Departments of 1 Physiology and Biophysics, 2 Surgery, and 3 Pediatrics, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005
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ABSTRACT |
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Human astrocytoma cells were studied using whole cell patch-clamp recording. An inward, amiloride-sensitive Na+ current was identified in four continuous cell lines originally derived from human glioblastoma cells (CH235, CRT, SKMG-1, and U251-MG) and in three primary cultures of cells obtained from glioblastoma multiforme tumors (up to 4 passages). In addition, cells freshly isolated from a resected medulloblastoma tumor displayed this same characteristic inward current. In contrast, amiloride-sensitive currents were not observed in normal human astrocytes, low-grade astrocytomas, or juvenile pilocytic astrocytomas. The only amiloride-sensitive Na+ channels thus far molecularly identified in brain are the brain Na+ channels (BNaCs). RT-PCR analyses demonstrated the presence of mRNA for either BNaC1 or BNaC2 in these tumors and in normal astrocytes. These results indicate that the functional expression of amiloride-sensitive Na+ currents is a characteristic feature of malignant brain tumor cells and that this pathway may be a potentially useful target for therapeutic intervention.
astrocytoma; glioblastoma; patch clamp; brain tumor cells
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INTRODUCTION |
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ASTROCYTE-DERIVED BRAIN TUMORS comprise a diverse group of neoplasms that differ in their morphology, their central nervous system location, their degree of invasiveness, their tendency for progression, and their growth characteristics. Primary malignant brain tumors are the most common cause of cancer-related death in children 16 years of age and younger. Approximately 3 in every 100,000 children under the age of 16 are diagnosed with brain tumors, according to the Central Brain Tumor Registry (14a). The most common childhood brain tumors are primitive neuroectodermal tumors (including medulloblastomas), astrocytomas, ependymomas, and choroid plexus papillomas (2, 11). Due to the infiltrative nature of many of these tumors or because of their location, total surgical excision is not always possible. Conventional adjunct treatment modalities include chemotherapy and radiation therapy. Because unifying molecular events underlying tumor development and progression are only beginning to be understood (4), there is a need to define tumor-specific markers so as to develop potential therapeutic regimens that will target exclusively these unwanted cells.
There is increasing evidence that ion channels may be intimately involved in the cellular pathophysiology related to cancer. Several different laboratories have demonstrated that the expression of certain oncogenes directly affects Na+ (12, 15, 19), K+ (20, 21, 29, 31), and Ca2+ (12-14) channel function. For example, the ras oncogenes, known to be involved in metastasis (6), influence nerve growth factor-induced neuronal differentiation and voltage-sensitive Na+ channel expression (18, 26). Moreover, cell adhesion (5), motility (24, 30), interaction with extracellular matrix (7), and proliferation (12, 25, 27, 31, 33) all involve ion channel activity. Ion channels make attractive candidates for investigation of tumor-specific protein expression because they can be rapidly identified using whole cell patch clamp. If a specific current is noted, there must be a channel containing an extracellular protein epitope through which the current passed. This epitope could serve as a specific antigen target for the tumor. Thus it may be possible to develop a dual attack strategy by specific antibody binding and subsequent immunization and inhibition of function.
The ever-expanding degenerin/epithelial Na+ channel (ENaC) superfamily of Na+ channels contains to date >20 proteins having a similar topology: short intracellularly located amino and carboxy termini, two predicted transmembrane-spanning domains, and a large extracellular loop (9, 17). All channels that have been examined electrophysiologically are cation selective and blocked by the diuretic drug amiloride (8, 9, 17). Recently, another branch of this superfamily, the human brain Na+ channel (BNaC) family, has been identified (16, 28). BNaC1 is located on chromosome 17q11.2-12 and BNaC2 on 12q12 (16). The two thus far identified members of this family, BNaC1 and BNaC2, are only expressed in brain. In normal brain, BNaC1 and BNaC2 have been localized primarily to neurons, although BNaC2 has been detected in choroid plexus (16). Neither transcript has been detected in ependymal cells, anterior commissure, or corpus callosum.
Electrophysiological studies of primary brain tumor cells and the role
of specific ion channels in growth control of gliomas are few. Both
K+ and
Cl channels have been
identified in astrocytoma-derived cell lines and, for the
Cl
channels, in primary
cell cultures obtained from human brain tumor resections (10, 34). In
this work, we identified the specific expression of an inward
amiloride-inhibitable Na+
conductance in high-grade glioma cells. This conductance was not found
in normal human astrocytes or in low-grade astrocytic tumors. This
conductance appears to be associated with the functional expression of
BNaC in these cells.
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EXPERIMENTAL PROCEDURES |
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Cell culture. Primary cultures from human gliomas were established from fresh brain tumor tissue obtained at the time of surgery. Primary human astrocyte cultures were established by disaggregating normal brain from patients undergoing surgery for intractable epilepsy. The tissue was transported from the operating room in sterile, ice-cold tissue culture medium, and necrotic and/or hemorrhagic regions were dissected away. For brain tumor cultures, the remaining tissue was minced, pipetted repeatedly to disassociate the cells, and plated onto a 35-mm tissue culture dish in DMEM mixed in equal proportions with Ham's F-12 (DMEM/F-12) supplemented with 10 mM HEPES (pH 7.4), 2 mM L-glutamine, and 20% fetal bovine serum. Normal brain was enzymatically disaggregated with 0.1% trypsin/0.53 mM EDTA and plated onto 150-cm2 flasks in DMEM/F-12 plus 20% fetal bovine serum, 1× mito-X, and 10 ng epidermal growth factor/ml as previously described (3). Established cell lines derived from human glioblastoma tumors (CH235, CRT, SKMG-1, U251-MG, U373-MG) are carried in the University of Alabama at Birmingham (UAB) Brain Tumor Research Laboratory facilities and were all used at passages >100. A portion of a freshly resected medulloblastoma was prepared as described above, and the disassociated cells were used immediately for electrophysiological recording and RNA extraction. Brain tumor tissue was obtained in accordance with the human tissue procurement procedures at the Medical Center as approved by the UAB Institutional Review Board (22 April 1998).
Whole cell patch-clamp studies.
Cultured cells were mechanically scraped from 35-mm culture dishes,
rinsed in serum-free RPMI medium, placed in the perfusion chamber, and
allowed to adhere to the glass bottom. Once the cells had adhered
(evidenced by lack of movement during bath superfusion), they were
examined electrophysiologically by whole cell patch clamp.
Micropipettes were constructed using a Narashigi PP-83 two-stage
micropipette puller. The tips of these pipettes had an internal
diameter of ~0.3-0.5 µm. When filled with an
electrolyte solution containing (in mM) 100 potassium gluconate, 30 KCl, 10 NaCl, 20 HEPES, 0.5 EGTA, and 4 ATP, as well as <10 nM free
Ca2+, at a pH of 7.2, the
electrical resistance of the tip was 1-3 M. The bath solution
was serum-free RPMI 1640 cell culture medium. The solutions approximate
the normal ionic gradients across the cell membrane. Pipettes were
mounted in a holder and connected to the head stage of an Axon 200A
patch-clamp amplifier affixed to a three-dimensional micromanipulator
system attached to the microscope. The pipettes were abutted to the
cells and slight suction applied. Seal resistance was continuously
monitored (Nicolet model 300 oscilloscope) using 0.1-mV electrical
pulses from an electrical pulse generator. After formation of seals
with resistances >1 G
, another suction pulse was applied to form
the whole cell configuration by rupturing the membrane within the seal
but leaving the seal intact. Successful completion of this procedure
was known by the sudden increase in capacitance with no change in seal
resistance. The magnitude of the capacitance increase is a direct
function of the membrane available to be voltage clamped (i.e., cell
size). Typically, this capacitance was between 5 and 10 pF.
Subsequently, the capacitive transients were compensated for with the
use of the capacitive and resistance circuits of the Axon 200A amplifier.
Once the whole cell configuration was obtained, the pipette solution and the cellular interior equilibrated within 30 s. the cells were then held at a membrane potential of 0 mV for 1 s between each test voltage. This procedure induced inward Na+ (at more hyperpolarized potentials) and outward K+ (at more depolarized potentials) currents to flow across the membrane. The currents were recorded digitally and filed in real time. The entire procedure was performed using a DOS Pentium computer modified for A/D signals with pCLAMP 6 software (Axon Instruments, Sunnyvale, CA). For any given cell line or tumor preparation, 4-12 cells were examined.
RNA extraction and RT-PCR. Total RNA
was extracted from tumor cell lines and freshly excised material using
guanidinium thiocyanate cell lysis and phenol chloroform extraction.
The integrity of the RNA was verified after electrophoresis through 1%
agarose-formaldehyde denaturing gels. Two micrograms of total RNA were
reverse transcribed at 42°C for 60 min using oligo(dT) as a primer
(Pharmacia) and avian myeloblastosis virus RT (Promega). PCR was
performed using Taq (Fisher)
polymerase with 0.2, 0.6, or 1 mM
MgCl2 added to the
Taq buffer and 2.5 µl of the RT-PCR
product, in a total volume of 50 µl. The primers, corresponding to
regions in either BNaC1 or BNaC2, were used at a concentration of 150 ng per reaction. BNaC1, forward and reverse primers, respectively, were
as follows: 5'-TGCAAGTTCA AAGGGCAG-3' and
5'-TGGCTGATGT CTTGCTGG-3' and corresponded to bases
511-528 and 1136-1153. The BNaC2, forward and reverse primers, respectively, were as follows: 5'-TGCTCTCCTG
CCACTTCC-3' and 5'-GCTTTGCTGG GGATCTTG-3' and
corresponded to bases 735-752 and 1366-1383. PCR was
performed beginning with a single cycle of 94°C for 4 min, followed
by cycles of 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min, for 30 cycles. This was followed by a single cycle of
72°C for 60 min to facilitate addition of T and A overhangs to the
PCR product. Parallel reactions were run using human -actin as a
control for the RT-PCR. Aliquots of each resulting reaction mixture
were run on a 2% agarose gel using
X174
Hae III cut DNA (Promega) as molecular
size markers. Products of correct molecular size were subcloned into
pCR-II using the TA cloning kit following the manufacturer's
instructions (InVitrogen). Recombinants were selected by blue/white
screening and restricted with EcoR I
(Promega). Plasmids containing inserts of correct size were then
subjected to automatic DNA sequencing (DNA Sequencing Facility, Iowa
State University).
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RESULTS |
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Whole cell patch-clamp experiments were performed on primary cultures
of cells derived from eight human brain tumors, five established human
glioblastoma cell lines, one freshly resected medulloblastoma, and two
separate populations of normal human astrocytes (see Table
1). All cells stained positively for glial fibrillary acidic protein. Representative voltage-clamp traces are
shown in Fig. 1 for a cell
obtained from a medulloblastoma. In the basal state, the current
records were characterized by large "ragged" inward currents
(Fig. 1A), and these currents were completely inhibited within 30 s following superfusion with 100 µM
amiloride (Fig. 1B). Figure
1C shows the difference current (i.e.,
amiloride-sensitive component), and it can be seen that only the inward
currents were inhibited by this drug. This result should be contrasted
to the lack of inward current and lack of effect of 100 µM amiloride
in juvenile pilocytic astrocytoma cells (Fig.
2) and normal astrocytes (Fig.
3). Comparable amiloride-sensitive inward
currents were seen in four of five continuous cell lines (U373-MG being
the exception), and in three primary cultures of freshly resected
glioblastoma multiforme cells (Table 1). None of the nonneoplastic
(i.e., normal) astrocytes or any of the four low-grade astrocytomas
displayed any measurable amiloride-sensitive current. These results
suggest the presence of an amiloride-inhibitable component to whole
cell currents in only the high-grade, highly invasive glioblastoma
multiforme tumors.
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To assess the range of amiloride efficacy on the constitutively activated inward currents, primary GBM cells (0968662, passage 3) were superfused sequentially with solutions containing 0.1, 1, 10, and 100 µM amiloride, subsequent to establishing the whole cell configuration. Amiloride concentrations of 0.1 and 1 µM had no measurable effect. Ten micromolar amiloride partially inhibited the current in one of three cells examined, and 100 µM amiloride consistently produced the maximum amount of current inhibition in these trials and in trials on the other tumor cell types studied. From these studies, we estimated a Ki (apparent equilibrium dissociation constant) of ~30 ± 20 µM for amiloride-mediated current inhibition in cells expressing this constitutively activated inward Na+ current.
To elucidate the molecular basis for this amiloride-sensitive current,
we next performed RT-PCR on total RNA extracted from each of these cell
samples. Specific primers were designed to amplify signals
corresponding to messages for either BNaC1 or BNaC2, the only two
amiloride-sensitive cation channels thus far identified in brain
tissue. The primer pairs for BNaC1 and BNaC2 predicted products of 643 and 649 bp, respectively. Examples of these reactions are shown in Fig.
4. The BNaC2 message was detected in
SKMG-1, medulloblastomas, and normal astrocytes. The BNaC2 message was
found in virtually all of the samples analyzed, with the notable
exceptions of two glioblastoma multiforme primary cultures and the one
anaplastic astrocytoma (see Table 1). In these particular cases, the
BNaC1 message was found instead. In the samples where the BNaC2 message
was detected, no BNaC1 product was found (and vice versa). Direct
sequencing of the PCR products confirmed their identity.
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DISCUSSION |
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We report the presence of amiloride-sensitive inward Na+ currents in human malignant brain tumor cells. These currents were seen in primary cultures of freshly resected tumors, as well as in established cell glioma lines. These currents were not present in normal astrocytes or in low-grade astrocytomas (e.g., pilocytic astrocytomas). Molecular biological analyses indicated the presence of either the BNaC1 or BNaC2 message in these cells. The level of expression was generally higher in the cells functionally expressing amiloride-sensitive current (for the same number of PCR cycles) but was, nonetheless, detected in all of the samples examined. The endogenous function and physiological role of the BNaC family of ion channels are not known. Their epithelial counterparts, the ENaCs, act as highly regulated Na+ channels in tissues designed to reabsorb Na+ (8, 9, 17). Like the degenerins and ENaCs, BNaCs may form regulated ion channels; such channels may be involved in cell volume regulation (1, 22, 23). The BNaCs may be involved in the small Na+ influx that occurs in neuronal cells and thus may contribute to the resting potential of the cell. Alterations in membrane potential, by either activating or inhibiting these channels, may have deleterious effects on cell survival (35).
It is difficult to detect currents following heterologous expression of either wild-type BNaC1 or BNaC2 (16, 28, 35). However, introduction of mutations into BNaC1, comparable with those found in the nematode degenerins that produce neurodegeneration, results in easily measurable amiloride-blockable currents, but with a reduced Na+ permselectivity (35). These mutations were made in the second membrane-spanning domain. Because the PCR primers used in our experiments did not cover this region, we cannot state with any certainty that the basal-activated currents measured in the malignant tumor cells were due to mutated BNaC. However, the existence of this current in highly proliferative human tumor cells is significant, in that it may play a crucial role in tumor cell progression. Because normal glial cells do not express this current, it is unlikely that inhibition of the current would be fatal to the malignant cells. However, if, as these studies suggest, the expression of these channels is restricted to malignant tumor cells, then these channels represent a specific target that is exclusive. It may be possible to devise treatments based on this protein.
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ACKNOWLEDGEMENTS |
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We thank Cathleen Guy for superb secretarial assistance.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37206.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. J. Benos, Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, MCLM 704, Birmingham, AL 35294-0005 (E-mail: benos{at}phybio.bhs.uab.edu).
Received 25 November 1998; accepted in final form 24 March 1999.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Adams, C. M.,
M. G. Anderson,
D. G. Motto,
M. P. Price,
W. A. Johnson,
and
M. J. Welsh.
Ripped pocket and pickpocket, novel Drosophila DEG/ENaC subunits expressed in early development and in mechanosensory neurons.
J. Cell Biol.
140:
143-152,
1998
2.
American Brain Tumor Association. [Online]
American Brain Tumor Association. http://www.abta.org
3.
Andreansky, S.,
L. Soroceanu,
E. R. Flotte,
J. Chou,
J. M. Markert,
G. Y. Gillespie,
B. Roizman,
and
R. J. Whitley.
Evaluation of genetically engineered herpes simplex viruses as oncolytic agents for human malignant brain tumors.
Cancer Res.
57:
1502-1509,
1997[Abstract].
4.
Ashley, D. M.,
and
D. D. Bigner.
Recent advances in the biology of central nervous system tumors.
Curr. Opin. Neurol.
10:
445-451,
1997[Medline].
5.
Asou, H.
Monoclonal antibody that recognizes the carbohydrate portion of cell adhesion molecule L1 influences calcium current in cultured neurons.
J. Cell. Physiol.
153:
313-320,
1992[Medline].
6.
Bading, H.,
D. D. Ginty,
and
M. E. Greenberg.
Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways.
Science
260:
181-186,
1993[Medline].
7.
Becchetti, A.,
A. Arcangeli,
M. R. Del Bene,
M. Olivotto,
and
E. Wanke.
Response to fibronectin-integrin interaction in leukaemia cells: delayed enhancing of a K+ current.
Proc. R. Soc. Lond. B Biol. Sci.
248:
235-240,
1992[Medline].
8.
Benos, D. J.,
M. S. Awayda,
I. I. Ismailov,
and
J. P. Johnson.
Structure and function of amiloride-sensitive Na+ channels.
J. Membr. Biol.
143:
1-18,
1995[Medline].
9.
Benos, D. J.,
C. M. Fuller,
V. G. Shlyonsky,
B. K. Berdiev,
and
I. I Ismailov.
Amiloride-sensitive Na+ channels: insights and outlooks.
News Physiol. Sci.
12:
55-61,
1997.
10.
Brismar, T.,
and
V. P. Collins.
Inward rectifying potassium channels in human malignant glioma cells.
Brain Res.
480:
249-258,
1989[Medline].
11.
Brown, K.,
T. B. Mapstone,
and
W. J. Oakes.
A modern analysis of intracranial tumors of infancy.
Pediatr. Neurosurg.
26:
25-32,
1997[Medline].
12.
Caffrey, J. M.,
A. M. Brown,
and
M. D. Schneider.
Mitogens and oncogenes can block the induction of specific voltage-gated ion channels.
Science
236:
570-573,
1987[Medline].
13.
Chen, C. F.,
M. J. Corbley,
R. M. Roberts,
and
P. Hess.
Voltage-sensitive calcium channels in normal and transformed 3T3 fibroblasts.
Science
239:
1024-1026,
1988[Medline].
14.
Collin, C.,
A. G. Papageorge,
D. R. Lowy,
and
D. L. Alkon.
Early enhancement of calcium currents by H-ras oncoproteins injected into Hermissenda neurons.
Science
250:
1743-1745,
1990[Medline].
14a.
Davis, F. G.,
S. Freels,
J. Grutsch,
S. Barlas,
and
S. Brem.
Survival rates in patients with primary malignant brain tumors stratified by patient age and tumor histological type: an analysis based on Surveillance, Epidemiology, and End Results (SEER) data, 1973-1991.
J. Neurosurg.
88:
1-10,
1998[Medline].
15.
Flamm, R. E.,
N. C. Birnberg,
and
L. K. Kaczmarek.
Transfection of activated ras into an excitable cell line (AtT-20) alters tetrodotoxin sensitivity of voltage-dependent sodium current.
Pflügers Arch.
416:
120-125,
1990[Medline].
16.
Garcia-Anoveros, J.,
B. Derfler,
J. Neville-Golden,
B. T. Hyman,
and
D. P. Corey.
BNaC1 and BNaC2 constitute a new family of human neuronal sodium channels related to degenerins and epithelial sodium channels.
Proc. Natl. Acad. Sci. USA
94:
1459-1464,
1997
17.
Garty, H.,
and
L. G. Palmer.
Epithelial sodium channels: function, structure, and regulation.
Physiol. Rev.
77:
359-396,
1997
18.
Gomez, M. P.,
G. Waloga,
and
E. Nasi.
Induction of voltage-dependent sodium channels by in vitro differentiation of human retinoblastoma cells.
J. Neurophysiol.
70:
1487-1496,
1993
19.
Grimes, J. A.,
S. P. Fraser,
G. J. Stephens,
J. E. G. Downing,
M. D. Laniado,
C. S. Foster,
P. D. Abel,
and
M. B. A. Djamgoz.
Differential expression of voltage-activated Na+ currents in two prostatic tumour cell lines: contribution to invasiveness in vitro.
FEBS Lett.
369:
290-294,
1995[Medline].
20.
Hemmick, L. M.,
T. M. Perney,
R. E. Flamm,
L. K. Kaczmarek,
and
N. C. Birnberg.
Expression of the H-ras oncogene induces potassium conductance and neuron-specific potassium channel mRNAs in the AtT20 cell line.
J. Neurosci.
12:
2007-2014,
1992[Abstract].
21.
Huang, Y.,
and
S. G. Rane.
Single channel study of a Ca2+-activated K+ current assoicated with ras-induced cell transformation.
J. Physiol. (Lond.)
461:
601-618,
1993[Abstract].
22.
Ismailov, I. I.,
B. K. Berdiev,
V. G. Shlyonsky,
and
D. J. Benos.
Mechanosensitivity of an epithelial Na+ channel in planar lipid bilayers: release from Ca2+ block.
Biophys. J.
72:
1182-1192,
1997[Abstract].
23.
Ji, H.-L.,
C. M. Fuller,
and
D. J. Benos.
Osmotic pressure regulates -rENaC expressed in Xenopus oocytes.
Am. J. Physiol.
275 (Cell Physiol. 44):
C1182-C1190,
1998
24.
Komuro, H.,
and
P. Rakic.
Selective role of N-type calcium channels in neuronal migration.
Science
257:
806-809,
1992[Medline].
25.
Leonard, R. J.,
M. L. Garcia,
R. S. Slaughter,
and
J. P. Reuben.
Selective blockers of voltage-gated K+ channels depolarize human T lymphocytes: mechanism of the antiproliferative effect of charybdotoxin.
Proc. Natl. Acad. Sci. USA
89:
10094-10098,
1992[Abstract].
26.
Mandel, G.,
S. S. Cooperman,
R. A. Maue,
R. H. Goodman,
and
P. Brehm.
Selective induction of brain type II Na+ channels by nerve growth factor.
Proc. Natl. Acad. Sci. USA
85:
924-928,
1988[Abstract].
27.
Nilius, B.,
and
W. Wohlrab.
Potassium channels and regulation of proliferation of human melanoma cells.
J. Physiol. (Lond.)
445:
537-548,
1992[Abstract].
28.
Price, M. P.,
P. M. Snyder,
and
M. J. Welsh.
Cloning and expression of a novel human brain Na+ channel.
J. Biol. Chem.
271:
7879-7882,
1996
29.
Repp, H.,
H. Draheim,
J. Ruland,
G. Seidel,
J. Beise,
P. Presek,
and
F. Dreyer.
Profound differences in potassium current properties of normal and Rous sarcoma virus-transformed chicken embryo fibroblasts.
Proc. Natl. Acad. Sci. USA
90:
3403-3407,
1993[Abstract].
30.
Silver, R. A.,
and
S. R. Bolsover.
Expression of T-type calcium current precedes neurite extension in neuroblastoma cells.
J. Physiol. Paris
85:
79-83,
1991[Medline].
31.
Teulon, J.,
P. M. Ronco,
M. Geniteau-Legendre,
B. Baudouin,
S. Estrade,
R. Cassingena,
and
A. Vandewalle.
Transformation of renal tubule epithelial cells by simian virus-40 is associated with emergence of Ca2+-insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers.
J. Cell. Physiol.
151:
113-125,
1992[Medline].
33.
Tsien, R. Y.,
T. Pozzan,
and
T. J. Rink.
T-cell mitogens cause early changes in cytoplasmic free Ca2+ and membrane potential in lymphocytes.
Nature
295:
68-71,
1982[Medline].
34.
Ullrich, N.,
and
H. Sontheimer.
Biophysical and pharmacological characterization of chloride currents in human astrocytoma cells.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1511-C1521,
1996
35.
Waldmann, R.,
G. Champigny,
N. Voilley,
I. Lauritzen,
and
M. Lazdunski.
The mammalian degenerin MDEG, an amiloride-sensitive cation channel activated by mutations causing neurodegeneration in Caenorhabditis elegans.
J. Biol. Chem.
271:
10433-10436,
1996