Acid-sensing Ion Channels in Malignant Gliomas*
Bakhrom K.
Berdiev
,
Jiazeng
Xia
,
Lee Anne
McLean
§,
James M.
Markert
¶,
G. Yancey
Gillespie¶,
Timothy B.
Mapstone
,
Anjaparavanda P.
Naren
**,
Biljana
Jovov
,
James K.
Bubien
,
Hong-Long
Ji
,
Catherine M.
Fuller
,
Kevin L.
Kirk
, and
Dale J.
Benos

From the Departments of
Physiology and Biophysics and
¶ Surgery, University of Alabama at Birmingham, Birmingham,
Alabama 35294 and the
Department of Neurosurgery, Emory
University, Atlanta, Georgia 30322
Received for publication, January 29, 2003
 |
ABSTRACT |
High grade glioma cells derived from patient
biopsies express an amiloride-sensitive sodium conductance that has
properties attributed to the human brain sodium channel family,
also known as acid-sensing ion channels (ASICs). This
amiloride-sensitive conductance was not detected in cells obtained from
normal brain tissue or low grade or benign tumors. Differential gene
profiling data showed that ASIC1 and ASIC2 mRNA were present in
normal and low grade tumor cells. Although ASIC1 was present in all of
the high grade glial cells examined, ASIC2 mRNA was detected in
less than half. The main purpose of our work was to examine the
molecular mechanisms that may underlie the constitutively activated
sodium currents present in high grade glioma cells. Our results show that 1) gain-of-function mutations of ASIC1 were not present in a
number of freshly resected and cultured high grade gliomas, 2) syntaxin
1A inhibited ASIC currents only when ASIC1 and ASIC2 were co-expressed,
and 3) the inhibition of ASIC currents by syntaxin 1A had an absolute
requirement for either
- or
-hENaC. Transfection of cultured
cells originally derived from high grade gliomas (U87-MG and SK-MG1)
with ASIC2 abolished basal amiloride-sensitive sodium conductance; this
inhibition was reversed by dialysis of the cell interior with Munc-18,
a syntaxin-binding protein that typically blocks the
interaction of syntaxin with other proteins. Thus, syntaxin 1A cannot
inhibit Na+ permeability in the absence of adequate
plasma membrane ASIC2 expression, accounting for the observed
functional expression of amiloride-sensitive currents in high grade
glioma cells.
 |
INTRODUCTION |
Primary intracranial neoplasms remain a significant cause of
mortality and morbidity in both children and adults. In patients with
malignant gliomas of World Health Organization Grades III and IV,
disease progression is uniformly rapid despite aggressive surgical and
adjunctive therapies. Median survival of optimally treated individuals
with the most aggressive of these tumors, glioblastoma multiforme, is
12 months, and this statistic has not varied for more than 30 years.
The ability to treat these tumors successfully has been hampered by a
fundamental lack of understanding of the control of the growth and
differentiation of glial cells, how the transformed phenotype evolves,
and how glioma cells modify their environment to support their
increased energy demands. We have identified a novel
amiloride-sensitive inward Na+ current that appears to be
constitutively activated in malignant gliomas but not in low grade or
normal astrocytes (1). In addition, glioma cells display up-regulation
of Cl
and K+ channels not found, at least
functionally, in normal glia (2, 3). Thus, it is reasonable to
hypothesize that ion transport systems specifically expressed by glioma
cells are intimately related to and indeed may define the unique growth
and migratory ability of these cells.
The main objective of the present study was to explore the molecular
mechanisms that underlie the constitutively activated Na+
currents present in high grade glioma cells. We assume, based on our
previous electrophysiological, pharmacological, and molecular biological studies, that the brain Na+ channels, also known
as acid-sensing ion channels
(ASICs),1 may comprise the
core conduction element of these channels. To date, six members of the
ASIC family have been cloned in mammals (4).
These channels share the common property of generating excitatory
currents in response to acidic pH when studied in heterologous expression systems, except for ASIC2b that, at least in its homomeric form, does not appear to respond to low pH (5). Moreover, ASIC4 is
inactive by itself and hence is not thought to encode a proton-gated ion channel (6, 7). Although the subunit composition of brain
Na+ channels in native tissues is unknown, evidence for
heteromultimeric channel formation with distinctive functional
characteristics has been obtained (7-10). These brain Na+
channels, like their epithelial counterparts, can be inhibited by
amiloride and its analogs, although with a much lower affinity (11). A
role for chemical pain sensation has been proposed for these channels in sensory neurons (12, 13), but their role in the brain
is obscure. Proton-activated neuronal currents have been identified in
different brain regions (13, 14). Hence, these channels may function as
acid pH sensors in normal brain and in pathophysiological states such
as ischemia or epilepsy where tissue acidification occurs (15, 16).
Gain-of-function mutations of ASIC1 and ASIC2 have also been detected
and have been proposed to participate in neurodegenerative disease (7, 17, 18). The function of these channels in glia remains a mystery, yet
functional amiloride-sensitive Na+ current expression
selectively characterizes high grade gliomas.
There are a number of potential mechanisms that may produce a
constitutive activation of this class of channel. We tested two
hypotheses. First, as a consequence of oncogenic transformation, gain-of-function mutations of ASIC1 may occur. Second, we tested the
hypothesis that syntaxin 1A may down-regulate ASIC1 activity by analogy
with the effects of syntaxin 1A on other ion channel activities, such
as voltage-dependent Ca2+ channels,
CFTR, and ENaC. Our results show that gain-of-function mutations
of ASIC1 are not present in a multitude of freshly resected and
cultured high grade gliomas (glioblastoma multiforme or GBM, World Health Organization classification Grade IV). However, syntaxin 1A inhibits ASIC currents, but only when both ASIC1 and ASIC2 are
co-expressed. Moreover, the inhibition of ASIC currents by syntaxin 1A
requires either
- or
-hENaC. Furthermore, message for ASIC2 could
be detected in 30-40% of high grade gliomas. We suggest that it is
the failure of syntaxin 1A to inhibit ASIC1 activity in the absence of
plasma membrane-localized ASIC2 that accounts for the functional
expression of amiloride-sensitive Na+ currents in
astrocytoma cells.
 |
MATERIALS AND METHODS |
DNA Constructs--
The cDNAs encoding full-length
-ENaC
subunits, ASIC1, and ASIC2 are described elsewhere (19-21). The human
ASIC 1a (ASIC1) and ASIC2a (ASIC2) cDNAs were a gift from Drs.
David Corey and Jaime Garcia-Añoveros of the Harvard Medical
School. The human ENaC subunits were given to us by Dr. Michael J. Welsh of the University of Iowa.
In Vitro Transcription and Translation--
cDNAs were
transcribed and translated in vitro using the TNT
transcription/translation system (Promega) as previously described (22). To test for protein-protein interaction between different ENaC
subunits and syntaxin 1A or between ASIC1 and ASIC2, we translated these constructs either with radioactive or nonradioactive methionine, immunopurified them, and reconstituted them in different combinations in proteoliposomes as previously described (22). Proteoliposomes were
solubilized in precipitation buffer that contained 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium
deoxycholate, and 0.1% SDS. All of the precipitation reactions were
carried out in this buffer. As a rule, antibodies directed against
nonlabeled proteins were used, and the presence of co-precipitated
radioactively labeled proteins were detected using SDS-PAGE
and autoradiography.
ASIC Expression in Xenopus Oocytes--
DNA samples were
in vitro transcribed using the SP6 or T3mMessage Machine
kits (Ambion, Austin, TX). The integrity of the cRNA was
assessed by running the samples under denaturing conditions on a
formaldehyde-agarose gel. RNA concentration and purity were determined
by UV spectrophotometry at 260 nm. The oocytes were removed from
appropriately anesthetized adult female Xenopus
laevis (Xenopus Express, Beverly Hills, FL) using
standard techniques (23). Follicular cells were removed by the addition
of collagenase to calcium-free medium as described (23). Defolliculated
oocytes were washed in OR-2 medium (82.5 mM NaCl, 2.4 mM KCl, 1.0 mM MgCl2, 1.8 mM CaCl2, and 5.0 mM HEPES, pH 7.4)
and allowed to recover overnight in half-strength Liebovitz medium at
18 °C. Groups of stage VI oocytes were injected with cRNA in a 50-nl
volume containing 12.5 ng of ASIC and/or ENaC subunits depending upon
the experiments. Standard two-electrode voltage clamp procedures were
performed at room temperature on the oocytes 24-72 h
post-injection.
The oocytes were impaled with two 3 M KCl-filled
electrodes, each having resistances of 0.5-2 M
. A Dagan TEV-200
voltage clamp amplifier was used. Two Ag-AgCl reference electrodes were connected to the bath by 3 M KCl, 3% agar bridges. The
regular perfused solution was ND96 (96 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 2 mM KCl, and 5 mM HEPES, pH 7.4). To prepare
ND96 having pH values below 6.0, MES replaced HEPES. To permit ASIC to
recover completely from desensitization following acidification, at
least 45 s elapsed prior to a subsequent challenge of the oocyte
with another acidic pH solution. The experiments were controlled by
pCLAMP 8.0 software (Axon Instruments, Burlingame, CA), and the
proton-sensitive currents measured at
60 mV were digitized. The
current-voltage (I-V) relationships were determined by changing the
clamp potential in 20-mV increments from
100 to +100 mV from a
holding potential of 0 mV. All of the animal care and experimental
protocols were approved by the University of Alabama at Birmingham
Institutional Animal Care and Use Committee (protocol number APN 020506241).
Planar Lipid Bilayer Experiments--
Planar lipid bilayers were
formed from a solution of a 2:1
diphytanoyl-phosphatidyl-ethanolamine:diphytanoyl-phosphatidylserine dissolved in n-octanol (total concentration, 25 mg/ml). The
membranes were spread onto a 200-µm diameter hole drilled in a
polystyrene cup. Membrane capacitance averaged 250-350
picofarads. The standard bathing solution was 100 mM
NaCl plus 10 mM MOPS buffer, pH 7.4. The lipids were
obtained from Avanti Polar Lipids (Alabaster, AL). All of the solutions
were filtered sterilized using 0.22-µm Sterivex-GS filters
(Millipore, Bedford, MA). Electrical connections and current
measurements were made as previously described (24). The voltage was
applied to the cis chamber, and the trans chamber was held at virtual ground. Oocyte membrane vesicles containing the
channels of interest were applied to a preformed bilayer with a glass
rod from the trans compartment with the potential held at
40 mV. Only membranes containing a single ion channel were used for
experiments. The data were analyzed as before (24).
Whole Cell Patch Clamp--
Micropipettes were constructed using
a Narashigi pp-83 two-stage micropipette puller. The tips of these
pipettes had internal diameters of ~0.3-0.5 µm and outer diameters
of 0.7-0.9 µm. When filled with an electrolyte solution containing
100 mM potassium gluconate, 30 mM KCl, 10 mM NaCl, 20 mM HEPES, 0.5 mM EGTA,
<10 nM free Ca2+, 4 mM ATP 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 ionic gradients across the cell membrane
in vivo. The 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 cells were viewed using a Nikon model TE200 inverted microscope
fitted with an ultraviolet light source. Under fluorescence,
transfected cells expressing jellyfish green fluorescence protein were
identified for whole cell patch clamp analysis.
The micropipettes were abutted to the cells, and slight suction was
applied. Seal resistance was continuously monitored using pCLAMP 8. After the formation of seals with resistances in excess of 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 produced a sudden increase in
capacitance with no change in seal resistance, indicative of the whole
cell configuration. The cells were then held at a membrane potential of
0 mV and clamped sequentially for 1600 ms each to membrane potentials
of
100 mV to +100 mV in 20-mV increments, returning to the holding
potential of 0 mV for 1 s between each test voltage. 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 8 software and with an A/D interface controlled by pCLAMP
(Axon Instruments, Sunnyvale, CA).
Antibodies, Immunoprecipitation, Gel Electrophoresis, and Western
Blots--
The anti-ASIC2a antibodies (Alamone, Jerusalem, Israel),
anti-ASIC1 antibodies (Chemicon International, Temecula, CA),
anti-
ENaC antibodies (raised against peptide;
CNTLRLDRAFSSQLTDTQLTNEL), and anti-syntaxin 1A antibodies (25) were
used for immunoprecipitation and Western blot detection. Crude membrane
fractions from the glioblastoma cell line SK-MG1 were prepared as
previously described (26). Immunoprecipitation and co-precipitation
from SK-MG1 cell lysate were performed using the size X protein A
immunoprecipitation kit from Pierce according to the manufacturer's
instructions. Briefly, affinity-purified ASIC2 antibody (280 µg) was
bound and cross-linked to protein A beads. After extensive washing, the beads were added to the SK-MG1 cell lysates (1.5 ml) and incubated overnight at 4 °C. The cells were lysed with 1.5 ml of 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.5, containing protease inhibitors.
Immunoprecipitated proteins were eluted and analyzed by electrophoresis
and Western blotting. Standard electrophoresis and blotting protocols
were followed. Briefly, the protein was run on 8% SDS-PAGE mini gels
with 4% stacking gels for about 1 h at 200 V in a Bio-Rad
Minisubcell apparatus. The gels were transferred onto polyvinylidene
fluoride, treated with 5% nonfat dry milk/Tris-buffered saline/Tween, and probed with the appropriate antibodies. Secondary antibody was conjugated to horseradish peroxidase, and visualization was performed with chemiluminescent reagents (Amersham Biosciences). Controls included substitution of nonimmune rabbit IgG for primary antibodies.
ASIC2 Transfections--
To determine whether expression of
hASIC2 could restore the normal astrocyte Na+ current
phenotype to astrocytoma cells, we transfected hASIC2 contained in a
eukaryotic expression vector (GW1-CMV, the kind gift of Dr. David
Corey, Massachusetts General Hospital) into the SK-MG1 (gift of Dr.
Gregory Cairncross, University of Calgary) or U87-MG (purchased from
ATCC) astrocytoma cell lines using LipofectAMINE (Invitrogen) for whole
cell patch-clamp analysis. Briefly, 5-10 µg of hASIC2 was mixed with
100 µl of serum-free Optimem (Invitrogen). In most experiments,
hASIC2 was co-transfected with 3 µg of a green fluorescence protein
reporter vector. Control cells were either untransfected or transfected
with GW1-CMV vector from which the hASIC2 insert had been removed by
cutting with EcoRI and religating the cut ends. The
DNA/Optimem mixture was mixed with 100 µl of Optimem plus 7.5 µl of
lipid and incubated at room temperature for 45 min, after which time a
further 800 µl of Optimem was added. Tumor cells plated onto glass
coverslips at 60-80% confluency were rinsed twice in Optimem and then
incubated for 6 h in the DNA/lipid Optimem mixture. At the end of
this incubation period, the medium was aspirated and replaced with
fresh Dulbecco's modified Eagle's medium plus 10% fetal bovine
serum. The cells were allowed to recover overnight and were used for
patch-clamp experiments over the course of the next 48 h.
Preparation of Syntaxin 1A and Syntaxin 3 Proteins--
Defined
regions of syntaxin 1A and syntaxin 3 (amino acids 1-266; referred to
as
C as they lack the C-terminal membrane anchor) were generated by
polymerase chain reaction. Restriction sites (EcoRI in
forward and XhoI in reverse), 5'overhangs (TATA), and a stop
codon were introduced into the primers. The PCR product was cloned into
pGEX5X-1 vector (Pharmacia Corp.) and transformed in a protease
deficient Escherichia coli strain (BL21-DE3). The GST fusion
protein (GST-Syn1A
C and GST-Syn3
C) was purified on glutathione-Sepharose beads (Pharmacia Corp.). The protein was eluted
using 20 mM reduced glutathione in phosphate-buffered
saline, pH 7.4, and dialyzed extensively at 4 °C with at least four
changes of cold phosphate-buffered saline. The protein was concentrated using a centrifugal filter device (Centriprep; 10,000 Dalton cut-off; Millipore). The protein was estimated using the BCA kit (Pierce) and
stored in small aliquots at
80 °C.
RNA Extraction--
Freshly excised human brain tissue or
primary cultured brain tumor cells (GBM or normal brain from temporal
lobe) were frozen and stored in liquid nitrogen by the University of
Alabama at Birmingham Neurosurgery Brain Tissue Bank under
Institutional Review Board approval X9804090300. The frozen
tissue was ground into a fine powder using a mortar and pestle, under
liquid nitrogen, after which 1 ml of Trizol (Invitrogen) containing 250 µg of glycogen was added. The Trizol/powder mixture was transferred
to a chilled glass/Teflon homogenizer and ground for 10 strokes while
on ice. The homogenate was sequentially passed through 25- and 26-gauge needles to further reduce cellular debris and then transferred to a
1.5-ml microcentrifuge tube. 200 µl of chloroform was added to
the homogenate, vortexed for 30 s to mix, and centrifuged at maximum speed (14,000 rpm) for 5 min using a tabletop centrifuge at
room temperature. The aqueous phase was transferred to a fresh 1.5-ml
tube, 500 µl of ice-cold isopropanol were added, and the RNA was
allowed to precipitate overnight at
20 °C. The precipitated RNA
was pelleted by centrifugation at maximal speed for 15 min at room
temperature, washed with 1 ml of 70% ethanol, and centrifuged for 5 min. The pellet was dissolved in 100 µl of RNase-free
H2O, and 100 µl of phenol:chloroform (1:1) was added and
mixed by vortexing. After centrifugation, the aqueous phase was
transferred to a fresh 1.5-ml tube, and 100 µl of chloroform was
added, mixed by vortexing, and centrifuged. After transferring the
aqueous phase to a fresh 1.5-ml tube, 10 µl of 7.5 mg/ml ammonium
acetate and 250 µl of 100% ethanol were added and mixed well, and
the RNA was allowed to precipitate overnight at
20 °C. The
precipitated RNA was pelleted by centrifugation, washed twice with 70%
ethanol, and resuspended in RNase-free H2O. The integrity
of the RNA was verified following electrophoresis through 1%
agarose-formaldehyde gels. All of the equipment (e.g.
homogenizers, mortar, pestle, etc.) were pretreated with RNase-Zap
(Ambion) and rinsed with diethyl pyrocarbonate-treated H2O prior to use. All of the human tissue acquisition and
protocols were reviewed and approved by the University of Alabama at
Birmingham Institutional Review Board (protocol numbers X021015002 and X980409003).
RT-PCR--
RT-PCR was performed using a OneStep RT-PCR kit
(Qiagen) according to the manufacturer's instructions, using 0.1-1
µg of total RNA as template. Custom primers specific to ASIC1 were
synthesized by Invitrogen and used at a final concentration of 0.6 µM. For example, the forward and reverse primers
5'-CCCGCATGGCAAAGAG-3' and 5'-GGCTCAGCAGGTAAAGTCC-3' corresponded to
bases 1109-1125 and 1572-1587 (plus 3 bases of the 3'-untranslated
region) of ASIC1, respectively. Reverse transcription was performed
using a single cycle of 50 °C for 30 min. This was followed by a
single cycle of 95 °C for 15 min, which inactivates the reverse
transcriptase while activating the HotStart Taq DNA
polymerase, followed by 40 cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 1 min, and finally a single 10-min cycle at
72 °C. Aliquots of each reaction mixture were electrophoresed on a
2% Nu-Sieve (FMC Corp.) agarose gel using PCR markers (Promega) to
determine molecular size. Products of the correct molecular size were
isolated from the gel using the QIAquick gel extraction kit (Qiagen)
and subcloned into the pCR-2.1 vector using the TOPO-TA cloning kit
(Invitrogen) following the manufacturer's instructions. Recombinants
were selected by blue/white screening and restricted with
EcoRI (Promega) to verify incorporation of insert of correct
size. ASIC1 sequences were verified by further restriction enzyme
digest analysis and automated DNA sequencing (DNA Sequencing Facility,
Iowa State University).
 |
RESULTS |
We previously reported that there was an amiloride-sensitive
component to whole cell inward currents in high grade, highly invasive
brain tumors (1). These currents were seen in primary cultures of
tumors as well as in established glioma cell lines. This
amiloride-sensitive current was not present in low grade brain tumors
or normal brain tissue. To verify these initial findings and to extend
these results to freshly resected GBM tumors, we performed whole cell
patch-clamp experiments on freshly excised normal astrocytes and
freshly resected and primary cultured brain tumor tissue samples (Fig.
1). In the basal state, the current records for both freshly resected and primary cultures of World Health
Organization Grade III and IV tumor cells were characterized by large
inward currents (Fig. 1A), and these results were completely inhibited following superfusion with 100 µM amiloride
(Fig. 1B). Fig. 1C shows the difference current
(i.e. the amiloride-sensitive component). These results
should be contrasted to the lack of effect of 100 µM
amiloride in normal astrocytes and Grades I and II astrocytoma cells.
The absolute magnitudes of the outward currents at +40 mV (Fig.
2A) and inward currents at
60 mV (Fig.
2B) in the absence and presence of amiloride for
normal astrocytes, different grade gliomas, medulloblastoma, and two
continuous GBM cell lines are summarized in Fig.
2. Although there was no discernible
pattern to the magnitudes of either the outward or inward currents,
amiloride only blocked inward currents in the more aggressive, higher
grade tumors (Grades III and IV and medulloblastoma). Amiloride
likewise blocked inward currents in SK-MG and U87-MG cells, both
originally derived from GBM. A summary of the current-voltage (I-V)
characteristics of both freshly resected normal astrocytes and GBM
cells is presented in Fig. 3. It is
apparent that the GBM cells are depolarized by an average of 31 mV
compared with normal astrocytes under these recording conditions. The
depolarized zero current membrane potential is due to the presence of
an enhanced Na+ conductance. These results confirm our
previous findings and establish that this amiloride-inhibitable
component to whole cell currents is present in freshly excised GBM
cells.

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Fig. 1.
Representative whole cell patch clamp
recordings from freshly isolated normal human astrocytes and GBM (World
Health Organization Grade IV) and primary cultures of different World
Health Organization Grade astrocytomas. The cells were
voltage-clamped between 100 and +100 mV in 20-mV increments from a
holding potential of 0 mV, and the resulting currents were recorded.
The cells were superfused with RPMI 1640 medium, and the pipette
contained 100 mM potassium gluconate, 30 mM
KCl, 20 mM HEPES, 0.5 mM EGTA, 4 mM
ATP, and <10 nM free calcium at a pH of 7.2. Top
traces, basal currents. Middle traces, currents
following superfusion with 100 µM amiloride. Bottom
traces, difference currents (amiloride-sensitive). This experiment
was repeated at least four separate times for each cell type.
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Fig. 2.
Summary of absolute outward (+40 mV;
A) and inward ( 60 mV; B) currents obtained
from a variety of gliomas and normal cells in the absence and presence
of 100 µM amiloride, using whole
cell patch clamp. At least four cells were measured from each
preparation.
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Fig. 3.
Summary I-V curves of freshly
resected normal astrocytes and GBM cells. Inward currents (at 60
mV) were 7.5 + 1.2 pA (normal) and 43.8 + 14.5 (GBM). Outward
currents (at +40 mV) averaged 42.2 + 2.4 and 47.2 + 12.5 pA for normal
and GBMs, respectively, in this set of experiments.
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|
Because these currents were inhibited by amiloride and because the
apparent amiloride inhibitory constant (Ki) was high
(i.e. ~10 µM), we hypothesized that the
channels underlying these currents were of the ASIC variety. Thus, to
begin to test this hypothesis, we performed RT-PCR on total RNA
extracted from human brain tissue samples obtained during craniotomy
for epilepsy (normal tissue, lanes 1-3) or primary GBM
resection (lanes 4-6) and from continuous cell lines
derived from anaplastic astrocytoma (lane 7), a gliosarcoma (lane 8),
and 7 different GBMs (lanes 9-15) (Fig.
4A). Specific primers were
designed to amplify a 482-bp product for ASIC1a and a 447-bp product
for ASIC2a. We also designed primers to identify ASIC 3, ASIC4, and
-,
-,
-, and
-hENaC subunits. The ASIC1a product was
detected in all of the samples (both normal and tumor), including the
pancreatic carcinoma cell line BxPC-3 (lane 16). In
contrast, the ASIC2a message was found in the three normal samples and
in only one of the freshly resected GBMs (lane 5) and in the
D54MG, SK-MG1, and U373MG cell lines (lanes 9,
11, and 15). Direct sequencing of the PCR
products confirmed their identity. ASIC 3 was present in many of the
glioma cell lines and in both normal tissue and GBM tumors, whereas
ASIC 4 was present in most of the fresh tissues samples but in none of the cultured cell lines.
-hENaC was found in the majority of the
tumor cell lines, in one freshly excised GBM tissue but not in any of
the normal samples.
-hENaC was found in one of the freshly resected
GBMs (lane 4) and in the pancreatic carcinoma cell line but
not in any other samples. Also, either
- and/or
-hENaC were
observed in most of the freshly excised GBM tissues as well as in the
tumor cell lines. The
-hENaC was also present in the normal tissue
as compared with
-hENaC.

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Fig. 4.
RT-PCR detection of ASIC/ENaC family members
in normal and GBM tissues and cell culture samples. All of the
reactions were negative for genomic DNA (PCR with RT). A ØX174Haell
molecular weight ladder was used in a 2% Nu-Sieve agarose gel.
A, RT-PCR of amiloride-sensitive Na+ channel
subunits. This entire experiment was repeated three times with
identical results. B, RT-PCR analysis of ASIC1 and ASIC2.
Primers for ASIC1 spanned bp 1091-1537 and bp 1109-1587 + 3'-untranslated region for ASIC2. N, freshly excised normal
tissue; G, freshly excised GBM; P, primary (first
passage) GBM cells; A, astrocyte; primary (first passage)
culture of normal human astrocytes.
|
|
Another independent set of experiments was performed to examine
specifically the distribution of ASIC1 and ASIC2 message between different tumor samples (Fig. 4B). Total RNA was extracted
from tissue obtained from resected GBM tumors (lanes labeled
G), primary cultures of GBM cells (lanes labeled
P), cultured normal human astrocytes (Astrocyte),
and continuous GBM cell lines. Examining in toto the data in
Fig. 4, ASIC1 mRNA was detected in all of the samples, both normal
and tumor. In contrast, ASIC2 message was found in the four normal
tissue samples, in 6 of 15 freshly resected and primary GBMs and in 4 of 12 GBM cell lines (SK-MG1, D54MG, U373MG, and LN229). These results
support the hypothesis that ASIC may be a component of the channels
that underlie the amiloride-sensitive currents seen in GBM. ASIC1 is
present in both normal astrocytes and GBM tissues, but ASIC2 and ASIC4
are present in normal astrocytes and not in the majority of high grade gliomas. Therefore, although the actual molecular constitution of the
observed amiloride-sensitive component found in high grade brain tumor
cells is not known, it may be comprised of a variety of subunits from
the degenerin/ENaC superfamily.
Electrophysiology of ASIC Expressed in Oocytes and Reconstituted
into Planar Bilayers--
We used Xenopus oocytes to
express ASIC1 and ASIC2 individually and in combination. Whole cell
acid-activated sodium currents, after expression of wild type ASIC1,
ASIC2, and ASIC1/2 in oocytes, are shown in Fig.
5A. The upper
panels show the actual currents measured during the voltage clamp
protocol. There was no measurable current when the extracellular pH
(pHO) was 7.5. However, upon reduction of pHO
to 4.0, there was a transient current response. Two currents are
routinely measured: the peak inward current (Ip) and the steady state current (Is), which was
measured 8 s after Ip.
Ip was greatest for ASIC1 and least for ASIC2.
The combination of ASIC1/2 resulted in a transient current signature
intermediate between ASIC1 and ASIC2. The reversal potentials for
Ip and Is were identical
within all three groups of oocytes, but the reversal potential for
ASIC2 and ASIC1/2 expressing oocytes was ~20 mV lower than for ASIC1
expressing oocytes (Fig. 5A, bottom panels). These results suggest that ASIC induces cation selective currents with
PNa/PK ranging from 6-20
depending upon whether the oocytes were injected with ASIC1, ASIC2, or
ASIC1/2 cRNA. Moreover, these functional results suggest that ASIC1 and
ASIC2 interact in such a way that the conductance characteristics are
altered, consistent with what has been suggested in earlier studies
(8).

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Fig. 5.
Acid-activated ASIC currents in
Xenopus oocytes. A, inward sodium
currents versus time were measured under voltage clamp
conditions. The oocytes were voltage-clamped from 100 mV to +100 mV
in 20-mV increments. The currents were activated by reduction in pH to
4.0. Between voltage clamp levels, the extracellular pH was returned to
pH 7.5 for 45 s prior to the next voltage clamp level and pH 4.0 exposure. Bottom panels, current-voltage relationships for
ASIC1, ASIC2, and ASIC1/2 expressing oocytes. ,
Ip; , Is (8 s after
Ip). Each experiment was repeated four times
with similar results. B, representative current traces of
gain-of-function mutation of ASIC2 G430F expressed in
Xenopus oocytes. The experimental conditions were the same
except that experiments were all conducted at pH 7.5. Bottom left
panel, representative current trace obtained at a voltage clamp
level of 100 mV in an oocyte expressing ASIC2 G430F. At pH 7.5 (time
0), a very large inward current could be measured. 100 µM
amiloride obliterates the current, and it is reversible. Bottom
right panel, summary current-voltage curve for the amiloride
sensitive component of the gain-of-function mutant (n = 4). The reversal potential was +3 mV.
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We have presented evidence that both wild type and gain-of-function
mutants of ASIC1 and ASIC2 can be incorporated and studied in planar
lipid bilayers (27). Well defined pH-activated (for the wild type)
single channels with a unitary conductance of ~18-20 picosiemens
were recorded. However, little work has been done with gain-of-function
ASIC mutants expressed in oocytes. Therefore, we expressed the G430F
mutation of ASIC2 in Xenopus oocytes and subsequently
assessed sodium currents (Fig. 5B). Voltage clamp traces in
the absence and presence of amiloride are shown in the upper
half of the figure. In contrast to the wild type channel, the
currents do not inactivate, they are constitutively active (even at
neutral pH), and the inward currents are sensitive to amiloride. The
bottom half of the figure shows the inhibition in response
to amiloride. These currents were recorded at
100 mV. Almost all of
the inward current was inhibited by amiloride. The reversal potential
for this gain-of-function mutation was +3 mV, indicating a somewhat
reduced PNa/PK as compared with the wild type
channel as in bilayers. This gain-of-function mutation was less
sensitive to reductions of external pH. No amiloride-sensitive currents
could be elicited in oocytes (n = 18, 3 frogs) injected with the gain-of-function ASIC1a mutant cDNA (i.e.
G433F). However, this mutant channel could be recorded in planar
bilayers and displayed the same biophysical and pharmacological
characteristics as the G430F ASIC2a channel (not shown). We have
previously shown that the protein actin, when added to the presumptive
cytoplasmic surface of ENaC incorporated into planar lipid bilayers,
can functionally interact with the channel reducing single channel
conductance as well as altering cation selectivity (28). Addition of
actin to the gain-of-function ASIC1 or ASIC2 channel had no effect on the single channel signatures. However, when vesicles were prepared from channels containing a mixture of gain-of-function ASIC1 plus ASIC2, actin reduced single channel conductance to ~9 picosiemens, and the channel was almost always open (not shown). Therefore, both our
bilayer and oocyte expression studies show that ASIC1 and ASIC2 can be
functionally studied, and more importantly, co-expression of ASIC1 plus
ASIC2 results in channels that are functionally distinct from their
homomeric counterparts.
Gain-of-function Point Mutations in ASIC1 Isolated from Brain Tumor
Samples--
To test the hypothesis that gain-of-function mutations
may underlie the appearance of an amiloride-sensitive conductance in malignant tumor cells, we isolated total RNA from malignant gliomas following resection or in established cell lines. The results of these
experiments are presented in Table I.
Using RT-PCR and the appropriately designed primers, we sequenced the
entire coding region of ASIC1 from these cells. From the sequence
analysis, we determined that there was no gain-of-function in positions that have previously been described, namely, Gly433 (ASIC1)
or Gly430 (ASIC2) (7, 17, 18). The results of these
experiments demonstrate that obvious gain-of-function mutations do not
occur in ASIC1. The ASIC1 primary sequence, for the most part, was
identical to the wild type channel. However, during these analyses we
did note some other random mutations in ASIC1, namely, F197S, D212G, E219K, and N395T. We constructed ASIC1 mutants containing these specific amino acid changes and expressed them in oocytes. None of
these mutant constructs displayed gain-of-function activity (not
shown). Thus, from these results we conclude that gain-of-function mutations in ASIC1 do not contribute to the constitutive
amiloride-sensitive sodium conductance measured in high grade glioma
cells.
SNARE Protein Interactions with ASIC--
Fig.
6 presents data obtained from our
previous differential gene profiling studies on human brains showing
that syntaxin 1A is present in normal and high grade glioma tumor
cells. A more extensive survey by RT-PCR demonstrates the presence of
syntaxin 1A in normal human astrocytes, freshly resected GBMs, and GBM cell lines (Fig. 7). Moreover,
-hENaC
appears to be present in all these samples as well. We tested the
hypothesis that syntaxin 1A may down-regulate ASIC activity by analogy
with the effects of syntaxin 1A on other ion channel activities such as
voltage-dependent calcium channels, CFTR, and ENaC (25,
29-32). The results of these experiments are shown in Fig.
8 and summarized in Table II. GST-Syn1A
C (0.175 µM) had no effect on the activity of channels comprised
of ASIC1 plus ASIC2 (either alone or in combination) when the
subunit of ENaC was not present (Fig. 8A). However, if
-hENaC was simultaneously reconstituted with ASIC1 plus ASIC2, the
same concentration of GST-Syn1A
C significantly inhibited channel
open probability (Fig. 8A). The concentration dependence of
this inhibitory effect is shown in Fig. 8B
(KD = 207 ± 31 nM). If the
syntaxin 1A-binding protein Munc-18 is added to the bath, subsequent
addition of GST-Syn1A
C had no effect (Fig. 8C). GST by
itself did not have any effect (Fig. 8D). Truncation of
either the N or C termini of
-ENaC rendered GST-Syn1A
C
ineffective (Table II). Interestingly,
-hENaC can substitute for
-hENaC. We conclude that syntaxin 1A can specifically down-regulate
ASIC activity only if ASIC1 plus ASIC2 and
(or
)-hENaC are
present. Essentially the same results were found in oocyte expression
studies, i.e. inhibition of AISC1 plus ASIC2 by syntaxin
required
-hENaC (Fig. 8E). This effect was specific for
syntaxin 1A, because syntaxin 3 was without effect (not shown). Based
on our molecular biological data (Figs. 4 and 6), it appears that in
normal cells ASIC1, ASIC2, and
(or
)-hENaC are present, but in
most tumor cells ASIC2 is lacking or attenuated. Thus, inhibition of
channel activity by syntaxin 1A would be relieved in tumor cells
because of the absence or reduced levels in the plasma membrane of one
necessary component of the complex, namely, ASIC2.

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Fig. 6.
Cluster diagram of selected ENaC and syntaxin
1A expressed in GBM samples compared with normal brain tissue.
Colors represent the relative levels of gene expression with the
brightest red indicating the highest level of expression and
the green depicting the lowest level or absence of
expression (these data were taken from supplemental information
associated with a previously published study (39) with
permission).
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Fig. 7.
RT-PCR analysis of syntaxin 1A in brain
tumors. RT-PCR of syntaxin 1A in various glioma and normal brain
tissue samples.
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Fig. 8.
The effects of GST-Syn
1A C on ASIC1 plus ASIC2 incorporated into
planer lipid bilayers. A, bilayers were bathed with
symmetrical 100 mM NaCl and 10 mM MOPS, pH 7.4;
the holding potential was 100 mV. These traces are
representative of at least four individual experiments. B,
concentration dependence of the inhibition effects of GST-Syn 1A C on
Po of ASIC1 + ASIC2 + -ENaC channel in planar
lipid bilayers. C, Munc-18 (0.5 µM) addition
prevents inhibitory effects of GST-Syn 1A C on ASIC1 + ASIC2 + -hENaC in planar lipid bilayers (n = 4).
D, GST by itself has no effect on ASIC1 + ASIC2 + -hENaC
(n = 4). E, ASIC1, ASIC2, and heteromeric
ASIC1/2 ± -hENaC following expression in oocytes. The currents
(Ip) were normalized to the values measured at
60 mV in the absence of syntaxin 1A. The currents were evoked by a
step decrease in pHo to 4.0. Co-expression of syntaxin 1A
with ASIC1 + ASIC2 + -hENaC resulted in significantly
(p < 0.01) lower mean currents
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Table II
Single channel characteristics of ASIC in the absence and in the
presence of syntaxin 1A in planar lipid bilayers
G, single channel conductance; Po, the
probability of the channel being in open state; Na/K, sodium and
potassium permeability ratio calculated under bionic conditions (100 mM NaCltrans/100 mM KClcis);
Ki Amil, Amiloride inhibitory constants; ND,
not determined. Bilayer bathing solution in the syntaxin 1A experiments
were buffered with 100 µM EGTA, and the levels of free
Ca2+ were 25-50 nM.
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Biochemical Evidence of ASIC1a, and ASIC2a, and
-ENaC
Interactions--
The results of our preliminary functional studies in
oocytes and bilayers imply an interaction between ASIC1, ASIC2, and
-ENaC. To provide biochemical evidence of such an interaction, we
did a co-precipitation study in a glioma cell line (SK-MG1). The
strategy was to immunoprecipitate ASIC2 and test for the
co-precipitation of ASIC1 and
-ENaC by Western blot. The results are
shown in Fig. 9. ASIC2 can be detected by
Western blot after precipitation with anti-ASIC2 antibodies, but not if
nonimmune IgG was used for precipitation (Fig. 9A),
demonstrating the specificity of the anti-ASIC2 antibodies. Fig. 9
(B and C) shows co-precipitation of ASIC1 and
-ENaC with ASIC2 and lack of co-precipitation if nonimmune IgG was
used for precipitation. These biochemical results demonstrate that
ASIC1, ASIC2, and
-ENaC can associate to form heteromeric complexes
in an intact glioma cell line.

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Fig. 9.
Analysis of the interaction between ASIC2,
ASIC1, and -ENaC. Crude membrane
fractions from SK-MG cells were used for precipitation and
co-precipitation. Anti-ASIC2 antibodies or nonimmune IgG cross-linked
with protein A beads were used for precipitation. Precipitated and
co-precipitated proteins were probed with anti-ASIC2 (A),
anti-ASIC1 (B), or anti- -ENaC antibodies (C).
ASIC2 can be detected by Western blot after precipitation with
anti-ASIC2 antibodies but not if nonimmune IgG was used for
precipitation (A). ASIC1 and -ENaC co-precipitated with
ASIC2 but not if nonimmune IgG was used for precipitation (B
and C).
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To provide additional evidence for these potential interactions,
co-immunoprecipitation experiments have been performed with in
vitro translated and proteoliposome-reconstituted ASIC1, ASIC2, and the voltage gated chloride channel 5 (ClC5) as a control peptide (Fig. 10A). Transcripts of
ASIC1 and ASIC2 were radioactively labeled (two left lanes).
These transcripts were immunopurified and reconstituted either with the
unlabeled conjugated ASIC partner or with unlabeled ClC5. The strategy
was to immunoprecipitate one partner to detect the presence of the
other by autoradiography. Our results demonstrated that ASIC1 and ASIC2
can indeed associate to form heteromeric complexes, and
co-precipitation between ASIC1 and ASIC2 is specific because of the
lack of co-precipitation of ASIC1 and ASIC2 with ClC5. We used the same
strategy to test for protein-protein interactions between
-hENaC and
syntaxin 1A, between
-hENaC and syntaxin 1A, and between
-hENaC
and syntaxin 1A (Fig. 10B). We used in vitro
translated proteins for these co-precipitation experiments because of a
lack of good anti-
-hENaC antibodies. In vitro translation allowed us to test co-precipitation between two proteins, even if only
one antibody is available, because the co-precipitated protein is
radioactively labeled and can be detected without antibodies. In Fig.
10B, the results show that immunoprecipitation with
anti-syntaxin 1A antibodies resulted in co-precipitation of both
-hENaC and
-hENaC but not
-hENaC.

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Fig. 10.
Analysis of interactions between ASICs and
different hENaC subunits with syntaxin 1A using co-precipitation.
A, in vitro (IN V) transcription and
translation of ASIC1 and ASIC2 were performed using either radioactive
(two left lanes) or nonradioactive methionine. Nonlabeled
ClC5 was used as a control peptide. Translated proteins were
immunopurified, reconstituted into liposomes in different combinations,
and solubilized before co-precipitation as described under "Materials
and Methods." To test for co-precipitation, antibodies directed
against nonlabeled protein were used, and the presence of
co-precipitated radioactively labeled ASIC was detected. In the first
of the two middle lines, protein from solubilized
proteoliposomes containing [35S]Met ASIC1 plus unlabeled
ASIC2 was immunoprecipitated (IP) using anti-ASIC2
antibodies; the next lane shows that ASIC2 is present in the
immunoprecipitate of a mixture of [35S]Met ASIC2 plus
unlabeled ASIC1 using anti-ASIC1 antibodies. The last two
lanes demonstrate that neither ASIC1 nor ASIC2 can be
co-precipitated with CLC5 demonstrating specificity of interaction
between ASIC1 and ASIC2. B, three different rENaC subunits
( , , and ) were transcribed and translated in vitro
in the presence of radioactive methionine. An asterisk was
added to the name of the channel subunits to mark radioactively labeled
components. Transcription products were analyzed using SDS-PAGE. Each
subunit was mixed with in vitro translated nonlabeled
syntaxin 1A, reconstituted in proteoliposomes, and solubilized before
precipitation as described under "Materials and Methods." Syntaxin
1A was precipitated using monoclonal anti-syntaxin 1A antibodies, and
the precipitates were analyzed for the presence of radioactively
labeled rENaC subunits using SDS-PAGE and autoradiography. Both
-hENaC and -hENaC, but not -hENaC, co-precipitate with
syntaxin 1A.
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Transfection of U87-MG and SK-MG-1 Tumor Cells--
If the
hypothesis is correct, namely, that the absence of an
amiloride-sensitive current in normal cells is due to the presence of
ASIC1, ASIC2,
(or
)-hENaC, and syntaxin 1A in the membrane and
that the basally activated amiloride-sensitive conductances seen in
high grade glioma cells is due to a relief of inhibition by syntaxin 1A
because of the absence of ASIC2, then transfecting ASIC2 back into a
tumor cell (displaying amiloride-sensitive currents) should
re-establish the nontumor phenotype (i.e. no inward
amiloride-sensitive current). The results of such experiments are shown
in Fig. 11A. These
experiments were performed in two different cultured glioma cell lines
(U87MG and SK-MG1) that were originally derived from GBMs. The cells
were transfected with an empty expression vector and green fluorescence
protein (left panels). Superfusion of 100 µM
amiloride abolished the inward current in these GBM-derived tumor
cells, as reported earlier (Ref. 1 and Fig. 2). In contrast, in
the right panels, tumor cells that were transfected with
both green fluorescence protein and the vector containing ASIC2
resulted in a phenotype in which there was no amiloride-sensitive
inward current. Likewise, inclusion of Munc-18, a syntaxin 1A-binding protein that blocks syntaxin interaction with other proteins (25), in
the patch pipette of ASIC2-transfected U87MG cells rapidly restored the
inward amiloride-sensitive Na+ currents (Fig.
11B). These results are consistent with the hypothesis that
SNARE protein interactions (specifically syntaxin 1A) with ASIC may
underlie the lack of channel activity seen in normal astrocytes and the
appearance of such activity in the malignant state.

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Fig. 11.
Representative whole cell patch-clamp
recordings from transfected U87-MG and SK-MG-1 cells. Voltage
clamp conditions were identical to those described in the legend to
Fig. 1. A, top traces, basal currents.
Middle traces, currents following superfusion with 100 µM amiloride. Bottom traces, difference
currents (amiloride-sensitive). At least six cells under each
experimental condition were measured. B, upper
panel, current record from a whole cell clamped ASIC2-trasfected
U87-MG cell. Munc-18 was added to the pipette solution. Middle
panels, current records from a whole cell clamped U-87-MG cell
that was pretreated with amiloride (100 µM). The pipette
solution also contained Munc-18. Lower panel, Average
current voltage relations of the current activated by Munc-18 compared
with the currents recorded after the same amount of time in the
presence of amiloride.
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Induction of Amiloride-sensitive Currents in Normal
Astrocytes--
In normal astrocytes, no amiloride-sensitive inward
currents are present. We hypothesize a tonic inhibition of these
currents by two independent, convergent mechanisms: inhibition by
PKC-
(33) and inhibition by syntaxin 1A. To test this hypothesis, we
performed whole cell patch clamp experiments on primary cultured normal
human astrocytes treated with chelerythrine chloride, a generalized PKC
inhibitor, and Munc-18 (to disrupt syntaxin 1A-protein interactions)
(Fig. 12). Chelerythrine treatment
alone or inclusion of Munc-18 in the patch pipette had no influence on
macroscopic currents. However, if astrocytes were pretreated with
chelerythrine prior to entering the whole cell mode with Munc-18
included in the pipette, activated, amiloride-sensitive inward currents
resulted. These results support the hypothesis that both PKC and
syntaxin 1A inhibit ASIC currents in astrocytes.

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Fig. 12.
Whole cell patch-clamp
recordings of normal human astrocytes. Conditions were the same as
described in the legend to Fig. 1. The cells used in the end rows were
pretreated with 10 µM chelerythrine chloride for 20-30
min prior to experimentation, and chelerythrine was included in both
the bath and pipette solutions throughout the experiment. The
experiments are representative of at least three under each
condition.
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DISCUSSION |
Our previous results indicated that an inward sodium conductance
is present in high grade gliomas but not in low grade tumors or normal
astrocytes (1). Moreover, messenger RNA analysis (either by gene
microarray or RT-PCR) indicated that ASIC1 message was present in
normal and GBM tissues and that ASIC2 mRNA was only present in
normal and not in the majority of glial cells examined thus far. In the
present work, we addressed several potential molecular mechanisms that
may account for the constitutive presence of an inward Na+
conductance in high grade glioma cells that is absent in normal astrocytes and lower grade astrocytic tumors. There are three main
conclusions that can be drawn from our experiments: 1) gain-of-function mutations of ASIC1 do not account for the constitutive activation of
sodium currents seen in high grade glioma tumor cells; 2) interactions between an ASIC-based channel complex and a SNARE protein (syntaxin 1A)
result in channel inhibition; and 3) the molecular composition of
functional brain amiloride-sensitive sodium channels is complex and may
include both brain and degenerin/ENaC subunits.
Gain-of-function mutations in both ASIC1 and ASIC2 have been described.
Using RT-PCR, we sequenced the entire coding region of ASIC1 from
malignant tumor cells obtained either from freshly resected tissue or
from established cell lines. From the sequence analysis, we determined
that there were no gain-of-function mutations in positions that have
been previously described (8, 17, 34). Although we detected
polymorphisms of ASIC sequence in positions other than those previously
described for gain-of-function mutations, these ASIC mutants were not
basally activated when heterologously expressed in Xenopus
oocytes. Thus, we postulate that gain-of-function mutations of ASIC1 do
not underlie the activated current seen in high grade gliomas. However,
if the amiloride-sensitive currents are derived from a combination of
subunits of ASIC and ENaC, as we suggest, our results do not exclude
the possibility of gain-of-function mutations occurring in other
channel components. Moreover, we posit but have not yet proved that
this complex of ASIC/ENaC components is by itself constitutively activated.
Observations from several different laboratories indicate that syntaxin
1A is expressed not only in the brain but also in epithelial cells such
as the gut and airway where it can interact with ion channels such as
CFTR (25) and ENaC (31, 32). The inhibition of CFTR by syntaxin 1A
requires a C-terminal membrane anchor of this SNARE (30). In
addition, the modulation of CFTR channel function involves a direct
binding interaction between the N-terminal tail of CFTR and the
membrane-proximal helical domain of syntaxin 1A (30). There is also a
large body of evidence indicating that syntaxin 1A can regulate
neuronal calcium channels (29, 35-37). Syntaxin 1A has been shown to
bind directly to the N-type calcium channel between loops 2 and 3 (also
referred to as the synprint peptide) (38). Functionally, syntaxin
regulates calcium channel activity at the level of gating rather than
by altering membrane trafficking. Interestingly, epithelial sodium channels have also been demonstrated to interact both physically and
functionally with syntaxin 1A (31, 32). Co-expression with ENaC and
syntaxin 1A in Xenopus oocytes has a regulatory effect on
the ability of this channel to conduct sodium. The mechanism of this
inhibition is not well understood, but it is known that the
-ENaC
subunit is required (32). There is disagreement as to whether the
inhibitory effect is due to altering the number of channels at the cell
surface or to an alteration of the gating properties of the channel
(31, 32). Our data demonstrate that syntaxin 1A can also affect ASIC.
Based upon our gene profiling RT-PCR and functional data, we
hypothesized that syntaxin 1A inhibits ASIC, that the C-terminal anchor
is not required and that ASIC1, ASIC2, and
-ENaC must be present
within the channel complex for this inhibition to occur. These results
were confirmed in our single channel bilayer experiments, indicating a
direct interaction between syntaxin and the channel. Moreover, our
RT-PCR results indicate that
-ENaC is not present in all tumors
(Fig. 2). However,
-ENaC has a widespread distribution. Our
functional studies do, in fact, indicate that
-ENaC can substitute
for
-ENaC in this regard. Biochemically, we show that syntaxin 1A
can immunoprecipitate both
- and
-ENaC but not
-ENaC (Fig.
10). Our results show that ASIC1 + ASIC2 +
(or
)-ENaC exists in
normal brain tissue or low grade tumors and that ASIC2 is not present
in more than half of the higher grade glioma cells that we have
examined. In the other subset of tumors where ASIC2 message is present,
we hypothesize that ASIC2 protein is not present in the plasma
membrane. Thus, in the absence of plasma membrane ASIC2, syntaxin 1A,
which is present in all cells of astrocytic origin (Fig. 7), cannot
inhibit channels comprised primarily of ASIC1. In the absence of
inhibitory inputs, the conductance would be constitutively active
because of the heterogeneous combination of ENaC/degenerin
components and/or low prevailing [Ca2+] (27). Our
biochemical and electrophysiological experiments are consistent with
this hypothesis.
Our working paradigm for amiloride-sensitive Na+ current
regulation in normal astrocytes and glial tumors is as follows. Two sodium channels, ASIC1 and ASIC2, are co-expressed in normal cells; however, in cells derived from high grade astrocytic tumors, ASIC2 is
either not expressed at all or is retained within the cell such that it
is absent from the plasma membrane. ASIC1 is present in the plasma
membrane of both normal and tumor cells. The consequence of this
difference in expression and cellular localization between ASIC1 and
ASIC2 is that astrocytic tumor cells can be characterized by a large
inward Na+ current that is sensitive to the Na+
channel blocker, amiloride. In contrast, in normal cells, this current
is not expressed; thus, the role of ASIC2 in this context is to
suppress the otherwise rampant Na+ conductance. Moreover,
in normal cells, two independent inhibitory pathways are operative:
inhibition by syntaxin 1A and inhibition by PKC-
(33). Addition of a
broad spectrum PKC inhibitor (chelerythrine chloride) to normal
astrocytes, in combination with a disruption of syntaxin
1A/Na+ channel interaction with Munc-18, results in the
appearance of a large, amiloride-sensitive Na+ conductance.
Although our results provide a plausible explanation for the
constitutive activation of sodium conductance seen in malignant glioma
cells, there is yet another possibility to consider, namely, that ASIC2
is not expressed in normal astrocytes. Our gene profiling studies were
performed on tissue specimens obtained in surgery for intractable
epilepsy (39). We recognize that there are limitations in the use of
any source of intact human tissue. Even though we obtained tissue
samples from white matter and not cortex and the number of astrocytes
exceeds the number of neurons by at least 1 order of magnitude in these
samples, these tissue specimens, nonetheless, contain other cell types
such as neurons. Even though RT-PCR results indicate the presence of
ASIC2 in primary cultures of human astrocytes (Fig. 4B), we
have not yet measured protein. Indeed, recent work from Johnson
et al. (16) using triple-labeling immunofluorescence suggest
that ASIC2 is not expressed in normal astrocytes. But even in the
absence of ASIC2, and thus the elimination of the syntaxin 1A
inhibitory arm, ASIC1-based amiloride-sensitive currents would still be
blocked because of the presence of PKC
I and
II in normal astrocytes, PKC
I and
II are not present in GBM (40). The experiments
presented in Fig. 12 are consistent with this postulate. Our findings
of the presence of constitutively activated, amiloride-sensitive sodium
channels in glial-derived tumor cells run counter to the suggestions of
Horimoto et al. (40), who show that transferring a
gain-of-function ASIC mutation into cancer cells using
tumor-specific promoters can produce cell death and suggest that
this may be a new strategy for cancer gene therapy. Although this may
be the case for certain somatic cell cancers, it cannot be so for brain
tumors because a functionally activated sodium conductance is operative
in the glial tumors that we have examined.
Thus, our work demonstrates that constitutive amiloride-sensitive
currents are a specific feature of the more aggressive brain tumors. At
present, little is known about the function of ASIC in the brain and
less about how the composition and activity of these channels are
regulated. Our results provide new insights into the cellular
mechanisms that control ASIC function in the brain and how these
controlling mechanisms integrate channel function with the other
physiologic demands of both normal and malignantly transformed glial
cells. Moreover, this work demonstrates that amiloride-sensitive sodium
channels cannot easily be classified based on simple biophysical
parameters, such as single channel conductance and/or sensitivity to
amiloride. This class of ion channel, both in the brain and in
epithelial tissues, appear to have a variable composition, and hence
tissue-specific differences in biophysical parameters may result from
different channel compositions in different tissues. In any case, the
novel findings concerning the regulation and biophysical properties of
ASICs that have been presented in this work are important in defining
the cell biology of brain tumor cells. Understanding the molecular
targeting or regulatory sites of ASIC will undoubtedly promote the
development of new strategies to circumvent the unwanted activation of
these channels and thus have important therapeutic ramifications.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge Drs. D. P. Corey and J. García-Añoveros (Department of Neurobiology,
Harvard Medical School, and Howard Hughes Medical Institute) for the
kind gift of ASIC DNA. ENaC DNA was a kind gift of Dr. M. J. Welsh
(Departments of Internal Medicine and Physiology and Biophysics, Howard
Hughes Medical Institute).
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant CA71933 and by funds from the Brain Tumor Foundation for
Children.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. Section
1734 solely to indicate this fact.
§
Present address: Novartis Pharmaceuticals Corporation,
Gaithersburg, MD 20878.
**
Present address: University of Tennessee Health Science Center,
Department of Physiology, Memphis, TN 38163.

To whom correspondence should be addressed: Dept. of Physiology
and Biophysics, The University of Alabama at Birmingham, 1918 University Blvd., MCLM 704, Birmingham, AL 35294-0005. Tel.:
205-934-6220; Fax: 205-934-2377; E-mail:
benos@physiology.uab.edu.
Published, JBC Papers in Press, February 12, 2003, DOI 10.1074/jbc.M300991200
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ABBREVIATIONS |
The abbreviations used are:
ASIC, acid-sensing ion channel;
CFTR, cystic fibrosis transmembrane
conductance regulator;
ENaC, epithelial Na+ channel;
GBM, glioblastoma multiforme;
MES, 4-morpholineethanesulfonic acid;
MOPS, 4-morpholinepropanesulfonic acid;
GST, glutathione
S-transferase;
RT, reverse transcriptase;
SNARE, soluble
N-ethylmaleimide-sensitive factor attachment protein
receptors;
PKC, protein kinase C.
 |
REFERENCES |
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