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
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ABSTRACT |
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patch clamp; amiloride; ion channels; acid-sensing ion channels
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.
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MATERIALS AND METHODS |
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Electrophysiological methods.
Micropipettes were constructed using a Narashigi pp-83 two-stage micropipette puller. The tips of these pipettes had an inside diameter of 0.30.5 µm and an outside diameter of 0.70.9 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 13 M
. 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
, 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:
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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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RESULTS |
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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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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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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 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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DISCUSSION |
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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 - and/or
-ENaC (4). Thus we hypothesize that, at a minimum, ASIC1/2 plus
(or
)-ENaC exist in the plasma membrane of normal astrocytes, whereas only ASIC1 plus
(or
)-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
, 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
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 toxins 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/P
of ASIC1 is >6, in contrast to that of the GBM pathway, namely, 0.150.36. 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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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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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