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CELLULAR AND MOLECULAR

Imatinib-Mesylate Blocks Recombinant T-Type Calcium Channels Expressed in Human Embryonic Kidney-293 Cells by a Protein Tyrosine Kinase-Independent Mechanism

Division of Pharmacology, Department of Neuroscience, Federico II, University of Naples, Naples, Italy

Received October 7, 2003; accepted December 10, 2003.

	Abstract

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The 2-phenylaminopyrimidine derivative imatinib-mesylate, a powerful protein tyrosine kinase (PTK) inhibitor that targets abl, c-kit, and the platelet-derived growth factor receptors, is rapidly gaining a relevant role in the treatment of several types of neoplasms. Because first generation PTK inhibitors affect the activity of a large number of voltage-dependent ion channels, the present study explored the possibility that imatinib-mesylate could interfere with the activity of T-type channels, a class of voltage-dependent Ca²⁺ channels that take part in the chain of events elicited by PTK activation. The effect of the drug on T-type channel activity was examined using the whole-cell patch-clamp technique with Ba²⁺ (10 mM) as the permeant ion in human embryonic kidney-293 cells, stably expressing the rat Ca_V3.3 channels. Imatinib-mesylate concentrations, ranging from 30 to 300 µM, reversibly decreased Ca_V3.3 current amplitude with an IC₅₀ value of 56.9 µM. By contrast, when imatinib-mesylate (500 µM) was intracellularly dialyzed with the pipette solution, no reduction in Ba²⁺ current density was observed. The 2-phenylaminopyrimidine derivative modified neither the voltage dependence of activation nor the steady-state inactivation of Ca_V3.3 channels. The decrease in extracellular Ba²⁺ concentration from 10 to 2 mM and the substitution of Ca²⁺ for Ba²⁺ increased the extent of 30 µM imatinib-mesylate-induced percentage of channel blockade from 25.9 ± 2.4 to 36.3 ± 0.9% in 2 mM Ba²⁺ and 44.2 ± 2.3% in 2 mM Ca²⁺. In conclusion, imatinib-mesylate blocked the cloned Ca_V3.3 channels by a PTK-independent mechanism. Specifically, the drug did not affect the activation or the inactivation of the channel but interfered with the ion permeation process.

Although the block in cell proliferation is the primary pharmacological effect of PTK inhibitors, which explains their promising use in cancer treatment, studies in vitro have shown that the first generation of these drugs induce marked changes in cell excitability and ionic homeostasis. The ability of these compounds to affect the activity of a large number of receptor-operated and voltage-dependent ion channels (Davis et al., 2001), such as the L- and N-types of voltage-dependent calcium channels (VDCCs) (Cataldi et al., 1996; Morikawa et al., 1998; Wijetunge et al., 2002), partly explains this phenomenon.

The inhibitory action of PTK inhibitors on VDCCs could be of special relevance in the case of low-voltage-activated (LVA) T-type calcium channels. In fact, these channels, which diverge from high-voltage-activated channels because of their peculiar permeation and gating properties (Cataldi et al., 2002; Perez-Reyes, 2003), are important functional partners of PTKs, for they cooperate with these proteins in a number of physiological processes. For example, T-type channels take part in the chain of events leading to cell proliferation and differentiation (Kuga et al., 1996; Richard and Nargeot, 1996) and are essential for the mitogenic response to the PDGF receptors (Wang et al., 1993). Therefore, the ability to block T-type channels could give a PTK inhibitor additional valuable properties that could optimize its pharmacological activity.

Given the relevance of imatinib-mesylate as an antineoplastic agent, the present article explored its potential effect in modifying the activity of T-type channels. In particular, by using the whole-cell patch-clamp technique on HEK-293 cells, stably expressing the recently cloned Ca_V3.3 isoform of T-type channels (Lee et al., 1999), specific experimental approaches were adopted to establish whether imatinib-mesylate acts on these channels indirectly through a PTK-dependent mechanism, or whether it directly interferes with channel gating or the permeation process. The results showed that imatinib-mesylate dose-dependently inhibited the activity of cloned Ca_V3.3 channels. However, this effect was not related to PTK inhibition but rather to the drug's interference with the ion permeation process.

	Materials and Methods

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Cell Culture. Stably transfected HEK-293 cells (courtesy of Dr. Perez-Reyes, Department of Pharmacology, University of Virginia, Charlottesville, VA), expressing the rat Ca_V3.3 subunit of T-type VDCC (Lee et al., 1999), were cultured in a humidified 5% CO₂ atmosphere using Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and nonessential amino acids; they were kept under constant selection with 1 g/l geneticin. For electrophysiological recordings, cells were plated on poly-L-lysine (30 µg/ml)-precoated glass coverslips and used 24 to 48 h after plating.

Western Blot Analysis. For Western blot analysis, confluent Ca_V3.3-expressing HEK cells grown onto 100-mm Petri dishes were collected by scraping and low-speed centrifugation and lysed by shaking at 4°C for 1 h in a lysis buffer containing, in the case of c-abl, 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 50 mM NaF, 0.5% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO₄, or in the case of PDGFr, 20 mM HEPES, pH 7.4, 50 mM NaF, 1 mM sodium azide, 1% Triton X-100, and 200 µM NaVO₄. Both in the case of c-abl and of PDGFr, proteases were blocked adding to the lysis buffer a protease inhibitor cocktail (Complete mini; Roche Diagnostics, Mannheim, Germany) constituted by aprotinin (0.1% final concentration), pepstatin (0.7 mg/ml final concentration), and leupeptin (1 µg/ml final concentration). Samples were cleared by centrifugation and supernatants were used for Western blot analysis. Protein concentration was determined using a commercially available kit (Bio-Rad protein assay; Bio-Rad, Hercules, CA), and 70 µg of the total cell lysate was loaded into a 6% polyacrylamide-0.1% SDS vertical gel and separated electrophoretically using an electrophoresis system (Mini-PROTEAN 3 electrophoresis cell; Bio-Rad). Proteins were then transferred electrophoretically from the gel to a nitrocellulose filter using a mini-trans blot cell (Bio-Rad).

After blocking the nonspecific binding sites of nitrocellulose membrane with 5% nonfat dried milk in a Tris-buffered saline solution (20 mM Tris base, 137 mM NaCl, pH 7.6 with HCl) supplemented with 0.1% Tween 20 for 2 h at room temperature (23 ± 2°C), the filters were incubated for 1 h at room temperature under constant agitation either with a monoclonal anti-c-abl antibody (1:300 final concentration) (Calbiochem, San Diego, CA) or a polyclonal anti-PDGFr- antibody (1:1000 final concentration) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Total cell lysates from SK-N-BE cells (courtesy of Dr. R. Sirabella, Department of Neuroscience, Federico II University of Naples, Naples, Italy) were used as positive controls for anti c-abl blots, whereas in the case of PDGFr- detection, total cell lysates obtained from starved IMR-92 fibroblasts after challenging with serum (courtesy of Dr. R. Ammendola, Department of Biochemistry, Federico II University of Naples) were used. The antibody-reactive bands were revealed using a commercially available chemiluminescent detection kit using horseradish peroxidase-labeled goat anti-mouse IgG as secondary antibodies (ECL Western detection kit; Amersham Biosciences Inc., Piscataway, NJ).

Electrophysiology. Experiments were performed by using the whole-cell configuration of the patch-clamp technique. The coverslips, where cells had been cultured, were placed into a laminar flow chamber (Warner Instrument, Hamden, CT) mounted on the stage of an Axiovert 25 inverted microscope (Carl Zeiss, Jena, Germany). The cells were continuously superfused by a gravity fed multilane system that allowed the rapid exchange of the perfusing solution. All the experiments were performed at room temperature (23 ± 2°C).

Ruptured patches were obtained by suction using fire-polished borosilicate electrodes having a final resistance of 3 to 5 M. The electrodes were backfilled with a CsCl-based internal solution containing 110 mM CsCl, 30 mM tetraethyl ammonium chloride, 10 mM EGTA, 2 mM MgCl₂, 10 mM HEPES, 8 mM glucose, 15 mM phosphocreatine, 5 mM ATP, and 1 mM cAMP (pH 7.4 adjusted with CsOH). Unless otherwise specified, the external solution contained 125 mM N-methyl-D-glucamine, 10 mM BaCl₂, 10 mM HEPES, and 1 mM MgCl₂ (pH 7.4 adjusted with HCl). The osmolarity of the external solution was adjusted to 300 mOsM by adding an appropriate amount of sucrose.

Test pulses were generated and the ensuing currents were collected with a 200 B patch-clamp amplifier (Axon, Union City, CA) driven by the P-Clamp 6 software running on a PC. Currents were filtered at 2 kHz with the amplifier's built-in Bessel filter, and leak currents were subtracted online with a P/4 protocol. Online corrections of membrane capacitance and series resistance were routinely performed by using the specific commands of the amplifier. Data were stored onto the hard disk of the PC. Offline analyses were then performed with the Clampfit 8.0 (Axon Instruments, Union City, CA) and SigmaPlot 5.0 (SPSS Science, Chicago, IL) software.

Cell capacitance was calculated upon the integration of current traces generated by cell membrane discharges in response to short (5-ms) square pulses of 5-mV amplitude (from –70 up to –65 mV), which were delivered immediately after rupturing the patch.

Drugs. Imatinib-mesylate was a generous gift from Dr. Buchdunger (Novartis Pharma, Basel, Switzerland). The drug was dissolved in water as a 10 mM stock solution and kept frozen at -20 C until use. Geneticin and ATP-sodium salt were obtained from Calbiochem, whereas CsOH was purchased from Aldrich Chemical Co. (Milan, Italy). Dulbecco's modified Eagle's medium, fetal calf serum, penicillin, streptomycin, and nonessential amino acids were purchased from Invitrogen (San Giuliano Milanese, Italy). All the other chemicals were of analytical grade and were purchased from Sigma (Milan, Italy).

Data Analysis. All the data have been reported as mean ± S.E.M. Statistical comparisons were performed with ANOVA followed by the Newman-Keuls post hoc test. The threshold for statistical significance was set at p < 0.05. Curve fitting was performed with the SigmaPlot 5.0 (SPSS Science) or N-fit (The University of Texas, Medical Branch at Galveston, Galveston, TX) software.

	Results

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Imatinib-Mesylate Blocks Ca_V3.3 Channels Acting from Outside the Cell. Membrane depolarization, induced by square voltage pulses, elicited large inward currents in Ca_V3.3-expressing HEK-293 cells that were bathed with a 10 mM Ba²⁺ solution. These currents displayed all the expected features that clearly differentiated Ca_V3.3 channels type from the other cloned members of the Ca_V3 family. In fact, they showed relatively slow kinetics with respect to activation and inactivation (Fig. 1, A and B). The fact that these currents were blocked by 500 µM Cd²⁺ (Fig. 1A), disappeared when Ba²⁺ ions were omitted from the extracellular solution (Fig. 1B), and were totally lacking in untransfected HEK-293 cells (Fig. 1C), indicated that they were genuine Ba²⁺ currents, flowing through the heterologously expressed T-type channels. Because we were interested in studying the consequences on Ca_V3.3 activity of PTK blockade by imatinib-mesylate, preliminary experiments were performed to assess whether Ca_V3.3-expressing HEK-293 cells do express the PTKs that are inhibited by this drug. Western blot analysis of total cell lysates showed a strong expression of the c-abl protein in this cell line (Fig. 1D), whereas, despite the strong signal observed in positive controls (serum-stimulated IMR-92 fibroblast), no detectable signal was observed when an anti-PDGFr- antibody was used (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Barium currents and c-abl expression in CaV3.3 expressing HEK-293 cells. A and B, inward currents elicited by a series of depolarizing steps in two different CaV3.3 expressing HEK-293 cells, bathed, on the left, with a 10 mM Ba2+ solution and, on the right, after a 3-min perfusion, with a 10 mM Ba2+ solution containing 500 µM Cd2+ (A) or a 120 mM N-methyl-D-glucamine Ba2+-free solution (B). Membrane potential was held at –90 mV, and 75-ms square pulses of increasing voltages (from –60 up to 30 mV in 10-mV increments) were delivered with a 10-s interpulse interval. C, reports the currents that were recorded with a similar depolarization protocol in an untransfected HEK-293 cell bathed with a 10 mM Ba2+ solution. The traces reported in A to C are representative of at least five other cells of each group recorded with the same experimental conditions. D, Western blot performed using an anti-c-abl antibody on total cell lysates from CaV3.3-expressing HEK-293 cells (left lane) and from human neuroblastoma SK-N-BE cells (right lane) is shown. Seventy micrograms of protein per lane were loaded. See Materials and Methods for further details.

Once established that Ca_V3.3-expressing HEK-293 cell are an appropriate experimental model, the hypothesis that imatinib-mesylate could affect T-type channel activity was explored monitoring the effect of increasing imatinib-mesylate concentrations (1–300 µM) on Ba²⁺ currents, evoked by square pulse depolarization (from -100 up to 0 mV). Test pulses were delivered with a 10-s interpulse interval to allow the inactivated channels a full recovery from the inactivation and, consequently, to prevent the progressive accumulation of Ca_V3.3 channels in the inactive state. Imatinib-mesylate induced a marked decrease in current amplitude that was largely reversible upon the drug washout (Fig. 2, A and B). Imatinib-mesylate-induced Ca_V3.3 blockade was clearly concentration-dependent. Indeed, when the concentration effect curve was fitted to a Hill function, an estimated IC₅₀ of 56.9 µM and a Hill coefficient of 1.27 were obtained (Fig. 2C). Moreover, to rule out the hypothesis that the elevated IC₅₀ needed for Ca_V3.3 inhibition was due to the poor penetration of the drug inside the cytoplasm, we examined whether 3 and 10 µM concentrations of imatinib-mesylate, which were closer to the IC₅₀ for PTK inhibition, affected channel activity after an overnight incubation. At both the concentrations tested, imatinib-mesylate was unable to reduce the amplitude of Ba²⁺ currents expressed as current density (pA/pF) after the normalization for cell capacitance (Fig. 2D).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Imatinib-mesylate blocks CaV3.3 channels. A, effect of 100 µM imatinib-mesylate on Ba2+ currents elicited by repeated step depolarization to 0 mV. Three superimposed current traces obtained in a representative cell taken from a group of five cells are shown. The first trace was obtained during the baseline perfusion with 10 mM Ba2+; the second at the nadir of Ba2+ current, during 100 µM imatinib-mesylate perfusion; and the third during the drug washout. The cells were held at –90 mV, and, as shown in the inset, Ba2+ currents were evoked by square pulse depolarizations (75 ms in duration) from –100 mV up to 0 mV, delivered at a 10-s interval. Cells were perfused with a 10 mM Ba2+ solution and, after 50 s of baseline recording, the perfusion was switched to a Ba2+ solution containing 100 µM imatinib-mesylate. The cells were perfused with imatinib-mesylate for 250 s. The drug was then washed out by switching back the perfusion to the 10 mM Ba2+ solution. B, time course of maximal inward Ba2+ currents in the same cell. Each plotted data point represents the maximal current value recorded in response to each of the consecutive voltage steps delivered according to the above-reported protocol. C, concentration-effect curve generated with the step depolarization protocol described above and different imatinib-mesylate concentrations (1–300 µM). Different groups of cells were exposed to the different imatinib-mesylate concentrations. Each data point represents the mean ± S.E.M. of percentage of decrease referred to the maximal inward current induced by each imatinib-mesylate concentration compared with the values recorded before drug addition. The number of cells in each group is reported in the figure. The values have been fitted using the Hill equation y = max/(1 + (x/IC50)n), where y is the inhibition of Ba2+ currents expressed as percentage of baseline current amplitude, max is the maximal inhibition, x is the imatinib-mesylate concentration in the bathing solution, and n is the Hill coefficient. D, mean ± S.E.M. of Ba2+ current densities recorded in CaV3.3-expressing cells that were exposed to an overnight incubation with 3 or 10 µM imatinib-mesylate and in their respective controls. The values were obtained by averaging the maximal values of the amplitude of inward barium currents that were evoked in each cell of each experimental group by a series of five consecutive voltage steps (from –100 to 0 mV, holding potential = -90 mV, interpulse interval = 10 s). The currents were normalized to the cell capacitance, extrapolated from the integration of the capacitative currents, which were elicited by brief subthreshold voltage steps (as detailed under Materials and Methods). Statistical significance has been assessed with the two tail Student's t test for unpaired data.

Moreover, to see whether the effect of imatinib-mesylate on Ca_V3.3 channels depended on PTK inhibition (in which case the drug should have acted intracellularly), we examined the impact on channel activity of 500 µM imatinib-mesylate concentration dissolved in the pipette solution and dialyzed into the cytoplasm. The results showed that intrapipette imatinib-mesylate neither induced a reduction in Ba²⁺ current density at the beginning of the recording (Fig. 3A), nor determined an acceleration in the rate of its spontaneous decrease over a 500-s period (Fig. 3B).

View larger version (17K): [in this window] [in a new window]   Fig. 3. Imatinib-mesylate does not block CaV3.3 channels when dialyzed into the cytoplasm through the patch pipette. A, mean ± S.E.M. of the Ba2+ current densities measured immediately after reaching the whole-cell configuration and after correcting for the membrane capacitance in a group of six cells patched with standard intracellular solution and in a group of six cells patched with a 500 µM imatinib-mesylate containing intracellular solution. B, time course of maximal current densities recorded in response to a series of square pulse depolarizations in the same two groups of cells in A. Each data point is the mean ± S.E.M. of the values recorded in the six cells of each group. As reported in the inset, CaV3.3 channels were activated by delivering a series of 75-ms steps (from –100 up to 0 mV) with an interpulse interval of 10 s. Current densities were obtained by normalizing to cell capacitance (expressed in pF and calculated as reported under Materials and Methods) the maximal Ba2+ current amplitudes obtained in response to each step depolarization.

Imatinib-Mesylate Does Not Affect Ca_V3.3 Channel Gating, but It Interferes with the Ion Permeation Process. To identify the molecular mechanism responsible for imatinib-mesylate effect on Ca_V3.3 channels, we examined whether the drug could reduce the ability of the channel to open, in response to a given depolarizing step, or increase its tendency to inactivate at a given membrane potential.

First, the effect of imatinib-mesylate on the voltage dependence of Ba²⁺ currents was examined. The delivery of a series of square depolarizing pulses of increasing amplitude elicited inward currents that appeared at membrane potential more positive than –50 mV, reached their maximum around –20 mV, and reverted at approximately +40 mV (Fig. 4A). When the same cells were exposed to 100 µM imatinib-mesylate for 180 s, the current amplitude was reduced by approximately 70%, at all voltages tested (Fig. 4A). The fact that the drug did not induce marked changes in the current to voltage (I/V) plots suggested that imatinib-mesylate-induced Ca_V3.3 channel blockade was not voltage-dependent (Fig. 4B). Given that the voltage dependence of activation curve tends to spontaneously drift leftward upon prolonged patch clamping of Ca_V3-expressing HEK-293 cells (Martin et al., 2000), the effect of imatinib-mesylate on the voltage dependence of Ca_V3.3 channel activation was determined by the following experiments. I/V plots were obtained from two different groups of cells: one exposed to vehicle and the other to 100 µM imatinib-mesylate. No difference in voltage dependence of activation was observed when the vehicle and imatinib-treated cells were compared (V_m = -25.03 ± 0.8 in control and –25.1.3 ± 1.29 in imatinib-mesylate-treated cells; k = 5.79 ± 0.6 in control and 5.19 ± 0.4 in imatinib-mesylate-treated cells) (Fig. 4C). These results suggested that imatinib-mesylate did not reduce the ability of Ca_V3.3 channels to open in response to membrane depolarization.

View larger version (27K): [in this window] [in a new window]   Fig. 4. Effect of imatinib-mesylate on the voltage dependence of Ba2+ currents in CaV3.3-expressing HEK-293 cells. A, Ba2+ currents elicited by a series of depolarizing square pulses of increasing voltage that were delivered to the same cell before and after a 180-s perfusion of 100 µM imatinib-mesylate. Membrane potential was held at –90 mV and a square pulse was delivered every 10 s. The step voltage was increased from –60 to +30 mV in consecutive 10-mV increments. The depicted cell is representative of a group of eight. B, I/V plot generated by the data collected in the same eight cells before and after the exposure to imatinib-mesylate. The data were normalized and expressed as percentage of the maximal inward current (Imax) recorded in the absence of the drug. C, voltage dependence of activation curves generated from the I/V plots. The I/V plots were obtained with a protocol similar to the one reported in A in a group of seven cells exposed to vehicle for 180 s before patching, and in another group of six cells exposed to 100 µM imatinib-mesylate for the same amount of time. The graph shows the mean ± S.E.M. of the ratios (expressed as a function of step voltages) between the conductance calculated for each step (G) and the maximal conductance attained in the whole experiment (Gmax). Both values obtained in the presence and in the absence of the drug are reported. Conductance was estimated as the ratio between the Imax, reached in response to each step, and the driving force that was calculated as the difference between the voltage of the step and the reversal potential (E - Erev) (Randall and Tsien, 1997). The values were fitted to the equation G/Gmax = 1/(1 - e(Vm - V)/k), where V is the voltage of the depolarizing pulse, Vm is the midpoint of activation, and k is the voltage step needed to induce an increase of e-times in whole cell conductance [e is the base of natural logarithms]). D, fractional blockade of Ba2+ currents induced by imatinib-mesylate expressed as a function of step voltages. For each voltage, Ba2+ currents recorded in the presence imatinib-mesylate were expressed as the percentage of the one detected before drug addition. To exclude any significant interference of channel activation, the analysis was restricted to voltages higher than –20 mV, because a constant number of channels can be assumed to be opened by the voltage step when the saturation of the voltage activation curve is reached (Woodhull, 1973).

According to the modulated receptor hypothesis of channel blockade (Hondeghem and Katzung, 1984), another mechanism that might determine a reduction in Ba²⁺ current amplitude via an interference with the gating apparatus is the preferential binding of the drug to the inactivated state of the channel and the consequent stabilization of this state.

The percentage of Ba²⁺ currents blocked by imatinib-mesylate did not increase with progressive increments in step voltages, as expected from a drug interacting with the inactive state of the channel (Fig. 4D). However, the fact that imatinib-mesylate-induced Ba²⁺ current inhibition was not voltage-dependent does not completely exclude the hypothesis that the drug could act on the inactivated state of Ca_V3.3 channels. Its binding kinetics could, in fact, be too slow to yield a significant interaction with those channels undergoing inactivation during the short pulses used to generate the I/V plots. To exclude this possibility, the effect of imatinib-mesylate on Ca_V3.3 channel activity was also studied by analyzing the inward Ba²⁺ currents evoked by step depolarization after long prepulses of increasing voltages. The steady-state inactivation curves, obtained in these conditions, did not differ in those cells exposed to vehicle or to 100 µM imatinib-mesylate (V_m = -57.9 ± 4.4 mV in control and –58.6 ± 4.2 mV in imatinib-treated cells; k = 7.65 ± 1.5 in control and 11.22 ± 1.3 in imatinib-treated cells) (Fig. 5, A and B).

View larger version (21K): [in this window] [in a new window]   Fig. 5. Lack of effect of imatinib-mesylate on the steady-state inactivation of Ba2+ currents in CaV3.3-expressing HEK-293 cells. A, steady-state inactivation curves obtained in two groups of five cells, one bathed with 10 mM Ba2+ and the other exposed to a 100 µM imatinib-containing Ba2+ solution. The current traces, obtained in a representative cell of each group, are reported in B. Steady-state inactivation curves were generated using the protocol described in the inset of B. In particular, CaV3.3 channels were inactivated by applying a series of long (5-s) prepulses of increasing amplitude (from –100 to +20 mV in 10-mV increments). The amount of ion channels still available for opening at the end of the prepulse was estimated by looking at the amplitude of the inward currents elicited by a brief depolarizing step up to 0 mV, delivered 5 ms after setting the membrane potential back to the holding potential value. Each data point is the mean ± S.E.M. of the maximal inward Ba2+ currents recorded in each cell during each step depolarization up to 0 mV. The data points were fitted to the Boltzman function IBa = (Imax/(1 + e(Vm - V)/k), where IBa is the amplitude of the inward Ba2+ current recorded after each step; Imax is the maximum value of inward Ba2+ current amplitude, reached during the entire experiments; Vm is the midpoint of inactivation; and k is the voltage needed to reduce e-times the amplitude of inward Ba2+ current amplitude (where e is the basis of natural logarithms).

Because imatinib-mesylate is ionized at physiological pH values, it could interact with fixed negative charges in Ca_V3.3 external vestibule, thus interfering with the channel permeation process. This idea can be tested, albeit indirectly, by looking at the impact of changes in extracellular Ba²⁺ concentration on the channel block induced by this 2-phenylaminopyrimidine derivative. In effect, if imatinib-mesylate had blocked the Ca_V3.3 channels by competing with Ba²⁺ for the access to the pore region, the reduction of Ba²⁺ concentration should have potentiated the drug's blocking activity. To test this hypothesis, cells were bathed in a 2 mM Ba²⁺ external solution and repeatedly stepped from –100 up to -20 mV before and after the addition of 30 µM imatinib-mesylate. The ability of 30 µM imatinib-mesylate to block Ca_V3.3 channels was significantly enhanced in a 2 mM Ba²⁺ solution. In fact, as opposed to the values recorded before the addition of the drug, Ba²⁺ current amplitude was reduced by 36.3 ± 0.9 and 25.9 ± 2.4% in 2 mM and in 10 mM Ba²⁺ solutions, respectively (p < 0.01 using analysis of variance followed by the Newman-Keuls test) (Fig. 6, A, B, and D). Because Ca²⁺ is the permeant ion in physiological conditions and a mechanism of channel blockade involving ion permeation could imply a different ability of the drug in blocking Ba²⁺ and Ca²⁺ currents, the effect of 30 µM imatinib-mesylate on the inward currents elicited by step depolarization from –100 to –20 mV with 2 mM Ca²⁺ in the bath was also assessed. In these experimental conditions, imatinib-mesylate induced a 44.2 ± 2.3% blockade of Ca²⁺ currents that was significantly different from what was observed with 2 mM Ba²⁺ (p < 0.05 using analysis of variance followed by the Newman-Keuls test) (Fig. 6, C and D).

View larger version (26K): [in this window] [in a new window]   Fig. 6. Effect on imatinib-induced CaV3.3 blockade of replacing Ca2+ for Ba2+ ions or changing Ba2+ concentration in the extracellular solution. A to C, representative current traces obtained in three different Ca 3.3-expressing HEK cells bathed with an extracellular solution containing either 10 mM Ba2+ (A), 2 mM Ba2+ (B), or 2 mM Ca2+ (C). As shown in Vthe inset, currents were elicited by a series of 75-ms step depolarizations, from -100 to -20 mV, delivered with a 10-s interpulse interval. For each cell, we reported a current trace acquired during the baseline perfusion with a drug-free extracellular solution, a current trace obtained at the nadir of 30 µM imatinib-induced current decrease, and a current trace recorded during the drug washout. The inset of each panel reports the time course of maximal inward current amplitude obtained in the respective cell. The traces reported in A to C are representative of at least five other cells for each group recorded with the same experimental conditions. D, mean ± S.E.M. of the percentage of decreases in inward current amplitude induced by 30 µM imatinib-mesylate respect to the values recorded before the addition of the drug attained in each of the three experimental groups are compared. The single asterisk indicates p < 0.05 versus 2 mM Ba2+, whereas the double asterisk indicates p < 0.01 versus 10 mM Ba2+.

	Discussion

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The main findings emerging from the present paper indicated that imatinib-mesylate blocks Ca_V3.3 T-type channels by acting in a PTK-independent mechanism. Specifically, it interfered with their permeation pathway without affecting the activation or the inactivation of Ca_V3.3 channels.

Several arguments support the conclusion that PTK inhibition is not responsible for the T-type channel blockade exerted by imatinib-mesylate. First, the IC₅₀ value for channel blockade was more than 100 times higher than the one needed for PTK inhibition (Buchdunger et al., 1996). This latter result could be explained by the inefficient intracellular diffusion of the drug owing to the short exposure of the cells during the brief patch-clamp experiments. However, the fact that micromolar concentrations of imatinib-mesylate were still ineffective when the cells were exposed to the drug for a prolonged time interval clearly excludes this possibility and strongly implies a mechanism that was independent of PTK inhibition. This idea is further supported by the evidence that imatinib-mesylate acted from outside the cell and remained ineffective when introduced into the cytoplasm through the recording pipette. As shown by the strong expression of c-abl found in these cells by Western blot analysis, the lack of efficacy of intrapipette imatinib-mesylate cannot be explained with the absence in the cytoplasm of Ca_V3.3-expressing HEK cells of the PTKs susceptible to the blocking action of this drug, and suggests that imatinib-mesylate was acting on an extracellular site. In particular, the main factor that determined a decrease in Ca_V3.3 channel activity was the drug's reversible binding to an extracellular site that caused the block of the permeation path of the channel itself. Because imatinib-mesylate is a charged organic cation, this notion is consistent with the classical evidence, which states that charged drugs and toxins may impinge on the outer vestibule of VDCC and disturb the access of Ba²⁺ ions to the pore region of the channel. The key role played by imatinib-mesylate in blocking Ca_V3.3 channel ion permeation was highlighted by the experiments performed with low Ba²⁺ concentrations. In fact, when an extracellular solution containing 2 mM Ba²⁺ was used, the extent of imatinib-induced Ca_V3.3 channel blockade was significantly higher than the one at 10 mM Ba²⁺. The channel blockade sensitivity to a decrease in the permeant ion concentration was as expected from a drug that competes with Ba²⁺ ions for the access to the pore region of the channel. A further argument supporting the idea that imatinib-mesylate exerts its Ca_V3.3 channel block activity due to an interference with the permeation process was provided by experiments performed using a 2 mM Ca²⁺-containing extracellular solution. In this experimental conditions the extent of drug-induced channel blockade was significantly higher, clearly suggesting that the nature of the ion competing with imatinib-mesylate could determine the effectiveness of the drug-induced channel blockade. Interestingly, Martin et al. (2000) obtained similar findings with the T-type blocker mibefradil that, besides interfering with other channel functions, displays similar permeation-blocking properties. Given a similar mechanism for imatinib-mesylate-induced Ca_V3.3 channel blockade, a relevant issue that emerges is that of specificity. In fact, it could be argued that, not differently from what observed for mibefradil, this drug could also interact with the pore region of other VDCC channel types. Indeed, preliminary observations from our laboratory confirm this idea because imatinib-mesylate proved to be effective also in blocking recombinant L-type channels in stably transfected Chinese hamster ovary cells (courtesy of Dr. F. Hofmann, Technische Universitat Munchen, Munich, Germany) coexpressing the rabbit Ca_V1.2, ₃ and ₂ subunits (M.C., unpublished data).

Contrary to other T-type channel blockers, i.e., mibefradil (McDonough and Bean, 1998; Gomora et al., 2000) and the neuroleptics pimozide and penfluridol (Santi et al., 2002), which do interfere with the channel inactivation process, imatinib-mesylate apparently does not. In fact, contrary to what is expected from drugs that bind to the channel in its inactive state, the extent of channel blockade did not increase despite the progressive increments in the step voltages that increased the inactivation rate of Ca_V3.3 channels. Furthermore, the hypothesis that the interaction of the drug with the inactivated state of the channel could be precluded by its slow binding kinetics can also be discarded, because no leftward shift in steady-state inactivation was observed when Ca_V3.3 channels were inactivated by long prepulses in the presence of the drug. Similarly, the activation process seems to be equally unaffected by imatinib-mesylate, for this 2-phenylaminopyrimidine derivative did not induce any changes in the channel voltage dependence of activation.

Therefore, it is worth underscoring the fact that PTK first generation inhibitors (genistein, lavendustin, and herbimycin) differ from imatinib-mesylate, for they reduce the activity of naive T-type channels in NG108-15 cells by a PTK-dependent mechanism (Morikawa et al., 1998).

Because T-type channels have a role in a number of physiological processes that are also regulated by the activity of PTKs, such as the control of cell growth (Kuga et al., 1996; Richard and Nargeot, 1996) and neuronal excitability (Huguenard, 1996; Perez-Reyes, 2003), the availability of compounds provided of T-type channel- and PTK-blocking properties could be of valuable clinical interest. For example, a pharmacological approach targeting T-type channels and PTKs simultaneously could be useful to prevent vascular remodeling as it occurs in arterial hypertension. The rationale behind this approach could be represented by the relevance of both PTKs and T-type channels in the pathogenesis of this condition. In fact, specific PTKs, as the PDGF receptors, which are activated by shear-stress (Hu et al., 1998), contribute to myointimal cell proliferation (Balasubramaniam et al., 2003) and represent the target of novel antivascular remodeling therapies (Waltenberger et al., 1999). On the other hand, T-type channels are expressed in proliferating myointimal cells (Kuga et al., 1996; Richard and Nargeot, 1996), and their pharmacological blockade with mibefradil prevents the development of neointimal hyperplasia in spontaneous hypertensive rats (Li and Schiffrin, 1996).

Similarly, the combined blockade of T-type channels and PTKs could also be useful in controlling pathological neuronal excitability, because they are both involved in this process. Specifically, T-type channels play a relevant role in regulating neuron excitability (Huguenard, 1996; Perez-Reyes, 2003) and take part in the process of epileptogenesis. The crucial involvement of T-type channels in seizures is, in fact, demonstrated by the finding that the antiseizure drug etosuximide blocks these channels (Coulter et al., 1990; Gomora et al., 2001) and by the observation that the thalamic neurons of genetic absence epilepsy rats of Strasbourg, an experimental model of absence epilepsy, show higher T-type current density than average (Tsakiridou et al., 1995). By contrast, Ca_V3.1 T-type channel knockout mice have shown to be resistant to thalamic spike-and-wave discharges, normally induced by the administration of GABA_B receptor agonists (Kim et al., 2001). The involvement of PTKs in the process of epileptogenesis is supported by the evidence that specific PTKs, such as PYK-2, are activated in response to seizures (Tian et al., 2000) and by the fact that the overexpression of selected PTK, such as Fyn (Kojima et al., 1998), raises the tendency to develop seizures in vivo, whereas the knocking out of the same kinases has opposite effects (Cain et al., 1995). Furthermore, the susceptibility of hippocampal slices in vitro to epileptic discharges, in response to electrical stimulation, can be raised by adding the PTK src into the recording pipette and can be decreased with the use of the src inhibitor PP2 (Sanna et al., 2000). Therefore, a drug that can simultaneously block T-type channels and specific PTKs could constitute a promising antiseizure agent.

Last, because T-type channels are selectively expressed in proliferating tumor cells, whereas their expression is lost after cell differentiation (Hirooka et al., 2002; Mariot et al., 2002), it has been suggested that they are also involved in tumor growth. Although it is still matter of controversy, this hypothesis suggests that T-type channel blockade could synergize with PTK inhibition and consequently determine an antineoplastic effect. Actually, drugs provided with T-type blocking properties such as mibefradil and pimozide have already shown to exert an antiproliferative effect on retinoblastoma and breast cancer cell lines in vitro (Bertolesi et al., 2002).

In conclusion, imatinib-mesylate has the ability to block Ca_V3.3 channels by a PTK-independent mechanism when used in the high micromolar range. More importantly, its T-type channel blocking ability could add to this important drug additional and useful pharmacological properties that could be exploited in the development of a new class of derivatives, which in addition to having the capability of blocking T-type channels could also retain the remarkable PTK inhibitory properties of imatinib-mesylate.

	Acknowledgements

 
We are indebted to Dr. Ed Perez-Reyes (Department of Pharmacology, University of Virginia, Charlottesville, VA) for kindly providing the stably transfected HEK-293 cells and to Dr. Elisabeth Buchdunger (Novartis Pharma, Basel, Switzerland) for the generous gift of imatinib-mesylate. Special thanks to Dr. Maurizio Taglialatela for the critical reading of the manuscript, Drs. Tommaso Russo and Nicola Zambrano for enlightening discussions on the data, and Drs. R. Ammendola and R. Sirabella for help in performing anti PDGFr- Western blot analysis. We also express appreciation to Dr. Paola Merolla and to Marcella Donato for the editorial help and to Vincenzo Grillo for technical support in performing the experiments.

	Footnotes

 
This work was supported by the following grants to L.A.: MIUR COFIN 2001, from the Ministero Italiano per l' Università e la Ricerca Scientifica; FIRB 2002 RBNE01E7YX_007, POP, and Legge 41 from Regione Campania; Programma Speciale art. 12bis comma 6, d. Igs 502/92 modificato 229/99 from Ministero della Salute and Regione Campania. M.R. is a Ph.D. student recipient of a fellowship grant from Ente Italiano Tabacchi.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.061184.

ABBREVIATIONS: PTK, protein tyrosine kinase; PDGF, platelet-derived growth factor; PDGFr, platelet-derived growth factor receptor; VDCC, voltage-dependent calcium channel; HEK, human embryonic kidney; I/V, current to voltage.

¹ Present address: Section of Pharmacology, Department of Neuroscience, University of Aneona, Italy.

Address correspondence to: Dr. Lucio Annunziato, Division of Pharmacology, Department of Neuroscience, Federico II University of Naples, Via Pansini no. 5, 80131 Naples, Italy. E-mail: lannunzi{at}unina.it

	References

Top Abstract Materials and Methods Results Discussion References

 

Arnold K (2001) After 30 years of laboratory work, a quick approval for STI571. J Natl Cancer Inst 93: 972.[Free Full Text]

Balasubramaniam V, Le Cras TD, Ivy DD, Grover TR, Kinsella JP, and Abman SH (2003) Role of platelet-derived growth factor in vascular remodeling during pulmonary hypertension in the ovine fetus. Am J Physiol 284: L826-L833.

Bertolesi GE, Shi C, Elbaum L, Jollimore C, Rozenberg G, Barnes S, and Kelly ME (2002) The Ca²⁺ channel antagonists mibefradil and pimozide inhibit cell growth via different cytotoxic mechanisms. Mol Pharmacol 62: 210-219.[Abstract/Free Full Text]

Buchdunger E, Cioffi CL, Law N, Stover D, Ohno-Jones S, Druker BJ, and Lydon NB (2000) Abl protein-tyrosine kinase inhibitor STI571 inhibits in vitro signal transduction mediated by c-kit and platelet-derived growth factor receptors. J Pharmacol Exp Ther 295: 139-145.[Abstract/Free Full Text]

Buchdunger E, Zimmermann J, Mett H, Meyer T, Muller M, Druker BJ, and Lydon NB (1996) Inhibition of the Abl protein-tyrosine kinase in vitro and in vivo by a 2-phenylaminopyrimidine derivative. Cancer Res 56: 100-104.[Abstract/Free Full Text]

Cain DP, Grant SG, Saucier D, Hargreaves EL, and Kandel ER (1995) Fyn tyrosine kinase is required for normal amygdala kindling. Epilepsy Res 22: 107-114.[CrossRef][Medline]

Capdeville R, Buchdunger E, Zimmermann J, and Matter A (2002) Glivec (STI571, imatinib), a rationally developed, targeted anticancer drug. Nat Rev Drug Discov 1: 493-502.[CrossRef][Medline]

Cataldi M, Perez-Reyes E, and Tsien RW (2002) Differences in apparent pore sizes of low and high voltage-activated Ca²⁺ channels. J Biol Chem 277: 45969-45976.[Abstract/Free Full Text]

Cataldi M, Taglialatela M, Guerriero S, Amoroso S, Lombardi G, Di Renzo G, and Annunziato L (1996) Protein-tyrosine kinases activate while protein-tyrosine phosphatases inhibit L-type calcium channel activity in pituitary GH3 cells. J Biol Chem 271: 9441-9446.[Abstract/Free Full Text]

Coulter DA, Huguenard JR, and Prince DA (1990) Differential effects of petit mal anticonvulsants and convulsants on thalamic neurones: calcium current reduction. Br J Pharmacol 100: 800-806.[Medline]

Davis M, Wu X, Nurkiewicz TR, Kawasaki J, Gui P, Hill MA, and Wilson E (2001) Regulation of ion channels by protein tyrosine phosphorylation. Am J Physiol 281: H1835-H1862.

Deininger MW and Druker BJ (2003) Specific targeted therapy of chronic myelogenous leukemia with imatinib. Pharmacol Rev 55: 401-423.[Abstract/Free Full Text]

Fabbro D, Parkinson D, and Matter A (2002) Protein tyrosine kinase inhibitors: new treatment modalities? Curr Opin Pharmacol 2: 374-381.[CrossRef][Medline]

Gomora JC, Daud AN, Weiergraber M, and Perez-Reyes E (2001) Block of cloned human T-type calcium channels by succinimide antiepileptic drugs. Mol Pharmacol 60: 1121-1132.[Abstract/Free Full Text]

Gomora JC, Xu L, Enyeart JA, and Enyeart JJ (2000) Effect of mibefradil on voltage-dependent gating and kinetics of T-type Ca(2+) channels in cortisolsecreting cells. J Pharmacol Exp Ther 292: 96-103.[Abstract/Free Full Text]

Hirooka K, Bertolesi GE, Kelly ME, Denovan-Wright EM, Sun X, Hamid J, Zamponi GW, Juhasz AE, Haynes LW, and Barnes S (2002) T-Type calcium channel alpha1G and alpha1H subunits in human retinoblastoma cells and their loss after differentiation. J Neurophysiol 88: 196-205.[Abstract/Free Full Text]

Hondeghem LM and Katzung BG (1984) Antiarrhythmic agents: the modulated receptor mechanism of action of sodium and calcium channel-blocking drugs. Annu Rev Pharmacol Toxicol 24: 387-423.[CrossRef][Medline]

Hu Y, Bock G, Wick G, and Xu Q (1998) Activation of PDGF receptor alpha in vascular smooth muscle cells by mechanical stress. FASEB J 12: 1135-1142.[Abstract/Free Full Text]

Huguenard JR (1996) Low threshold calcium current in central nervous system neurons. Annu Rev Physiol 58: 329-348.[CrossRef][Medline]

Kim D, Song I, Keum S, Lee T, Jeong MJ, Kim SS, McEnery MW, and Shin HS (2001) Lack of the burst firing of thalamocortical relay neurons and resistance to absence seizures in mice lacking alpha(1G) T-type Ca²⁺ channels. Neuron 31: 35-45.[CrossRef][Medline]

Kojima N, Ishibashi H, Obata K, and Kandel ER (1998) Higher seizure susceptibility and enhanced tyrosine phosphorylation of N-methyl-D-aspartate receptor subunit 2B in fyn transgenic mice. Learn Mem 5: 429-445.[Abstract/Free Full Text]

Kuga T, Kobayashi S, Hirakawa Y, Kanaide H, and Takeshita A (1996) Cell cycle-dependent expression of L- and T-type Ca²⁺ currents in rat aortic smooth muscle cells in primary culture. Circ Res 79: 14-19.[Abstract/Free Full Text]

Lee JH, Daud AN, Cribbs LL, Lacerda AE, Pereverzev A, Klockner U, Schneider T, and Perez-Reyes E (1999) Cloning and expression of a novel member of the low voltage-activated T-type calcium channel family. J Neurosci 19: 1912-1921.[Abstract/Free Full Text]

Li JS and Schiffrin EL (1996) Effect of calcium channel blockade or angiotensin-converting enzyme inhibition on structure of coronary, renal and other small arteries in spontaneously hypertensive rats. J Cardiovasc Pharmacol 28: 68-74.[CrossRef][Medline]

Lyseng-Williamson K and Jarvis B (2001) Imatinib. Drugs 61: 1765-1774.[CrossRef][Medline]

Mariot P, Vanoverberghe K, Lalevee N, Rossier MF, and Prevarskaya N (2002) Overexpression of an alpha 1H (Cav3.2) T-type calcium channel during neuroendocrine differentiation of human prostate cancer cells. J Biol Chem 277: 10824-10833.[Abstract/Free Full Text]

Martin RL, Lee JH, Cribbs LL, Perez-Reyes E, and Hanck DA (2000) Mibefradil block of cloned T-type calcium channels. J Pharmacol Exp Ther 295: 302-308.[Abstract/Free Full Text]

McDonough SI and Bean BP (1998) Mibefradil inhibition of T-type calcium channels in cerebellar Purkinje neurons. Mol Pharmacol 54: 1080-1087.[Abstract/Free Full Text]

Morikawa H, Fukuda K, Mima H, Shoda T, Kato S, and Mori K (1998) Tyrosine kinase inhibitors suppress N-type and T-type Ca²⁺ channel currents in NG108-15 cells. Pflueg Arch 436: 127-132.[CrossRef][Medline]

Perez-Reyes E (2003) Molecular physiology of low-voltage-activated t-type calcium channels. Physiol Rev 83: 117-161.[Abstract/Free Full Text]

Randall AD and Tsien RW (1997) Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels. Neuropharmacology 36: 879-893.[CrossRef][Medline]

Richard S and Nargeot J (1996) T-type calcium currents in vascular smooth muscle cells: a role in cellular proliferation? in Low-Voltage-Activated Channels (Tsien RW, Clozel JP, and Nargeot J eds) pp 123-132, Adis International, Chester, UK.

Sanna P, Berton F, Cammalleri M, Tallent MK, Siggins GR, Bloom FE, and Francesconi W (2000) A role for Src kinase in spontaneous epileptiform activity in the CA3 region of the hippocampus. Proc Natl Acad Sci USA 97: 8653-8657.[Abstract/Free Full Text]

Santi CM, Cayabyab F, Sutton KG, McRory JE, Mezeyova J, Hamming KS, Parker D, Stea A, and Snutch TP (2002) Differential inhibition of T-type calcium channels by neuroleptics. J Neurosci 22: 396-403.[Abstract/Free Full Text]

Tian D, Litvak V, and Lev S (2000) Cerebral ischemia and seizures induce tyrosine phosphorylation of PYK2 in neurons and microglial cells. J Neurosci 20: 6478-6487.[Abstract/Free Full Text]

Tsakiridou E, Bertollini L, de Curtis M, Avanzini G, and Pape HC (1995) Selective increase in T-type calcium conductance of reticular thalamic neurons in a rat model of absence epilepsy. J Neurosci 15: 3110-3117.[Abstract]

Waltenberger J, Uecker A, Kroll J, Frank H, Mayr U, Bjorge JD, Fujita D, Gazit A, Hombach V, Levitzki A, and Bohmer FD (1999) A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling. Circ Res 85: 12-22.[Abstract/Free Full Text]

Wang Z, Estacion M, and Mordan LJ (1993) Ca²⁺ influx via T-type channels modulates PDGF-induced replication of mouse fibroblasts. Am J Physiol 265: C1239-C1246.

Wijetunge S, Dolphin AC, and Hughes AD (2002) Tyrosine kinases act directly on the alpha1 subunit to modulate Ca(v)2.2 calcium channels. Biochem Biophys Res Commun 290: 1246-1249.[CrossRef][Medline]

Woodhull AM (1973) Ionic blockage of sodium channels in nerve. J Gen Physiol 61: 687-708.[Abstract/Free Full Text]

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