Movatterモバイル変換

[0]ホーム
URL:

Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
Thehttps:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

NIH NLM Logo
Log inShow account info
Access keysNCBI HomepageMyNCBI HomepageMain ContentMain Navigation
pubmed logo
Advanced Clipboard
User Guide

Full text links

Wiley full text link Wiley Free PMC article
Full text links

Actions

Share

.2004 Oct 15;78(2):224-34.
doi: 10.1002/jnr.20240.

Role for calcium-activated potassium channels (BK) in growth control of human malignant glioma cells

Affiliations

Role for calcium-activated potassium channels (BK) in growth control of human malignant glioma cells

Amy K Weaver et al. J Neurosci Res..

Abstract

Voltage-dependent large-conductance Ca(2+)-activated K(+) channels, often referred to as BK channels, are a unique class of ion channels coupling intracellular chemical signaling to electrical signaling. BK channel expression has been shown to be up-regulated in human glioma biopsies, and expression levels correlate positively with the malignancy grade of the tumor. Glioma BK channels (gBK) are a splice variant of the hslo gene, are characterized by enhanced sensitivity to [Ca(2+)](i), and are the target of modulation by growth factors. By using the selective pharmacological BK channel inhibitor iberiotoxin, we examined the potential role of these channels in tumor growth. Cell survival assays examined the ability of glioma cells to grow in nominally serum-free medium. Under such conditions, BK channel inhibition by iberiotoxin caused a dose- and time-dependent decrease in cell number discernible as early as 72 hr after exposure and maximal growth inhibition after 4-5 days. FACS analysis shows that IbTX treatment arrests glioma cells in S phase of the cell cycle, whereupon cells undergo cell death. Interestingly, IbTX effects were nullified when cells were maintained in 7% fetal calf serum. Electrophysiological analysis, in conjunction with biotinylation studies, demonstrates that serum starvation caused a significant translocation of BK channel protein to the plasma membrane, corresponding to a two- to threefold increase in whole-cell conductance, but without a change in total gBK protein. Hence, expression of functional gBK channels appears to be regulated in a growth-factor-dependent manner, with enhanced surface expression promoting tumor cell growth under conditions of growth factor deprivation as might occur under in vivo conditions.

Copyright 2004 Wiley-Liss, Inc.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
BK channel blockers inhibit glioma cell growth in a dose-dependent manner after growth factor withdrawal. D54 glioma cells were treated with TEA or IbTX for 5 days.A: Relative cell numbers were measured after 5 days of treatment in the presence or absence of 7% FBS and normalized to the respective values at day 1.B: Dose-dependent inhibition of cell growth by IbTX after 5 days of treatment in media lacking serum.C: Images of cells after 5 days under serum-free conditions with (right) or without (left) IbTX. *P < 0.05.
Fig. 2
Fig. 2
BK channel inhibition affects tumor cell growth in a time-dependent and irreversible manner. Five-day growth curves were obtained from cells receiving no treatment or IbTX in media containing 7% FBS (A) or serum-free (SF) media (B).C: Cells were treated as described above in the absence of serum. After 3 days of treatment, cells were washed and cultured in media lacking serum for the remainder of the experiment. Cell number was determined daily with a Coulter counter.
Fig. 3
Fig. 3
Whole-cell BK currents are up-regulated after serum withdrawal.A: Representative whole-cell currents evoked after voltage steps from 0 to +160 mV from a holding potential of -40 mV. All traces have been normalized to whole-cell capacitance to compare currents under different conditions regardless of cell size. Representative traces evoked with the same protocol as described above in the presence of 100 nM IbTX illustrating that sensitivity to drug has remained the same. Subtracted traces showing specifically the IbTX-sensitive BK current.B: Bar graph illustrating an approximately two-fold increase in the whole-cell specific conductance of the BK current. Specific conductances were determined by dividing current (I) by the driving force (E - EK) and normalizing these values to whole-cell capacitance (pF).C: An I/Imax plot comparing cells cultured in media with or without serum illustrates that increases in current size were not due to changes in [Ca2+]i, which would have caused a leftward shift in the activation curve.
Fig. 4
Fig. 4
BK channel protein is translocated to the membrane after chronic serum withdrawal.A: Western blot analysis reveals a prominent band at ∼120 kDa, which is consistent with the expected BK channel protein. Actin was used as a loading to control in order to analyze qualitatively the BK protein levels in control (right lane) and serum-free (left lane) cultured cells.B: Densitometric analysis of the blot shown in A reveals no difference in protein levels between control and serum-free cells.C: Blot from biotinylation study indicating an approximate twofold increase in BK protein in the membrane-bound fraction lane of the serum-free cells. HEK-293 cells were used as a negative control, and the blot was probed with actin to ensure that membrane fractions were not contaminated with cytoplasmic protein.D: Densitometric analysis of three biotinylation assays revealed a 77% increase in the level of BK channel protein expressed on the plasma membrane as represented by the membrane-bound fraction.
Fig. 5
Fig. 5
Immunostaining revealing translocation of BK channels to the membrane of serum-starved cells. Cells maintained in the presence (A-C) or absence (D-F) of 7% FBS were fixed and probed with the BK channel antibody G18 (A,D) or DAPI (B,E). C,F: Merge of previous image with G18 (green), and DAPI (blue). The majority of cells grown in media containing serum showed prominent immuno-reactivity for BK around the nucleus of the cell (A), indicative of protein in the ER or Golgi, whereas cells grown in the absence of serum (D) were virtually devoid of the same perinuclear staining, with more labeling occurring on the plasma membrane.
Fig. 6
Fig. 6
FACS analysis of cells stained with ethidium homodimer. Representative example of an FACS analysis of cells cultured in media lacking serum (A) and treated with IbTX (B). Ethidium homodimer was used to label dying cells, those cells that occupy the upper left quadrant in the analysis plot.C: Analysis of the percentage of cells stained positively for ethidium homodimer after 5 days of treatment compared with control. Results were taken from five experiments, all performed in triplicate.D: Cell death caused by IbTX was dose dependent.
Fig. 7
Fig. 7
Cell cycle analysis of cells treated with IbTX. Representative FACS assays of cells left untreated (A) and treated with 100 nM IbTX (B). Propidium iodide, which stains DNA, was used to analyze DNA content of each cell, which is directly dependent on cell cycle stage. Using Modfit software, we were able to translate fluorescence into DNA content and thus cell cycle stage for each cell. Cells from five independent experiments, performed in triplicate, were used to determine cell cycle stage of cells receiving IbTX treatment.C: Relative percentages of cells in G1/G0 phase of the cell cycle after 5 days of serum-free (control) and serum-free + 100 nM IbTX (IbTX) conditions.D: Relative percentages of cells in S phase of the cell cycle under the same conditions as for C. *P < 0.05.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Arcangeli A, Bianchi L, Becchetti A, Faravelli L, Coronnello M, Mini E, Olivotto M, Wanke E. A novel inward-rectifying K+ current with a cell-cycle dependence governs the resting potential of mammalian neuroblastoma cells. J Physiol. 1995;489:455–471. - PMC - PubMed
    1. Ashkenazi A, Ramachandran J, Capon DJ. Acetylcholine analogue stimulates DNA synthesis in brain-derived cells via specific muscarinic receptor subtypes. Nature. 1989;340:146–150. - PubMed
    1. Attali B, Wang N, Kolot A, Sobko A, Cherepanov V, Soliven B. Characterization of delayed rectifier Kv channels in oligodendrocytes and progenitor cells. J Neurosci. 1997;17:8234–8245. - PMC - PubMed
    1. Baran I. Calcium and cell cycle progression: possible effects of external perturbations on cell proliferation. Biophys J. 1996;70:1198–1213. - PMC - PubMed
    1. Basrai D, Kraft R, Bollensdorff C, Liebmann L, Benndorf K, Patt S. BK channel blockers inhibit potassium-induced proliferation of human astrocytoma cells. Neuroreport. 2002;13:403–407. - PubMed

Publication types

MeSH terms

Substances

Related information

Grants and funding

LinkOut - more resources

Full text links
Wiley full text link Wiley Free PMC article
Cite
Send To

NCBI Literature Resources

MeSHPMCBookshelfDisclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.

[8]ページ先頭
©2009-2025 Movatter.jp