Cation selectivity and inhibition of malignant glioma Na+ channels by Psalmotoxin 1

James K. Bubien,1 Hong-Long Ji,1 G. Yancey Gillespie,2 Catherine M. Fuller,1 James M. Markert,2 Timothy B. Mapstone,3 and Dale J. Benos1

1Department of Physiology and Biophysics, and 2Department of Surgery, Division of Neurosurgery, University of Alabama at Birmingham, Birmingham, Alabama 35294; and 3Department of Neurosurgery, Emory University, Atlanta, Georgia 30322

Submitted 9 February 2004 ; accepted in final form 6 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
Psalmotoxin 1 (a component of the venom of a West Indies tarantula) is a 40-amino acid peptide that inhibits cation currents mediated by acid-sensing ion channels (ASIC). In this study we performed electrophysiological experiments to test the hypothesis that Psalmotoxin 1 (PcTX1) inhibits Na+ currents in high-grade human astrocytoma cells (glioblastoma multiforme, or GBM). In whole cell patch-clamped cultured GBM cells, the peptide toxin quickly and reversibly inhibited both inward and outward current with an IC50 of 36 ± 2 pM. The same inhibition was observed in freshly resected GBM cells. However, when the same experiment was performed on normal human astrocytes, the toxin failed to inhibit the whole cell current. We also determined a cationic selectivity sequence for inward currents in three cultured GBM cell lines (SK-MG-1, U87-MG, and U251-MG). The selectivity sequence yielded a unique biophysical fingerprint with inward K+ conductance approximately fourfold greater than that of Na+, Li+, and Ca2+. These observations suggest that PcTX1 may prove useful in determining whether GBM cells express a specific ASIC-containing ion channel type that can serve as a target for both diagnostic and therapeutic treatments of aggressive malignant gliomas.

patch clamp; amiloride; ion channels; acid-sensing ion channels


THE ION CHANNEL SUPERFAMILY ENaC/Deg (epithelial Na+ channels/degenerin) contains over 60 proteins having a similar topology: short intracellularly located NH2 and COOH termini, two transmembrane-spanning domains, and a large extracellular loop (2). All family members are cation-selective ion channels and can be inhibited by amiloride (1, 17). One branch of this superfamily comprises the brain Na+ channels (BNaC), also known as acid-sensing ion channels (ASIC) (12, 21). The six mammalian members of this family cloned so far are primarily expressed in the brain and in sensory organs. Individual members of this family coassemble to form heteromeric channels with different properties and are postulated to be involved in a variety of cellular responses including nociception and mechanosensation (5, 20). Data from our laboratory have revealed that all high-grade glioma cells, derived from either freshly resected tumors or established cell lines, express a constitutively active, amiloride-sensitive inward Na+ current displaying characteristics consistent with at least some of the properties of ASIC (3, 4, 25). In contrast, an amiloride-sensitive Na+ current cannot be detected in astrocytes obtained from normal human brain tissue or from glioma cells derived from low-grade or benign tumors.

Peptide toxins derived from the venom of poisonous animals such as snakes, spiders, coelenterates, fish, and other species have been important tools in developing strategies to inhibit ion conductance pathways (6, 9, 10, 16, 18). Escoubas et al. (7, 8) described the only compound to date that can inhibit ASIC with high affinity, namely, Psalmotoxin 1 (PcTX1), a peptide derived from the venom of a West Indies tarantula. In experiments reported in this article, we tested the hypothesis that PcTX1 can inhibit currents in high-grade glioma cells. We found that these currents are indeed inhibited by PcTX1. Thus epitopes of ASIC may serve as useful targets for therapeutic intervention as has been so successfully done for other major diseases (14, 24).

One desirable prerequisite for a glioblastoma multiforme (GBM)-specific cellular "target" is that the protein expresses epitopes on the extracellular surface of the tumor cells. Ion channels (by definition) must have a portion of the protein outside the cell surrounding the conductive pore. Also, ion channels can be identified and characterized by a variety of biophysical properties such as conductance, kinetic activity, response to agonists, inhibition by pharmacological agents, and ionic selectivity. The development of the patch-clamp technique for the investigation of ion channel biophysics and cellular regulation of ionic permeability has provided a means whereby specific ionic conductors can be identified and examined extensively in individual cells in culture or in cells isolated directly from resected malignant glioma tissue. Because ion channels present extracellular epitopes, their ionic currents can be examined with relative efficiency by using patch-clamp technology. We have found that GBM cells express a characteristic cation channel and have investigated the possibility that GBM cells may express a unique cation channel that is not expressed by normal human brain astrocytes.

Therapies designed to target malignant glioma cells must be specific for only the tumor cells. Therefore, we chose to develop multiple criteria for comparative analysis (malignant/nonmalignant) in an attempt to isolate a specific target. In the present study, we used PcTX1 current inhibition and cationic selectivity to show that a unique cation channel is expressed by GBM cells and that these channels are absent in normal human astrocytes. Thus, at least in accordance with these criteria, it appears as though a PcTX1-sensitive cation channel may provide an exclusive therapeutic target.


    MATERIALS AND METHODS
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Cell culture. Primary cultures of normal human astrocytes and freshly resected GBM astrocytes, obtained from neurosurgical operations for epilepsy and tumor resections, respectively, along with continuous tumor cell lines (SK-MG-1, U251-MG, and U87-MG), were maintained in a 37°C humidified atmosphere containing 5% CO2 with Dulbecco’s modified Eagle’s medium (DMEM). This medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. All protocols for obtaining and culturing human malignant glioma tissue samples were reviewed and approved by the University of Alabama at Birmingham’s Institutional Review Board (IRB Protocols X030403011, X011120003, and X020919005).

Electrophysiological methods. Micropipettes were constructed using a Narashigi pp-83 two-stage micropipette puller. The tips of these pipettes had an inside diameter of ~0.3–0.5 µm and an outside diameter 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+, and 4 mM ATP at a pH of 7.2, the electrical resistance of the tip was 1–3 M{Omega}. The initial bath solution was serum-free RPMI 1640 cell culture medium. These solutions approximate the usual ionic gradients across the cell membrane. The pipette was mounted in a holder connected to the head stage of an Axon 200A patch-clamp amplifier affixed to a three-dimensional micromanipulator system attached to the microscope. The pipette tip was abutted to the cell, and slight suction was applied. Seal resistance was continuously monitored (Nicolet model 300 oscilloscope) using 0.1-mV electrical pulses from an electrical pulse generator. After formation of a seal with a resistance in excess of 1 G{Omega}, 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 indicated by a 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 10 and 20 pF for both normal astrocytes and GBM astrocytes. The cells were then held at a membrane potential of –60 mV and clamped sequentially for 800 ms each to membrane potentials ranging between –160 and 40 mV in 20-mV increments, returning to the holding potential of –60 mV for 1 s between each test voltage. This procedure caused inward Na+ currents (at more hyperpolarized potentials) and outward K+ currents (at more depolarized potentials) to flow across the membrane. These currents were digitally recorded and filed in real time. The entire procedure was controlled by a DOS Pentium computer modified for analog-to-digital (A/D) signals with pCLAMP 6 software (Axon Instruments, Sunnyvale, CA). Data were analyzed off-line at a later time. A minimum of three different cells were utilized for each experimental manipulation.

Once the whole cell configuration was established, the capacitance was balanced and initial currents were measured as control. To characterize the ion selectivity of the whole cell currents and calculate the relative permeability for various cations, we subsequently changed the bath solution (by perfusing the entire chamber) to solutions A, B, C, D, and E, which contained Na+, Li+, Ca2+, K+, and N-methyl-D-glucamine (NMDG+), respectively, as the major cation. Bath solution A contained (in mM) 140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 5 dextrose. Bath solution B contained (in mM) 140 LiCl, 1 MgCl2, 10 HEPES, and 5 dextrose. Bath solution C contained (in mM) 75 CaCl2, 1 MgCl2, 10 HEPES, 5 dextrose, and 75 mannitol. Bath solution D contained (in mM) 140 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, and 5 dextrose. Bath solution E contained (in mM) 140 NMDG-Cl, 1 MgCl2, 10 HEPES, and 5 dextrose. The pH of the solutions was adjusted with NaOH, LiOH, Ca(OH)2, KOH, or Mg(OH)2, respectively, to 7.5.

The absolute permeability coefficients for monovalent and divalent cations were retrieved by fitting the whole cell current-voltage curves with the Goldman-Hodgkin-Katz current equation (13) using Origin version 7:

where R, T, and F have their usual meanings, z is the valence of the cation, I represents the amiloride-sensitive current carried by the cation X+, PX is the absolute permeability coefficient for the cation X+, and [X+]out and [K+]in represent the bath and pipette cation concentrations, respectively (13).

Preparation of normal astrocytes. Brain tissue samples (nonmalignant), obtained from patients undergoing surgery for epilepsy, were minced, washed in RPMI cell culture medium, and transferred to a 15-ml culture tube. The cells were dissociated using a solution of 1 ml of collagenase (200x) plus 4 ml of trypsin (10x) diluted in 195 ml of phosphate-buffered saline. Minced tissue was suspended in this digestive solution at 37°C and agitated using a magnetic stirring bar. The cell suspension was removed after 20 min of digestion and centrifuged to remove debris. The pelleted viable cells were then resuspended and cultured. Because the tissue samples were obtained from epileptic patient resections, there was no possibility that the astrocytes could be contaminated with GBM cells. Also, the only viable cells in culture from these resections are astrocytes, as determined in independent experiments using astrocytic or neuron-specific markers. GBM cells were obtained from high-grade freshly resected gliomas maintained in a similar manner. Also, three continuous GBM cell cultures (SK-MG-1, U251-MG, and U87-MG) were used.


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PcTX1 inhibits currents in GBM cells. The active component of the venom of a West Indies tarantula Psalmopoeus cambridgei is a 40-amino acid peptide containing 6 cysteines. Escoubas et al. (7, 8) originally showed that PcTX1 was specific for rat ASIC1a. We (4) showed previously that glioma cells express ASIC.

PcTX1 inhibits plasma membrane ionic current in human glioma cells. We tested the hypothesis directly that PcTX1 can inhibit plasma membrane ionic current in human cells. In these experiments, we used a continuous glioma cell line originally derived from a human GBM, namely, SK-MG-1 cells. The results of these initial experiments are shown in Fig. 1. In these experiments, an individual SK-MG-1 cell was whole cell patch-clamped, and transmembrane currents were recorded at 11 different clamp potentials to evaluate the current-voltage relationship. Also, we recorded currents in real time by continuously applying positive and negative pulses and returning to the holding potential between each test potential. Typical results of these voltage-clamp protocols are shown in Fig. 1. As shown at the top of Fig. 1, the holding potential was –60 mV and the cell was pulsed to –120 mV, then to +60 mV, and back to the holding potential at 1.25-s intervals. After stable currents were recorded, 1 nM PcTX1 was superfused over the cell. A very rapid inhibition was seen such that within 30 s, virtually all of the current was inhibited. The records at the bottom of Fig. 1 show typical whole cell clamp currents before (left) and after (right) application of PcTX1.



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Fig. 1. Effect of Psalmotoxin 1 (PcTX1) on whole cell currents in SK-MG-1 cells. Top: real-time inhibition of currents after addition of PcTX1. Bottom: steady-state whole cell currents over a wide voltage-clamp range before (basal) and after treatment with 1 nM PcTX1.

 
Figure 2 displays a dose-response curve for PcTX1-induced inhibition of inward Na+ currents (measured at –120 mV) in SK-MG-1 cells (log scale). From these data, an apparent equilibrium inhibition constant (Ki) of 36 ± 2 pM was obtained.



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Fig. 2. Dose-response curve showing inhibitory effects of increasing concentrations of PcTX1 on inward Na+ currents in SK-MG-1 cells. Inset: whole cell current records showing the efficacy of PcTX1 (top) and the inability of the control peptide (bottom) to inhibit the currents.

 
Because GBMs are, by nature, a heterogeneous group of tumors, we tested the effects of PcTX1 on currents obtained in a number of different tumor cells maintained in continuous culture. The findings were consistent in that PcTX1 inhibited endogenous currents in each GBM cell line (1 example is shown in the inset to Fig. 2). However, it is possible that transformed cells in culture may express ion channels that are not expressed by GBMs in situ. Therefore, we obtained freshly resected GBM tissue, isolated the tumor cells, and performed the same electrophysiological and PcTX1 analyses. Figure 3A shows that 10 nM PcTX1 can completely block inward Na+ currents in a whole cell clamped, freshly resected GBM cell with approximately the same affinity as was found in the cultured GBM cells. The control or scrambled peptide was without effect (Fig. 3B). When normal human astrocytes were whole cell patch clamped, ionic currents could also be recorded; however, 10 nM PcTX1 did not affect these whole cell currents (n = 8; not shown).



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Fig. 3. A: whole cell clamp of a cell from a freshly resected glioblastoma multiforme (GBM). PcTX1 (10 nM) inhibited the inward current (middle). The current that was inhibited by PcTX1 is shown at right. B: the same experiment was performed on a different GBM astrocyte. This cell was superfused with a control peptide composed of the same amino acids in the same proportions but in a different sequence. This control peptide had no effect on the currents (middle), and thus there was no difference in the currents (right).

 
Astrocytes derived from high-grade gliomas have a unique plasma membrane ionic selectivity. To characterize more completely the PcTX1-sensitive current, we assessed ionic selectivity in whole cell clamped astrocytes derived from three high-grade glioma cell lines (SK-MG-1, U-87-MG, and U-251-MG). All of the cells examined had substantial inward and outward currents when normal ionic gradients for Na+, K+, and Cl were used in the pipette and bath solutions. Subsequently, the bath solution was changed by replacing the normal solution with solutions that had Li+, Ca2+, or K+ substituted for Na+. The three solution changes were repeated no less than three times on each of the three cell lines examined. The findings were indistinguishable in each of the cell lines. The currents were similar when Na+, Li+, and Ca2+ were in the bath solution. However, when Na+ was substituted with K+ in the bath solution, in every case large inward currents were measured at hyperpolarizing voltage-clamp potentials. A typical experiment on a U87-MG cell is shown in Fig. 4. The summarized current magnitudes for each of the cell lines studied are shown in the current-voltage relationships depicted in Fig. 5, A–C. Also, the ability of amiloride (100 µM) to inhibit the K+-conducted inward current is shown in Fig. 5D. These observations suggest that GBM cells conduct K+ in the inward direction with a far greater efficacy (2.5 to 6.8 times depending on the cell line) than they conduct Na+. This fact is not physiologically relevant; however, this characteristic is useful in our effort to determine whether this specific conduction pathway is or is not present in the plasma membrane of normal human astrocytes. Permeability coefficients derived from the Goldman-Hodgkin-Katz current equation for each of the cations on each of the cell lines are given in Table 1.



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Fig. 4. A representative selectivity sequence performed on a single whole cell clamped GBM astrocyte showing the large inward current increase observed in every GBM cell studied when K+ was substituted for Na+ in the bath solution. The currents were similar when Na+, Li+, and Ca2+ were in the bath solution.

 


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Fig. 5. Summary data in the form of current-voltage relationships for the selectivity sequences performed on 3 different cell lines derived from gliomas (A, U87-MG; B, U251-MG; and C, SK-MG). D: inhibition of the inward current when K+ was the charge carrier, indicating that the inward K+ current was being conducted by acid-sensing ion channels (ASIC).

 

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Table 1. Permeability coefficients for each cation in the three glioma cell lines tested

 
Because the inward K+ currents in GBM cells were amiloride sensitive, we hypothesized that these K+ currents would also be inhibited by PcTX1. Therefore, GBM cells were superfused with PcTX1 in the continued presence of K+ (Fig. 6A). This toxin rapidly inhibited the inward currents carried by K+, as shown at the far right. The same experiment was also performed on normal (nonmalignant) human brain astrocytes. The findings (Fig. 6B) were that the plasma membrane conductance did not increase when K+ was substituted in the bath solution. Because no increase in current was observed, there was no current to inhibit with PcTX1. Thus the evidence suggests that the ionic conductance present in tumor cells is absent from normal brain astrocytes.



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Fig. 6. A: whole cell current records from a GBM astrocyte showing the increased inward current generated when K+ was substituted for Na+ in the bath solution (middle) and the subsequent inhibition of the current by PcTX1 while the conductive species remained K+ (right). B: whole cell current records from a nonmalignant human astrocyte showing that K+ failed to alter the plasma membrane ionic permeability (middle). The difference current (right) shows the lack of any increased permeability, without regard to whether the main external cation was Na+ or K+.

 
Normal human astrocytes do not express whole cell current similar to that found in GBM cells. We (4) previously reported that amiloride-sensitive currents could be induced in normal astrocytes by disrupting simultaneously two inhibitory mechanisms that are tonically active in normal astrocytes. Inhibition of syntaxin 1A with Munc-18, combined with inhibition of protein kinase C{beta} (PKC{beta}), induces a plasma membrane Na+ permeability of approximately the same magnitude as that found constitutively in tumor cells. Because we do not precisely know the subunit composition of these induced amiloride-sensitive channels in normal astrocytes or in GBM cells, we assessed the PcTX1 sensitivity and this ionic selectivity of the induced current in normal human astrocytes and then compared the properties with those found in GBM cells.

Normal human astrocytes were whole cell clamped using normal ionic gradients described in MATERIALS AND METHODS. Under these conditions, the cells had minimal inward currents (Fig. 7A) that could be either minor leak currents across the seal or minimal currents carried by Na+ through K+ channels, because there can be some small but finite ability of Na+ to permeate the plasma membrane through other channel types under the relatively strong voltage clamps that were used for these experiments. However, no significant currents that could be attributed to any type of amiloride-sensitive channels were present in any of the normal astrocytes that were examined for this study under basal conditions.



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Fig. 7. A: a typical current set obtained from a whole cell clamped nonmalignant human astrocyte. The cells were pretreated with LY-379196 to inhibit PKC{beta}. This record illustrates that inhibition of PKC{beta} was insufficient to activate ASIC currents. Records showing this current phenotype were obtained immediately (<5 s) after formation of the whole cell configuration. B: currents from the same cell ~2 min after the basal records were obtained. The increased currents were activated as the Munc-18 contained in the pipette solution diffused into the cell via the membrane breach that created the whole cell configuration. C: activated currents were substantially inhibited by 100 mM amiloride, consistent with the properties of ASIC. D: average current-voltage relationships obtained using nonmalignant astrocytes and the "disinhibitory" treatment combination.

 
When these normal astrocytes were treated with agents that inhibit endogenous proteins that can themselves inhibit ENaC/Deg channels (i.e., LY-379196, 100 nM) to block PKC{beta} and Munc-18 (8 pM in the pipette solution) to inhibit syntaxin 1A, currents with electrical characteristics similar to those seen in GBM cells under basal conditions were activated. Figure 7B shows the effect of these treatments on a normal human astrocyte. In the presence of both inhibitors, inward currents were activated and the current-voltage relationship was shifted to the right. These currents were subsequently inhibited with 100 µM amiloride (Fig. 7C). These experiments demonstrate that normal human astrocytes contain functional amiloride-sensitive channels and extend our previous results that showed the presence of ENaC/DEG mRNA in these cells (4). The question we addressed using PcTX1 and selectivity measurements was, Is this induced conductive pathway in normal astrocytes identical to that expressed by GBM cells?

K+ does not increase inward conductance in normal human astrocytes. We first assessed the selectivity of the basal currents of normal human astrocytes to test our hypothesis that the induced conductance in normal astrocytes was not identical to that endogenously expressed by high-grade gliomas. Figure 6 shows that when the bath solution Na+ was replaced with K+, the whole cell currents across the plasma membrane of a normal human astrocyte were not altered and no larger inward current appeared. Thus, using selectivity criteria, we were unable to demonstrate the presence of the functional ENaC/Deg-mediated currents that we observed in malignant human astrocytes.

However, further characterization was required to determine whether the induced current was mediated by the same channel species that is present in GBM cells or by a similar channel possibly composed of different ENaC/Deg orthologs. We therefore induced amiloride-sensitive currents in normal human astrocytes by treatment with Munc-18 and a specific PKC{beta} inhibitor. In contrast to GBM cells, PcTX1 had no effect (Fig. 8). Subsequently, the bath solution Na+ was changed to K+. The change in the external cation inhibited the induced currents. This finding was exactly opposite of what was observed in GBM cells. Thus, in accordance with two criteria, refractivity to PcTX1 and differing Na+/K+ ionic selectivity ratios, we conclude that the induced ENaC/Deg currents in normal astrocytes are different from those basally expressed by GBM cells. We deduce, therefore, that GBM cells express a unique ionic conductor.



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Fig. 8. Whole cell current records showing a normal human astrocyte, which has very little plasma membrane ionic conductance (A). However, a conductance could be activated in these cells by inhibition of PKC and syntaxin (B). The ASIC1-specific ligand (PcTX1) had no inhibitory effect on the activated currents (C). When Na+ was substituted for with K+ (D), the conductance was no longer present. Reintroduction of Na+ into the bath solution restored the activated whole cell conductance (E). The selectivity of the conductance and the inability of PcTX1 to inhibit it are two independent criteria that are consistent with the hypothesis that nonmalignant human astrocytes do not express ASIC1-containing ion channels.

 

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Toxins have been instrumental for understanding structure/function relationships of ion channels as well as very useful for therapeutic intervention in a number of diseases (11, 15, 19, 22, 23). Examples of such toxins include tetrodotoxin and saxitoxin (voltage-sensitive Na+ channels), charybdotoxin, iberiotoxin, and apamin (Ca2+-activated K+ channels), chlorotoxin (Cl channels), and {omega}-canotoxin and {omega}-agatoxin (voltage-dependent Ca2+ channels). Previous studies on the crude venom and purified peptide component from the tarantula P. cambridgei (the Trinidad chevron) demonstrated high specificity of PcTX1 toward rat ASIC1, a member of the non-voltage-gated ENaC/Deg ion channel superfamily. PcTX1 has limited homology with other known spider toxins, but it does share a conserved cysteine distribution with that found in other spider and cone snail peptides. In this study, we have shown that PcTX1 effectively inhibits basally active cation currents in malignant astroglioma cells while leaving ionic currents in normal (i.e., nonmalignant) human astrocytes unaffected.

A constitutive amiloride-sensitive inward Na+ current exists in high-grade glioma cells (4). These inward Na+ currents persist in primary cultures of freshly resected high-grade gliomas as well as in established cell lines derived from high-grade astrocytomas. These inward Na+ currents are not present in normal astrocytes or in low-grade astrocytomas (e.g., pilocytic astrocytomas). However, the composition of the channels responsible for this inward Na+ conductance is unknown. We hypothesize, on the basis of both functional and biological evidence, that this channel is heteromeric and is composed of ASIC1 and some ENaC components, most likely {delta}- and/or {gamma}-ENaC (4). Thus we hypothesize that, at a minimum, ASIC1/2 plus {gamma} (or {delta})-ENaC exist in the plasma membrane of normal astrocytes, whereas only ASIC1 plus {gamma} (or {delta})-ENaC is present in the plasma membrane of malignant glioma cells (4). In the absence of ASIC2 in the plasma membrane, syntaxin 1A, which is present in all cells of astrocytic origin, cannot inhibit channels composed primarily of ASIC1; hence, the channel would be active. Moreover, we have previously shown that PKC{beta}, which is present in normal cells but not in GBM cells, inhibits both ASIC1 and the currents constitutively active in GBM cells (3). The fact that PcTX1 can inhibit inward currents in GBMs supports the hypothesis that channels mediating this current are composed, at least in part, of ASIC1 and do not contain any ASIC2. Even when inward currents are activated in normal astrocytes by the combination of PKC{beta} inhibitors and Munc-18 (3, 4), PcTX1 is without effect. This observation is consistent with the hypothesis that amiloride-sensitive channels in normal astrocytes are different than those in high-grade gliomas.

PcTX1 is a peptide toxin. Because it is a rather complex peptide, some uncertainty remains as to whether this ligand will be directly useful as a therapy for the treatment of malignant gliomas. Our findings thus far have indicated that the PcTX1 binds to an epitope on the surface of malignant human astrocytes. The binding is implied because the toxin inhibits ionic conduction through a specific channel protein complex. We have not yet performed any histological studies to confirm the presence of the toxin on the cell surface. We have observed that upon washout of the toxin, the currents return; therefore, the toxin’s interaction with the channel is not permanent. This makes histological studies problematic. The reversibility of the effect is also of concern when postulating the use of the toxin as a therapy.

All amiloride-sensitive ion channels characterized to date, including cloned members of the ENaC/Deg superfamily, have a Na+ permeability equal to or greater than that of K+, never less (see Table 2). This appears not to be the case for the amiloride-inhibitable ion conductance pathway in high-grade glioma cells. Moreover, this pathway is exquisitely sensitive to inhibition by the spider toxin PcTX1, a molecule heretofore thought to be specific for rat ASIC1 (8). It is unlikely that the current pathway in GBM cells is composed exclusively of ASIC1, for several reasons. First, the GBM current is constitutively active, in contrast to ASIC1, which must be activated by either a sudden reduction in extracellular pH (8) or a reduction in intracellular Ca2+ (4, 8). Second, ASIC1 inactivates, whereas the GBM currents are always active (3). Third, the P/Pof ASIC1 is >6, in contrast to that of the GBM pathway, namely, 0.15–0.36. Thus we propose that the amiloride-sensitive current measured in GBM cells represents a unique ion channel undoubtedly composed of different ENaC/Deg subunits, one of which (ASIC1) is an essential component.


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Table 2. Summary data for amiloride-sensitive ion channels

 
Another unresolved question is the cell physiological function of the ion channel complex with which PcTX1 interacts. It is possible that the malignant cells require the channel complex for a specific function such as migration. At this time, it is equally likely that the malignant cells can survive and migrate without utilizing the PcTX1-sensitive ionic conductance and that transiently blocking this channel would have no long-lasting detrimental effect on the malignant astrocytes. However, these possibilities do not rule out the use of this toxin as a therapeutic agent. There are both advantages and disadvantages of using a complex peptide for therapy. For example, the amino acid sequence could be modified to produce a related peptide that interacts more strongly, or even irreversibly, with the ion channel complex. Another possibility is that the peptide could be bound to a cytotoxic agent and used to deliver such an agent to the malignant cell specifically. This toxin may eventually be used in surgery to help guide resection; i.e., a fluorescent molecule may be attached to identify a group of abnormal cells or show malignant cells upon neuroimaging that could help differentiate between necrosis and active tumor. One of the negative possibilities that must be investigated is the potential antigenicity of the toxin. Use of a complex peptide could induce a strong immune response that could result in inflammation or limit any repetitive use of the toxin in afflicted individuals.

One of the most important criteria for any possibility of success for an eventual treatment strategy is the requirement for specificity for malignant cells only. The ASIC-containing conductive pathway may meet these criteria. However, there are some inconsistencies that must be resolved. One inconsistency is that mRNA for ASIC1 can be identified in normal human astrocytes. For this reason we sought to increase the number of independent functional criteria that can be used to assess the question of specificity. The unique selectivity profile described in these experiments complements the PcTX1-mediated current inhibition because PcTX1 inhibits the inward K+ in GBM cells, and we could not find a K+ conductance in normal cells. Thus, using two independent criteria, we have demonstrated the specificity (for malignant astrocytes) of a set of ion channels that must express an extracellular epitope that could be a target for therapeutic intervention. Our data show that PcTX1 is a specific inhibitor for GBM cells. Thus it is possible that PcTX1 is a potential carrier for cytotoxic agents that could be used to kill the residual malignant cells at the time of surgery.

From data that were not shown, we observed that PcTX1 inhibition was readily reversible when the toxin was washed from the bath solution. This observation means that any potential treatment using this specific ligand would be transient, because in situ, the ligand would eventually unbind from the target and be washed away. We have not yet determined whether PcTX1 would be a suitable ligand. More experiments must be performed before this determination can be made. Nonetheless, because we can demonstrate efficacy by inhibition of current, PcTX1 remains a viable candidate as a carrier ligand and offers some hope that a suitable therapy can be devised that can improve the currently poor prognosis for the treatment of patients with glioblastomas multiforme.


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This research was supported by National Cancer Institute Grants CA-10195 and CA-97247 and by funds from the Brain Tumor Foundation for Children.


    ACKNOWLEDGMENTS
 
We thank Cathy Langford and Melissa McCarthy for excellent work in preparing and culturing GBM and normal astrocytes, and Eli Lilly Corp. for the kind gift of LY-379196.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. K. Bubien, Dept. of Physiology and Biophysics, Univ. of Alabama at Birmingham, 1918 Univ. Blvd., MCLM 726, Birmingham, AL 35294 (E-mail: bubien{at}uab.edu)

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.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
1. Benos DJ. Amiloride: a molecular probe of sodium transport in tissues and cells. Am J Physiol Cell Physiol 242: C131–C145, 1982.[Abstract]

2. Benos DJ and Stanton BA. Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J Physiol 520: 631–644, 1999.[Abstract/Free Full Text]

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