©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation by Spermine of Native Inward Rectifier K Channels in RBL-1 Cells (*)

(Received for publication, October 10, 1995; and in revised form, January 8, 1996)

Laura Bianchi (1)(§)(¶) Mary-Louise Roy (1)(§) Maurizio Taglialatela (2) David W. Lundgren (3) Arthur M. Brown (1) Eckhard Ficker (1)(**)

From the  (1)Rammelkamp Center for Education and Research, Case Western Reserve University School of Medicine, MetroHealth Campus, Cleveland, Ohio 44109-1998, the (2)Department of Neuroscience, Section of Pharmacology, 2nd School of Medicine, University of Naples ``Federico II'', 80131 Naples, Italy, and the (3)Department of Pediatrics and Biochemistry, Case Western Reserve University School of Medicine, MetroHealth Campus, Cleveland, Ohio 44109-1998

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Polyamines have been shown to participate in the rectification of cloned inwardly rectifying potassium channels, a class of potassium channel proteins that conducts inward current more readily than outward current. Here, basophil leukemia cells were used to determine the effects of polyamines on a native, inwardly rectifying potassium current. Rat basophil leukemia cells were cultured in the presence of two different polyamine biosynthesis inhibitors, and both the electrophysiological properties and the polyamine levels were monitored. Treatment with alpha-difluoromethylornithine, a specific ornithine decarboxylase inhibitor, resulted in no significant change of electrophysiological properties. In contrast, treatment with 5`-{[(Z)-4-amino-2-butenyl]methyl-amino}-5`-deoxyadenosine (MDL73811), an inhibitor of S-adenosylmethionine decarboxylase, resulted in increased outward currents through inwardly rectifying potassium channels while intracellular putrescine was markedly increased and spermidine and spermine levels were decreased. Fluctuations of intracellular polyamine concentrations as imposed by MDL73811 were directly translated in an altered cell excitability. Based on these results we conclude that the rectification properties of native inwardly rectifying potassium channels are largely controlled by intracellular spermine.


INTRODUCTION

Rectification of cloned inwardly rectifying potassium channels (IRKs) (^1)depends on two processes: (a) a fast voltage-dependent block of the open channel pore by internal Mg ions(1, 2) , and (b) a much slower voltage-dependent block of the open channel by cytoplasmic polyamines, in particular spermidine and spermine(3, 4, 5) . The block by polyamines (PAs) is important, as it controls the shape of the current-voltage relationship in strong IRKs such as IRK1, HIR, or hIRK(6, 7, 8) , where outward currents reach a maximum with increasing depolarizations and then shut down completely. PA block creates a physiologically important region of negative slope conductance at potentials just above the potassium equilibrium potential, E(K), which first limits and then terminates the stabilizing effects of strong IRKs on the membrane potential(9) . Weakly rectifying IRKs, such as ROMK1, are more than 4 orders of magnitude less sensitive to spermidine or spermine, and their mild rectification properties are mainly controlled by Mg ions(10, 11, 12) .

PA block is not restricted to IRKs. Block at micromolar concentrations has been reported for a subfamily of glutamate receptors where the rectification properties of Ca-permeable forms of the alpha-amino-3-hydroxy-5-methyl-isoxazolepropionate receptor are controlled by intracellular spermine(13, 14, 15) . In addition, intracellular polyamines were found to bind to and modulate voltage-gated and Ca-gated K channels(16, 17) .

Putrescine, spermidine, and spermine are essential for cell growth and differentiation(18) . Membrane proteins that depend on polyamines for proper function are exposed to a wide range of intracellular polyamine concentrations, since biosynthesis is regulated by growth factors, mitogens, and hormones. Polyamine biosynthesis is usually enhanced during cell growth but may also be increased in brain after excessive electrical stimulation of neurons or after epileptic episodes(19, 20) . Pharmacological manipulations that inhibit polyamine biosynthesis result in decreased growth rates and/or cell death. The importance of polyamines in malignant cell growth has made their biosynthetic enzymes prime targets for therapeutic interventions leading to the development of potent inhibitors for key enzymes in polyamine metabolism. The most widely studied inhibitors include alpha-difluoromethylornithine (DFMO) for ornithine decarboxylase, which catalyzes the formation of putrescine from L-ornithine, and 5`-{[(Z)-4-amino-2-butenyl]methyl-amino}-5`-deoxyadenosine (MDL73811), which inhibits S-adenosylmethionine decarboxylase, the enzyme that provides aminopropyl groups for the synthesis of spermidine and spermine from putrescine(21) .

In the present study, DFMO and MDL73811 were used as pharmacological tools to manipulate the internal polyamine content of rat basophil leukemia (RBL-1) cells. RBL-1 cells were selected because their major membrane current is an inwardly rectifying K conductance (22) . This conductance has been shown recently to originate from a strong inward rectifier, rIRK1, with 94% homology to the cloned mouse IRK1 channel(6, 23) . Here, we show for the first time that the rectification properties of native IRK channels are controlled by intracellular spermine and can be changed by pharmacological manipulation of the intracellular spermine level.


EXPERIMENTAL PROCEDURES

Materials

Putrescine, spermidine, spermine, and 1,7-diaminoheptane were from Sigma. RPMI 1640 medium, penicillin-streptomycin, and fetal calf serum were from Life Technologies, Inc. DFMO and MDL73811 were gifts from Marion Merrell Dow, Inc.

Cell Culture and Treatments

RBL-1 cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum, streptomycin (10 µg/ml), and penicillin (10 units/ml) and maintained at 37 °C in 5% CO(2). RBL-1 cells were replated either at low densities (5-8 times 10^5 cells/ml of medium) on glass coverslips for electrophysiological recordings or at a 5 times higher density into 6-well culture plates for determination of intracellular polyamines. For treatment with polyamine biosynthesis inhibitors, the culture medium was replaced 24 h after plating by medium supplemented with appropriate inhibitor concentrations. DFMO and MDL73811 were used in aqueous stocks. During treatment the culture medium was replaced daily. Control cell cultures were handled the same way. Prior to electrophysiological recordings or HPLC measurements cells were allowed to recover for at least 8 h after the last medium change.

Determination of Intracellular Polyamine Levels

Cellular polyamine concentrations were determined as described previously(24) . In brief, cells were washed twice with ice cold phosphate-buffered saline, scraped from wells, and frozen at -80 °C. Thawed samples were sonicated, and proteins were precipitated with perchloric acid. After the addition of 1,7-diaminoheptane as internal standard, samples were neutralized with K(2)CO(3), and polyamines were derivatized with 5-dimethylamino-napthalene-1-sulfonyl chloride.

Derivatized polyamines were partially purified with Sep-Pak C(18) cartridges. Samples were further fractionated by HPLC, employing a Partisil-10 ODS column, utilizing one of two elution methods. The first method employed acetonitrile in water as mobile phase. The column was equilibrated with 56% acetonitrile. One minute after sample injection a linear gradient to 78% acetonitrile over 10 min was carried out. The gradient was then increased to 86% acetonitrile over 10 min, followed by 90% acetonitrile for 15 min. The second procedure, which provides enhanced resolution of putrescine (25) , utilized as mobile phases 92.5% acetonitrile, 7.5% methanol (Solvent A) and 10 mM monopotassium phosphate (pH 4.4, Solvent B). First, the column was equilibrated with 35% Solvent A and 65% Solvent B. One minute after sample injection a linear gradient to 60% Solvent A was carried out. The gradient was then increased to 90% Solvent A over 5 min and held there for an additional 20 min. Column effluent was monitored with a fluorescence detector using excitation and emission filters of 305-395 and 435-650 nm, respectively.

Authentic putrescine, spermidine, and spermine standards were carried through the entire procedure to establish column retention times and calibration curves for each polyamine. Concentrations were expressed as pmol of polyamine/µg of DNA and given as means ± S.E. DNA concentrations were measured according to the method of Burton (26) .

Electrophysiology

Membrane currents were measured in the whole cell version of the patch clamp technique (31) using an Axoclamp 1B amplifier (Axon Instruments, Foster City, CA). Pipettes had resistances of 2-5 M when filled with (in mM): 130 potassium aspartate, 10 NaCl, 4 CaCl(2), 2 MgCl(2), 10 HEPES, and 10 EGTA (pH 7.4). In some experiments spermine (0.1 or 1 mM) was added to the pipette solution. The extracellular bathing solution had the following composition (in mM): 130 NaCl, 4.4 KCl, 2 CaCl(2), 2 MgCl(2), 10 HEPES, and 5 D-glucose (pH 7.4). All recordings were done at room temperature. The seal resistance was usually higher than 10 G. The input resistance of cells included in the analysis was 2-6 G. Capacitive currents were evoked by small voltage clamp pulses of -5 mV from a holding potential of 0 mV and compensated by the analogue circuit of the amplifier. The readout of the compensation circuit was taken as an estimate of the membrane capacitance and used to normalize current amplitudes and calculate current densities. Such capacitance measurements give an estimate of cell size, since the specific membrane capacity is assumed to be rather constant at 1 µF/cm^2. Current recordings were not corrected for leak. If not otherwise stated, they were filtered at 1 kHz and sampled at 5 kHz for off-line analysis. PClamp programs were employed for data acquisition and analysis. Data were analyzed after stable whole cell recordings were obtained, generally after about 2 min. Data are given as mean values ± S.E.


RESULTS

Whole cell patch clamp recordings in RBL-1 cells reveal large inwardly rectifying K currents that are blocked by external Ba in the micromolar concentration range (Fig. 1A). With 4.4 mM potassium in the extracellular bath solution, current amplitudes at -140 mV were on average -786.5 ± 61.4 pA (n = 20). Since the IRK current is by far the dominating membrane current expressed in RBL-1 cells the small outward current component flowing through these channels is easily identified. Fig. 1B illustrates a typical steady state I-V relationship measured in RBL-1 cells. Outward currents are seen in a potential range slightly more positive than the reversal potential for potassium ions (from -70 to -30 mV; E(K) = -87 mV) with a maximum at -55 mV. At more positive potentials the outward current becomes smaller, producing a negative slope conductance and a characteristic ``hump'' in the I-V relationship. This negative slope conductance observed in the I-V relationship is characteristic for outward currents flowing through ``strong'' inward rectifier channels (Fig. 1B). Similar IRK currents have been reported in RBL cells (22, 23) .


Figure 1: Macroscopic potassium currents in RBL-1 cells. A, currents elicited by voltage steps from -140 to +80 mV in 20-mV increments. Holding potential was -60 mV. Lower panel, block induced by 50 µM Ba. B, current-voltage (I-V) relationship of steady state currents (from cell shown in A) measured at the end of the voltage command in control (closed circles) and 50 µM Ba (open squares) containing solution. Amplitudes between -70 and -15 mV are plotted in 5-mV increments. Note, cell was selected for a large outward ``hump'' (current density at -55 mV is 0.54 pA/pF) to illustrate that 50 µM Ba are sufficient to block outward as well as inward currents through IRK channels.



Polyamines Regulate Outward Currents through IRK Channels

DFMO and MDL73811 were used to manipulate intracellular polyamine concentrations in RBL-1 cells. Treatment of RBL-1 cells with 500 µM DFMO for 2 days resulted in a decrease of the internal spermidine concentration by 85%, from 765 to 114 µM (Fig. 2C; Table 2). At the same time the internal spermine content more than doubled, from 252 to 567 µM, while putrescine concentrations remained fairly constant between 5 and 10 µM. In contrast, treatment of RBL-1 cells with 50 µM MDL73811 resulted in a 50-100-fold increase of the intracellular putrescine content to about 500 µM. This concentration change was accompanied by a prominent decrease of both spermidine and spermine concentrations from 765 and 252 µM to 121 and 31 µM (n = 5), respectively ( Table 2and Fig. 2C; see also Table 1).


Figure 2: Outward currents through IRK channels are increased in cells treated with MDL73811. A, steady state I-V relationships measured in untreated RBL-1 cells (solid triangle, n = 3) and in cells treated for 2 days with either 500 µM DFMO (open triangle, n = 4) or 50 µM MDL73811 (closed circles, n = 5). I-V relations were determined in response to voltage clamp pulses from -140 mV to +80 mV from a holding potential of -60 mV. Whole cell currents were normalized with respect to cell capacitances. Data were given as mean values ± S.E. B, outward part of I-V relationships shown in (A) at higher resolution. C, intracellular polyamine content of control cells (left column, n = 12) and cells exposed for 2 days to either 500 µM DFMO (middle column, n = 4) or 50 µM MDL73811 (right column, n = 7). Upper, middle, and lower panels show putrescine (PUT), spermidine (SPD), and spermine (SPM) content, respectively. Note that the y axis is broken in plot illustrating putrescine contents. Concentrations are given as means ± S.E.







The effects on the rectification properties of IRK currents were analyzed by studying I-V relationships in DFMO- and MDL73811-treated RBL-1 cells (Fig. 2, A and B). Treatment of RBL-1 cells with 500 µM DFMO produced no obvious alterations in the I-V relationships even though the internal spermidine concentration had decreased (Fig. 2A). Current amplitudes measured at -55 mV were -0.074 ± 0.073 pA/pF in control (n = 33) and -0.032 ± 0.033 pA/pF in DFMO-treated cells (n = 16). However, when RBL-1 cells were treated with 50 µM MDL73811 to decrease both the internal spermine and spermidine concentrations much larger outward current amplitudes were observed between -70 and -15 mV (Fig. 2A). Current amplitudes at -55 mV were 1.54 ± 0.13 pA/pF (n = 26; Fig. 2B) with slightly smaller inward currents. At test potentials of -140 mV the current density was -28.52 ± 1.67 pA/pF for cells treated with 50 µM MDL73811 (n = 26). In control cells, -35.35 ± 3.05 pA/pF (n = 35) were measured (Fig. 2A). A similar reduction of inward current amplitudes could be observed for all other groups treated with either DFMO or combinations of DFMO and MDL73811 (Table 1) and might reflect a decrease in protein biosynthesis imposed by MDL73811 and DFMO on rapidly growing cells.

Passive electric properties such as input resistance or resting membrane potential were not different between control and MDL73811- or DFMO-treated cells. The resting membrane potential was -66.6 ± 0.62 mV in control cells (n = 33), -69.27 ± 0.33 mV in cells treated with 50 µM MDL73811 (n = 29) and -68.31 ± 0.52 mV in cells treated with 500 µM DFMO (n = 16). One parameter showing a statistically significant deviation, however, was cell capacitance. Treatment with 50 µM MDL73811 increased the cell capacitance from 24.01 ± 1.27 pF (n = 20) to 33.65 ± 2.73 pF (n = 20; Table 2). The reported differences in cell size do not invalidate our conclusion that outward currents through IRK channels are increased under MDL73811 treatment, since all electrophysiological data are normalized with respect to cell capacitance and therefore cell size.

To define the pharmacological conditions that could give us maximal outward currents we also examined cells treated for 48 h with 500 µM MDL73811 and cells treated with a combination of 50 µM MDL73811, 500 µM DFMO or 500 µM MDL73811, 500 µM DFMO. Data analyzed with respect to polyamine content and electrophysiological properties are summarized in Table 1. Treatment of RBL-1 cells with 50 or 500 µM MDL73811 proved to be most effective, resulting in a large increase in outward current through inward rectifier channels in response to a pronounced decrease of internal spermine concentrations (Table 1).

To estimate the turnover in the intracellular polyamine pool the time course of the developing MDL73811 effect was examined (Fig. 3A). RBL-1 cells were treated for various times with 50 µM MDL73811, and changes in outward currents were monitored at -55 mV. In 50 µM MDL73811 outward currents reached a plateau after about 3 days. These data are contrasted with outward currents in cells cultured in the presence of 500 µM DFMO, where outward currents fluctuate around base line (Fig. 3A). Although these experiments indicate that the maximal outward current was developed after about 3 days, most of the data presented in this paper were acquired from cells exposed for only 2 days for two reasons: (a) short exposure times were used to minimize the cytotoxicity reported for MDL73811, and (b) after 2 days cell densities were most suitable for electrophysiological experiments in this fast growing cell line.


Figure 3: Time- and concentration-dependent effects of MDL73811 and DFMO on outward currents through IRK channels. A, time-dependent changes of outward current amplitudes recorded at -55 mV after exposure of RBL-1 cells for various times to either 50 µM MDL73811 (solid squares) or 500 µM DFMO (solid circles). Current amplitudes were normalized with respect to cell capacitances. Each data point represents mean ± S.E. of six to eight cells. B, normalized outward current amplitudes at -55 mV plotted against MDL73811 concentrations. RBL-1 cells were exposed to MDL73811 for 2 days. Each data point represents mean ± S.E. of 4-10 cells. Solid line shows fit to data points with the following equation: y = I(max)/(1 + X/K), where X is the MDL concentration and n the Hill coefficient. 54 nM MDL73811 increased outward currents through IRK channels by 50%.



To exclude nonspecific drug effects the dose-response relationship was analyzed by treating RBL-1 cells for 48 h with different concentrations of MDL73811. Outward currents at -55 mV were increased by 50% with 54 nM MDL73811 (Fig. 3B). This is about 10 times lower than the K(i) value of about 600 nM found for MDL73811 and rat S-adenosylmethionine decarboxylase in an in vitro assay(27) . Similar experiments were done to determine concentration-dependent effects of MDL73811 on intracellular polyamines. After exposing RBL-1 cells for 48 h to different MDL73811 concentrations, cells were harvested, and the polyamine content was analyzed by means of HPLC. Mean values of intracellular polyamine levels were plotted against MDL concentrations and fitted by Hill equations. The estimated IC values were 62.0, 45.1, and 5.9 nM for putrescine, spermidine, and spermine, respectively (Fig. 4).


Figure 4: Concentration-dependent effects of MDL73811 on intracellular polyamine content. Polyamine concentrations were measured as described under ``Experimental Procedures'' and plotted against MDL73811 concentrations. Cells were exposed to MDL73811 for two days prior to experiments. Upper panel shows changes in putrescine content (PUT, n = 3); middle panel shows spermidine content (SPD, n = 4); lower panel shows spermine content (SPM, n = 4). Each data point represents means ± S.E. Solid line shows fit to data points with the following equation: y =I(max)/(1 + X/K), where X is the MDL concentration and n the Hill coefficient. Note that putrescine concentrations increase, while spermidine and spermine concentrations decrease with increasing MDL73811 concentrations.



Intracellular Application of Spermine Decreases Outward Currents through IRK Channels

If the increase in outward currents under MDL73811 treatment is mainly due to a decline in intracellular spermine, then addition of spermine to the cytoplasm of spermine deprived RBL-1 cells should restore the outward current pattern of untreated control cells. Fig. 5A shows steady state I-V relations recorded immediately after establishing the whole cell configuration in RBL-1 cells treated for 2 days with MDL73811 (con MDL) and 37 min after perfusion with a pipette solution containing 100 µM spermine. During the time course of the experiment with spermine added to the cytoplasm via the patch pipette, the outward current ``hump'' that could be blocked completely by 50 µM Ba (n = 5; inset to Fig. 5A) decreased gradually with time. Since the diffusion rate of spermine from the pipette reservoir into the cell is limiting, it is assumed that recordings made immediately after breaking into the cell (t = 0) are similar to those made without spermine in the pipette solution (as shown in Fig. 3, A and B). The steady increase in intracellular spermine concentrations under such recording conditions is reflected in gradually decreasing outward currents. Outward currents were stable when cells were perfused with standard pipette solution, whereas a time- and concentration-dependent decline in outward currents was observed by addition of spermine to MDL73811-treated cells (Fig. 5B). The observed decrease could be characterized by fitting monoexponential equations. Time constants were 34 min for perfusion of cells with 100 µM exogenous spermine (n = 3) and 98 s in experiments done with 1 mM spermine in the recording pipette (n = 10; three independent experiments). When inward currents were monitored at -140 mV during the time course of such experiments, they proved to be stable as expected for application of a strongly voltage-dependent blocker such as spermine (Fig. 5B, lower panel).


Figure 5: Intracellular perfusion of spermine reduces outward currents through IRK channels increased by treatment with MDL73811. A, I-V relationships determined in response to voltage ramps of 150 ms (from -120 to +60 mV; holding potential, -60 mV; dV = 1.2 mV/ms) in a cell treated for 2 days with 50 µM MDL73811 immediately after gaining access to the cell interior (con MDL) or 37 min after the start of intracellular perfusion in the whole cell mode with 100 µM spermine added to the internal recording solution (SPM 100 µM). Inset shows block of outward currents induced by 50 µM Ba in an MDL73811-treated cell. B, normalized outward current amplitudes at -55 mV versus time spent in the whole cell mode of the patch clamp technique. Illustrated are cells perfused with intracellular control solution (n = 2; + center symbols) and cells perfused with solutions supplemented with either 100 µM spermine (n = 3; closed symbols) or 1 mM spermine (n = 10; open symbols). Individual symbols represent individual cells. Time-dependent changes of current amplitudes in control cells were fitted by a linear equation. Amplitude changes in spermine-treated cells could be described by monoexponential functions. Time constants were 34 min and 98 s for cells treated with 100 µM or 1 mM spermine, respectively. Lower panel, normalized steady state inward current amplitudes recorded at -120 mV versus time in whole cell mode. Cells shown were internally perfused with either 100 µM (open squares/circles) or 1 mM spermine (closed squares).



Outward Currents through Inward Rectifier Channels Stabilize Membrane Potentials of RBL-1 Cells

Inward rectifier currents mediate the resting K conductance in RBL-1 cells. Increasing amounts of outward current conducted by such channels should have a highly stabilizing effect on the resting membrane potential. This was demonstrated using whole cell recordings done under current clamp conditions immediately after gaining access to RBL-1 cells treated with 50 µM MDL73811. Voltage responses were limited in amplitude to -40 mV for depolarizing current injections up to +5 pA. On average, cells were depolarized by 20.36 ± 1.28 mV (n = 3) with a +5 pA current injection (Fig. 6B). The same experiment was repeated a few minutes later after spermine included in the recording pipette had equilibrated with the cell interior. Subsequent current injections resulted in voltage responses of up to +10 mV. On average cells were now depolarized by 63.7 ± 2.45 mV (n = 3) with a +5 pA current injection, while voltage responses evoked by hyperpolarizing current injections were not significantly altered (Fig. 6D). The diffusion of spermine from the pipette into the cell interior was continuously monitored during such experiments with current ramps recorded under voltage clamp conditions (Fig. 6, A and C). Outward currents were gradually reduced by increasing internal spermine concentrations, and correspondingly, the cells became more sensitive to depolarizing stimuli as shown with current-clamp experiments.


Figure 6: Outward currents through IRK channels stabilize membrane potential in RBL-1 cells. A, I-V relationship determined under voltage clamp conditions in response to voltage ramps of 150 ms (from -120 to +60 mV; holding potential, -60 mV; dV = 1.2 mV/ms) in a cell treated for 2 days with 50 µM MDL73811. The voltage ramp shown was recorded immediately after getting access to the cell. B, membrane voltage versus current injected into cell shown in panel A under current clamp conditions (V-I relationship). V-I relationship was determined in response to current injections between -25 and +5 pA starting from resting membrane potential. C, I-V relationship determined under voltage clamp conditions in response to voltage ramps of 150 ms (from -120 to +60 mV; holding potential, -60 mV; dV = 1.2 mV/ms) for the cell shown in panel A 4 min after the start of intracellular perfusion with 1 mM spermine in the recording pipette. Note block of outward current ``hump.'' D, V-I relationship determined under current clamp conditions for cell in panel C. Note the large changes of membrane potential following on positive current injections.




DISCUSSION

This study indicates that the negative slope conductance of native IRK channels in RBL-1 cells is largely conferred by intracellular spermine. Our experiments strengthen the concept previously developed in experiments with heterologous expressed channel and receptor proteins that polyamines are widely used cytoplasmic gating molecules (3, 4, 5, 13, 14, 15, 16, 17) .

Polyamine biosynthesis blockers were used to manipulate the polyamine content of RBL-1 cells. Treatment with DFMO led to a reduction in spermidine levels while the internal spermine content actually increased. Usually, a small reduction in spermine is reported upon treatment with DFMO, since spermidine stores are no longer replenished (see (18) ). We suspect that this difference is due to the short exposure time to DFMO (2 days) and the rather low concentrations used. On the other hand, this experimental design proved that an increase in spermine levels could compensate completely for the dramatic reduction observed in spermidine levels. I-V relationships measured both for DFMO treated and control cells were identical, although the combined cellular content for spermine and spermidine was reduced from 1020 to 680 µM. A direct interference of DFMO with IRK channels is unlikely, since it has been shown that ornithine by itself has negligible effects on recombinant IRK1 channels(5) .

In contrast, MDL73811 led to a decline of both spermidine and spermine and to the accumulation of large amounts of putrescine. While the decrease in spermidine levels was of the same magnitude as that observed with DFMO, spermine levels were increased by DFMO but decreased by MDL73811. Since significant increases in outward currents were observed only following MDL73811 treatment, we can conclude that intracellular spermine is the major determinant of outward currents in RBL-1 cells. The proposed higher efficiency of spermine in blocking native IRK channels is further supported by data on cloned IRK channels. The affinity of polyamines for IRK1 channels correlates strictly with the number of charges putrescine (+2), spermidine (+3), and spermine (+4) carry(28) .

The interaction of the various polyamines and Mg at their common binding site(s) in the channel pore is not very well defined. This has to be taken into account for all experiments done with MDL73811 in which the internal putrescine concentration is increased by about 100 times. The voltage-dependent block exerted by putrescine on IRKs, however, is rather shallow(4) . A significant interaction of high internal putrescine concentrations with IRKs should be reflected in a reduction of IRK inward currents in a voltage range between -80 and -100 mV, where the block by spermine and spermidine is weak. Inward currents in MDL73811-treated cells, however, were not different from currents in DFMO-treated cells. For this reason the interaction of putrescine with the polyamine-Mg binding site must be rather weak, as predicted from experiments with cloned IRK channels where the affinity for putrescine was found to be much lower than that for the higher charged polyamines(3, 4) .

A significant problem with the use of enzyme inhibitors such as MDL73811 is the possibility that the observed changes in outward currents are due either to cytotoxic side effects (29) or to nonspecific drug interactions. A decreased cell viability should become evident in more depolarized resting membrane potentials of MDL73811-treated cells. There were no signs of cytotoxicity as measured by analysis of resting membrane potentials. In general, MDL73811 is considered a very effective and specific inhibitor of polyamine biosynthesis(21) . Three arguments are in favor of a specific interaction of MDL73811 with its enzyme target, S-adenosylmethionine decarboxylase, under our experimental conditions: (a) the MDL73811 effect developed slowly with time as expected for manipulations of metabolic turnover rates; (b) 54 nM MDL73811 were sufficient to increase outward currents by 50% (this sensitivity is even higher than the reported ``in vitro'' K(i) of MDL73811 for S-adenosylmethionine decarboxylase (600 nM)); and (c) exogenous spermine could be used to replace authentic intracellular polyamines depleted by the pharmacological regimen.

Moreover, the reported data show clearly that pharmacologically induced changes in polyamine metabolism are directly translated into physiological response schemes. In RBL-1 cells, IRKs are mainly used to clamp the membrane potential at negative values to maintain a large driving force for Ca influx, essential for stimulus-secretion coupling in nonexcitable RBL-1 cells(30) . Low concentrations of intracellular spermine allow for large outward currents, thereby stabilizing negative membrane potentials. High internal spermine concentrations, in contrast, destabilize RBL-1 cells and lower the threshold for membrane depolarizations. Any spermine concentration change is therefore directly translated into an altered cell excitability.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants NS 23877 and HL 36930 (to A. M. B.), a grant from the American Heart Association, Northeast Ohio Affiliate (to M-L. R.), and a grant from the A. Von Humboldt Foundation (to E. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
These two authors contributed equally to this study.

A Ph.D. student at the Department of Physiology, University of Florence, Italy.

**
To whom correspondence should be addressed: Rammelkamp Center for Education and Research, MetroHealth Campus, Case Western Reserve University School of Medicine, 2500 MetroHealth Drive, Cleveland, OH 44109-1998. Tel.: 216-778-8977; Fax: 216-778-8282.

(^1)
The abbreviations used are: IRK, inwardly rectifying K channel; PA, polyamine(s); DFMO, alpha-difluoromethylornithine; MDL73811, 5`-{[(Z)-4-amino-2-butenyl]methyl-amino}-5`-deoxyadenosine; RBL, rat basophil leukemia cell line; HPLC, high pressure liquid chromatography; pF, picofarads.


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