Kohl T., 2011), and posttranslational modifications in the Golgi complex, e.g. glycosylation (Napp J., 2005). Moreover, alternative splicing was suggested to contribute to the functional properties ofKv10.1 (Ramos Gomes F., 2010) but its’ role is not yet completely understood.

Alternative splicing of KCNH1 gene results in at least two distinct transcripts - E65 and E70-in addition to the full length forms (Fig. 1). E65 and E70 splice variants were discovered in melanoma cell lines and were named according to their expected weights in kDa. These splice variants are produced by exon skipping: while full length Kv10.1 mRNA is made of11 exons, E65 and E70 lack 4th to 9th and 4th to 7th exons respectively. Therefore, such isoforms have no transmembrane domains. Also E65 and E70 do not elicit any current when expressed alone in Xenopus oocytes (Ramos Gomes F., 2010). Splice isoforms co-express with KvlO.l in certain cell types: E70 was found in the brain, SH-SY5Y; E65 was detected in DU145, IGR39, and PNT2 cancer cell lines. Both E65 and E70 were shown to colocalize with the full length channel when expressed in the HEK 293 cells. Although, splice variants do not generate functional channels themselves, they may physically interact with Kv10.1 and modulate its’ activity.

Fully functional Kv10.1 channel requires N-linked glycosylation in at least two positions: core glycosylation at Asn-388 may be essential for correct folding and stability of the polypeptide, while complex glycosylation at Asn-406 is necessary for proper trafficking and function of the channel. Attachment of core or complex oligosaccharides determines 2 distinct cellular variants of Kv10.1 - of—110 and 130 kDa respectively (Napp J., 2005). The amount of complex glycosylated Kv10.1 decreases in presence of E70 isoform (Romaniello V., unpublished), and co-expression ofE65 causes the reduction of the overall Kv10.1 quantity in the cell according to immunoblotting experiments (Ramos Gomes F.,2010).

Besides, E65 splice variant rectifies Kv10.1 current in the same way as the injections of progesterone or mitosis-promoting factor, and, consequently, E65 may induce maturation (Ramos Gomes F., 2010; Pardo L.A., 1998). Molecular mechanisms of interactions between Kv10.1 and its splice isoforms should be further investigated.

illustration not visible in this excerpt

Figure 1. KCNH1 pre-mRNA can be alternatively spliced into the full length Kv10.1, the E65 isoform, which lacks exons 4-9, or the E70 isoform, which lacks exons 4-7.

KvlO.l channel has characteristic electrophysiological properties. Its’ activity is strongly dependent on holding potential and usually demonstrates rectification at very positive voltages. A specific feature of the channel is so-called Cole-Moore shift, gradual decrease of activation speed after hyperpolarizing prepulse potentials. During long-lasting depolarizing pulses KvlO.l does not undergo inactivation. (Pardo LA, 2OO8). In this project we aimed to investigate influence ofE65 and E7O splice variants on these electrophysiological characteristics ofKvlO.l channel, dose- dependence and specificity of such effects.

MATERIALS AND METHODS

Synthesis of capped RNA

Capped RNA (cRNA) is an analog of mRNA which is able for translation in eukaryotic cells due to presence of a 7-methyl guanosine cap at the 5’-end. It was synthesized for heterologous expression ofKvlO.l and its’ splice isoforms in Xenopus oocytes and subsequent electrophysiological recordings.

In order to obtain cRNA we used pSGEM vectors with subcloned template DNA for KvlO.l, E65 or E7O proteins. The quality of the pSGEM-KvlO.l, pSGEM-E65 and pSGEM-E7O plasmids was checked in a control digestion with Xhol and Spel enzymes (Fig. 2A).

illustration not visible in this excerpt

Figure 2. cRNA production. (A) Control digestion and (B) linearization of pSGEM-KvlO.l, pSGEM-E65 and pSGEM-E7O vectors. (C) Obtained concentrations of cRNA.

Sense cRNA was produced with the T7 mMessage mMachine kit. pSGEM constructs (lOpg each) were linearized with Sfil enzyme in a total volume of 5Opl at 5O°C overnight. The digestion was terminated with adding 2.5pl O.5M EDTA, 5pl 3M Na acetate and lOOpl ethanol. Samples were cooled at -2OOC for 2 hours, DNA was pelleted by centrifugation (l6.4OO x g 4°C 2O min), washed with 7O% ethanol, dried and resuspended in lOpl dH2O. Linearization efficiency was examined on O.8 agarose gel (Fig. 2B).

The concentration of linearized DNA was determined photometrically by measuring absorption of the samples at 26O nm. The ratio A26O/A28O was used to estimate DNA purity, and the A26O/A28O value about l.8 indicated lack of protein contamination in the samples.

Transcription reaction reagents were assembled at room temperature in the order and amount indicated in the kit protocol. Then T7 reaction was incubated 2 hours at 37OC for the maximum yield of cRNA. The template DNA was removed by digestion with lpl of TURBO DNase at 37°C for l5 min.

RNA was recovered with lithium chloride precipitation. For this we added to each sample 3Opl of nuclease-free water and 3Opl of LiCl precipitation solution. Afterwards reactions were kept at -2OOC for 3Omin, centrifuged at l6.4OO x g 4OC for l5min, washed with 7O% ethanol. Each sample of obtained cRNA was resuspended in l5pl of DEPC water. Concentrations of cRNA were determined with IMPLEN nanophotometer (Fig. 2C). Then samples were aliquoted and stored at -8O°C.

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Details

Pages
16
Year
2013
ISBN (eBook)
9783656520641
ISBN (Book)
9783656524229
File size
1.2 MB
Language
English
Catalog Number
v263279
Institution / College
University of Göttingen – Max Plank Institute for Experimental Medicine
Grade
1.3
Tags
effect kv10

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Title: Effect of E65 and E70 splice isoforms on electrophysiological properties of Kv10.1