(Received for publication, October 10, 1995; and in revised form, January 8, 1996)
From the
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 -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.
Rectification of cloned inwardly rectifying potassium channels
(IRKs) ()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
, 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
-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 -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.
Derivatized
polyamines were partially purified with Sep-Pak C 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) .
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
= -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.
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/(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 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/(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.
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).
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.
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 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.