From the
The effect of spermine, a low molecular mass aliphatic amine
with positive charges, on the strongly inwardly rectifying muscarinic
K
Inwardly rectifying K
In the case of the cardiac I
In the present
study, we demonstrate that submicromolar concentrations of spermine and
spermidine, low molecular mass aliphatic amines with positive charges
(21, 22) , when applied to the intracellular side of
patch membrane, reversibly reduce the open probability of K
Fig. 1A shows the effect of spermine (10
µ
M) on K
The effect of spermine (100 n
M) on K
Restoration
of the relaxation required relatively high concentrations of spermine
(300 n
M to 3 µ
M) compared with the IC
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
Then, the time constant of relaxation (
On-line formulae not verified for accuracy
On-line formulae not verified for accuracy
For the data at hyperpolarized
potentials a similar estimation was difficult. No effect of spermine on
the steady-state K
The possible interaction between spermine and
Mg
Neither the unitary
current amplitude nor P
Spermine makes the P
Spermine and Mg
It is likely
that the polyamine-bound state is an independent state distinguished
from the Mg
Addendum-After submission of
this manuscript, Lopatin et al. (Lopatin, A. N., Makhina, E.
N., and Nichols, C. G. (1994) Nature
372, 366-369) and Ficker et al. (Ficker, E.,
Taglialatela, M., Wible, B. A., Henley, C. M., and Brown, A. M. (1994)
Science
266, 1068-1072) reported that
intracellular polyamines gate the classical inward rectifiers through
the aspartate in the M
We thank Dr. Ian Findlay (University of Tours, Tours,
France) for his suggestions during the work and critical reading of
this manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(K
) channel was examined in rabbit
atrial myocytes. In inside-out patch membranes, the single channel
current-voltage relationship of K
channels activated by
guanosine 5`-3- O-(thio)triphosphate became linear in the
absence of intracellular Mg
. The open probability
( P
) of the channels did not show significant
voltage dependence under these conditions. Spermine specifically
reduced P
of outwardly flowing K
channel currents without affecting the unitary current amplitude
at depolarized potentials, but had no effect on inward K
currents under hyperpolarization. This voltage dependence of
P
of K
channels in the presence of
spermine resembled that normally observed in the whole cell or open
cell-attached configurations. Spermine (300 n
M to 3
µ
M) also restored the relaxation of K
currents which had been lost in the inside-out configuration. The
effect of spermine was concentration-dependent with IC
of
10 n
M at +40 mV. The order of potency of polyamines
in reducing P
at +40 mV was spermine
spermidine > putrescine > ornithine; arginine had no significant
effect. Intracellular Mg
antagonized the effect of
spermine. Neither the single channel conductance nor P
of the ATP-sensitive K
channel, a weak inward
rectifier, was affected by spermine. Because submillimolar
concentrations of spermine and spermidine are available in the cytosol
of most cells, these substances may be the unidentified intracellular
gating factors for strong inward rectifiers such as K
and
I
channels.
channels permit the flow
of K
ions more efficiently in the inward direction
(1) . This unique property allows these channels to clamp the
resting potential of cell membrane near the K
equilibrium potential ( E
) without preventing
the generation of action potentials
(1) . Inward rectification
is believed to result from the voltage-dependent blockade of the
outward movement of K
ions by intracellular
Mg
ions
(Mg
)
(
)
and/or the voltage-dependent intrinsic gating of the
channel
(2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14) .
Inwardly rectifying K
channels are a heterogenous
group, including members with different degrees of rectification
(1) . Cardiac background inward rectifier K
(I
) channels
(15) and muscarinic
K
(K
) channels
(16) inwardly
rectify strongly and can be classified as strong inward rectifiers,
whereas the rectification of the ATP-sensitive-K
(K
) channel
(17) and the renal
ATP-regulated K
channel (ROMK1)
(18) is much
weaker (weak inward rectifiers). The distinct extent of rectification
has been attributed to the difference of the individual channels in
their sensitivity to Mg
and/or
their gating properties
(4, 7, 11, 12, 18, 19) .
channel,
Mg
instantaneously reduces
outward currents which flow at potentials positive to
E
, and the channel's intrinsic gating
further decreases the outward current in a time-dependent manner,
leading to stronger rectification
(13) .
Mg
also paradoxically permits
some outward flow of K
ions at positive membrane
potentials by hampering closure of the gate
(11, 13) .
Upon hyperpolarization to potentials more negative than
E
, the Mg
blockade is nearly
instantaneously relieved, followed by a time-dependent increase in
inward currents through the gating process
(13) . The I
channels in the open cell-attached patch in
Mg
-free solution gradually lose
the time-dependent change in the open probability
( P
); as a result, P
remains
relatively high at potentials positive to E
,
suggesting that an unidentified diffusible intracellular substance may
regulate the intrinsic gating
(11) . Similar phenomena have also
been noticed with the cardiac K
channel, another strong
inward rectifier
(20) . Thus, such a substance might universally
contribute to the gating of strong inward rectifiers.
channels at potentials positive but not negative to
E
. In contrast, the weak inward rectifier
K
channel was insensitive to these substances. Because
spermine and spermidine are known to exist in the submillimolar range
in the cytosol of various cell types
(21, 22) , these
substances may contribute to the intrinsic gating of strong inward
rectifiers.
Chemicals
GTP (sodium salt), acetylcholine
chloride, and ATP (potassium salt) were purchased from Sigma;
GTPS, Boehringer Mannheim;
L-arginine and polyamines,
Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All of the other
chemicals were of the highest purity available and obtained from
commercial sources.
Solutions
Control bathing solution contained (in
m
M): 136.5 NaCl, 5.4 KCl, 1.8 CaCl, 0.53
MgCl
, 0.33 NaH
PO
, 5.5 glucose, and
5.5 HEPES-NaOH buffer (pH 7.4). The composition of the high
K
, low Cl
solution was (in
m
M): 10 taurine, 10 oxalic acid, 70 glutamic acid, 25 KCl, 10
KH
PO
, 11 glucose, 0.5 EGTA, and 10 HEPES-KOH
(pH 7.3-7.4). The internal side of inside-out patch membranes was
perfused with the internal solution composed of (in m
M): 140
KCl, 5 EGTA-KOH, and 5 HEPES-KOH (pH 7.3) with 2 or 0.3 m
M
MgCl
: the free Mg
concentration in each
of the solutions was calculated to be 1.4 m
M or 204
µ
M, respectively
(23) . Mg
-free
internal solution was (in m
M): 140 KCl, 5 EDTA-KOH, and 5
HEPES-KOH (pH 7.3). In some experiments, this solution was supplemented
with 1 m
M ATP to suppress K
channel openings.
Polyamines and
L-arginine were dissolved in distilled water at
10 m
M, dispensed into small aliquots, and stored at -20
°C. On use, they were diluted with the Mg
-free
internal solution to the desired concentrations and applied to the
intracellular side of the inside-out patch membranes. Pipette solution
contained (in m
M): 140 KCl, 1 CaCl
, 1
MgCl
, and 5 HEPES-KOH (pH 7.4).
Preparations of Isolated Atrial Myocytes
Single
atrial myocytes were enzymatically isolated from adult female Japanese
White rabbits (Kitayama Labes Co., Ltd., Nagano, Japan) as described
previously
(24) . Using a Langendorff apparatus, the heart was
perfused in a retrograde manner through coronary arteries at 37 °C
for 10 min with 16 mg of collagenase (Yakult, Tokyo, Japan) in 100 ml
of nominally Ca-free bathing solution. The heart was
then stored for use the same day in the high K
, low
Cl
solution at 4 °C. To isolate single cardiac
myocytes, a small piece of tissue was dissected from the atrium and
gently agitated in the recording chamber filled with the control
bathing solution. Quiescent relaxed myocytes with clear striations were
used for experiments.
Current Measurement and Data Analysis
Single
channel currents were measured at room temperature (25 °C)
using the inside-out variant of the gigaohm seal patch clamp technique
(25) . The tips of the patch electrode were coated with Sylgard
and fire-polished. The tip resistance of the electrodes was 5-8
megaohms. Channel activity was measured by a patch clamp amplifier
(EPC-7, List, Darmstadt, Germany) and monitored throughout experiments
with a high gain digital storage oscilloscope (VC-6025, Hitachi, Tokyo,
Japan). A continuous record of channel currents was stored for
subsequent analysis on videocassette tapes using a PCM converter system
(VR-10B, Instrutech Corp., New York). For analysis, data were
reproduced, low pass-filtered at 1 kHz (-3 db) by an 8-pole
Bessel filter (Frequency Devices, Haverhill, MA), sampled at 5 kHz and
analyzed off line on a computer (Macintosh Quadra 700, Apple Computer
Inc., Cupertino, CA). To measure P
of the
channels, the threshold for judging the open state was set at half of
the single channel amplitude
(26) . Statistical data are
expressed as mean ± S.E.
channels in an inside-out patch
membrane of a rabbit atrial myocyte. At -60 mV, GTP
S (10
µ
M) applied to the intracellular side of the patch
membrane in the presence of 1.4 m
M free
Mg
promptly induced inward
K
currents
(27) : N
P
( N is the number of the channels in a patch;
P
is the open probability of each channel) was
increased from 0.001 to 0.099. Although this activation was
irreversible after washout of the nucleotide
(27) , only small
outward currents were observed at +40 mV because of the
Mg
blockade
(10, 12) . On removal of
Mg
, the unitary amplitude of
outward K
channel currents increased to
1.1 pA
( N
P
= 0.112). Spermine
applied to the intracellular side of the patch membrane promptly
inhibited the outward but not the inward K
channel
current under these conditions ( N
P
= 0.001 at +40 mV and 0.106 at -60 mV). A few
openings of channels were still observed in the presence of spermine.
Compared with the previous part of the current trace recorded at
+40 mV in the presence of Mg
,
flickery openings of K
channels with smaller unitary
current amplitude were hardly observed under this condition. Following
washout of spermine, the frequency of openings recovered. This recovery
was promoted by transient hyperpolarization of the patch membrane
probably because of accelerated dissociation of spermine at these
potentials (see below).
Figure 1:
Spermine concentration-dependently
inhibits outward but not inward muscarinic K currents
by decreasing the open probability without changing the unitary current
amplitude. A, effect of intracellular spermine (10
µ
M) on outward and inward K
currents in an
inside-out patch membrane of rabbit atrial myocytes. Experiments were
conducted in symmetrical 150 m
M K
solutions
with 0.5 µ
M acetylcholine in the patch pipette. The
protocol of perfusion of intracellular side of the patch membrane and
the change in holding membrane potentials are indicated above and below
the current trace, respectively. Arrowheads indicate the zero
current levels. Time and current scales are shown beneath the trace.
Larger and smaller spikes observed at +40 mV in the presence of
spermine were occasional openings of K
and K
channels, respectively. The very large spikes seen on changing
membrane potentials are the artificial capacitative currents introduced
by rapid voltage jumps. B, concentration-dependent inhibitory
effect of spermine on outward K
currents at +40 mV.
Panel a, after K
channels were irreversibly
activated by GTP
S, indicated concentrations of spermine was
applied to the intracellular side of the patch membrane at +40 mV
in the absence of intracellular Mg
. The
N
P
of the K
channels,
where N is the number of the channels in a patch, whereas
P
is the open probability of each channel, was
0.096, 0.084, 0.046, and 0.014 in the presence of 0, 10, 30, and 100
n
M spermine, respectively. An arrowhead indicates the
zero current level. The bars and numbers below the
current trace correspond to the numbers in the graph ( panel
c). Panel b, high speed current traces obtained from the
same patch as panel a under the condition indicated to the
right of each current trace. Arrowheads indicate the
zero current level. Panel c, the current amplitude histogram
obtained from the same current record as panel a. The numbers
in the graph correspond to those in panel a and indicate the
portions of the record from which each histogram was constructed.
Arrowheads indicate the first and second levels of channel
opening. Bin width is 0.02 pA, and the number of points in each bin is
expressed as a percentage of the total number of points recorded, which
was 199,876-210,031. Panel d, concentration-dependent
effect of spermine on the unitary current amplitude ( open
circles) and N
P
( closed
circles). Both data were normalized to the values obtained in the
absence of spermine. Symbols and bars indicate the
mean ± S.E.; the number of observations is four to seven for
each point. Lines were drawn by eye. Experiments were conducted at
+40 mV. Unitary current amplitude at 1 and 10 µ
M was
measured by eye, because very few openings of K
channels
were available in this concentration range.
Spermine suppressed outward Kchannel currents recorded at +40 mV in a
concentration-dependent manner (Fig. 1 B, panel a)
with the IC
of
10 n
M
(Fig. 1 B, panel d). Different from
Mg
blockade of the channel
(10, 12) ,
the effect of spermine was primarily due to a decrease in the frequency
of channel opening (Fig. 1B, panel b) and not a decrease
in the unitary current amplitude (Fig. 1 B, panel
c). Although the detailed kinetic analysis was not possible
because of the absence of one-channel patches in this series of
experiments, the traces in Fig. 1 B, panel b, suggest
that spermine provoked long closures of the channel (Fig. 1 B,
panel b).
channels was examined at different membrane potentials
(Fig. 2). In the cell-attached configuration, K
channels showed the characteristic inward-rectifying single
channel current-voltage relationship ( open squares in
Fig. 2A). The almost identical current-voltage
relationship was obtained in inside-out patches when K
channels were activated by GTP
S in the presence of 1.4
m
M free Mg
(data not
shown). It became linear on removal of
Mg
( open circles in
Fig. 2 A). Under the
Mg
-free condition,
P
showed little voltage dependence over a wide
range of potentials; it decreased by only
20% at +60 mV
( open circles in Fig. 2 B)
(20) . This
voltage dependence is much weaker than observed in the whole cell
configuration
(19, 28) or in the open cell-attached
patch perfused with Mg
-free solution
(10, 12) : under these conditions, P
steeply decreased at least severalfold as membrane was
depolarized across E
(
0 mV in this condition). When
spermine (100 n
M) was applied, the
N
P
at potentials positive to
E
became less than 30% of the maximum value
available at hyperpolarized potentials ( closed circles in
Fig. 2B), whereas the unitary current amplitude was not
altered (closed circles in Fig. 2 A).
N
P
could not be precisely measured
between -30 mV and +30 mV because of the limited
signal-to-noise ratio in this range of potentials. It is, nevertheless,
evident that spermine can make P
measured in the
inside-out configuration steeply voltage-dependent in a range of
potentials around E
. This was also the case when
the external K
concentration was decreased to 70, 35,
and 5 m
M in the presence of 150 m
M intracellular
K
(in each case, E
calculated
from the Nernst equation is -20, -37, and -87 mV,
respectively) (data not shown).
Figure 2:
Effect of spermine on the unitary current
amplitude and open probability of K channels at different
membrane potentials. A, current-voltage relationship in
cell-attached configuration ( open squares) and in the
inside-out configuration in the absence ( open circles) and
presence ( closed circles) of spermine. Experiments in the
inside-out configuration were conducted in symmetrical 150 m
M
K
solutions. The intracellular surface of patch
membranes was perfused with the Mg
-free internal
solution containing 1 m
M ATP to suppress openings of K
channels. Because ATP made spermine somewhat less potent,
spermine was used at 100 n
M. The unitary current amplitude was
determined from the current amplitude histogram, whereas that at
positive potentials in the cell-attached configuration was measured by
eye. Symbols and bars indicate the mean ± S.E.
Most of the standard error values are smaller than the graphic symbols.
The number of observations at each point is 7-10. B,
voltage-dependent change in N
P
of
K
channels in the presence and absence of 100 n
M
spermine. ATP (1 m
M) was added to the intracellular side of
patch membranes. N
P
was measured in
the absence ( open circles) and presence ( closed
circles) of spermine. N
P
is
expressed as a percentage of the value at -60 mV. Symbols and bars indicate the mean ± S.E. The number of
observations at each point is 7-10. Lines were drawn by
eye.
In the whole cell configuration, an
abrupt depolarization of the membrane from potentials negative to
Eto those positive to E
is
followed by a time-dependent decay of an outward K
channel current. On the other hand, an instantaneous jump of the
membrane potential in the opposite direction, from a positive to a
negative potential, results in a time-dependent increase in an inward
K
current
(28) . These phenomena occur because the
P
of K
channels relatively slowly
relaxes to a new steady-state level following the abrupt change in the
membrane potential. This relaxation is, however, barely observed in the
cell-free condition even in the presence of
Mg
(10, 28) .
Spermine may restore the relaxation in the inside-out configuration
because it made the P
of K
channels
voltage-dependent (Fig. 2 B). In the absence of spermine
(control), the time-dependent change in N
P
during the command voltage steps to +40 mV was negligible
(Fig. 3 A). As expected, spermine (300 n
M to 3
µ
M) induced the time-dependent decay of
N
P
, resulting in a clear
time-dependent decrease in ensemble average outward currents. As
progressively higher concentrations of spermine was applied, the decay
became faster; the decay in the ensemble average current at +40 mV
could be fitted by a single exponential with the time constant of 9.9,
1.3, 0.38, and 0.18 s in the absence and presence of 300 n
M, 1
µ
M, and 3 µ
M spermine, respectively. On
repolarization from +40 to -60 mV, however, a time-dependent
increase in inward currents was less evident. This was probably due to
a fast kinetics of the channels at this potential from the following
two reasons: 1) there was no detectable change in a mean current level
during the 4-s interval between the command steps, and 2) spermine (up
to 10 µ
M) did not show any significant inhibitory effect
on inward currents when membrane potential was continuously held at
-60 mV. Spermine did not cause any significant instantaneous
inhibition of the outward currents when the membrane was depolarized
from -60 to +40 mV: the ratio of the average instantaneous
outward to the steady-state inward currents did not alter throughout
the experiment. The gradual decrease in the
N
P
of inward currents, which is more
clearly seen in the ensemble average currents, therefore indicates a
certain rundown of the channel during this experiment.
value (
10 n
M) (Fig. 1 B, panel d).
Thus, it was examined whether the kinetics observed here is really
comparable with the data obtained from the steady-state analysis.
Because there was virtually no relaxation observed in the absence of
spermine, and because the relaxation induced by spermine could be
fitted by a single exponential, the interaction between spermine and a
K
channel was assumed to occur as follows,
) is as follows,
Figure 3:
Spermine
evokes the relaxation of K channels. A, the
time-dependent change in K
currents in the presence or
absence of 300 n
M, 1 µ
M, and 3 µ
M
spermine. K
channels in an inside-out patch membrane were
preactivated with 10 µ
M GTP
S and then Mg
was removed from the intracellular side of the patch membrane. As
shown at the top, 1-s duration command steps to +40 mV
were applied every 5 s from a -60 mV holding potential. Reading
from left to right, progressively higher
concentrations of spermine (300 n
M to 3 µ
M) was
applied to the intracellular side of the patch membrane. The membrane
was depolarized
30-40 times in each condition. The first and
second rows show representative current traces. In each of the traces,
the base-line current was subtracted with a computer. Arrow heads
indicate the zero current level. Time and current scales are shown
right beneath the second row. The third row shows the ensemble average
current obtained from 20 successive pulses under each condition. The
zero current level is indicated by a straight line. Each of the
ensemble average current traces could be fitted by a single exponential
both at +40 and -60 mV. At -60 mV, the time constant
obtained with the best fit was
0 s in the control; 0.0015 s in the
presence of 300 n
M spermine; 0.0034 s, 1 µ
M;
0.0032 s, 3 µ
M. The values obtained at +40 mV are
given in the text. B, the plot of the reciprocal of time
constant measured at +40 mV against the concentration of spermine.
Except for a single data point at 3 µ
M, data were well
fitted by a straight line:
= 2.5
10
[ S] + 0.065, where
is the time constant, and [ S] is the concentration
of spermine.
The equilibrium dissociation constant ( M) of
the spermine-channel complex ( K) is as
follows.
currents at -60 mV,
nevertheless, indicates that [ S]
is much
smaller than
at this potential. Thus, if approximating
is
1/
, the fitting data indicate that
is in the order of
10
s
, whereas
[ S]
is much smaller than this (see
Fig. 3
legend). Therefore, the rate constants of spermine binding
are strongly voltage-dependent. This could be the mechanism underlying
the observed steep voltage dependence of the affinity of spermine and
of the steady-state P
of the channel in the
presence of spermine (Figs. 1 B, panel d, and 2 B).
on K
channels
was next examined (Fig. 4). As in the upper trace and
the inset in Fig. 4 A, 204 µ
M
Mg
reduced the unitary current
amplitude to approximately a half of the control. Under this condition,
spermine (100 n
M to 10 µ
M)
concentration-dependently reduced the P
of
K
channels. Compared with the data from the same patch in
the absence of Mg
( lower
trace), the effect of spermine, however, appeared to be somewhat
attenuated. In the presence and absence of 204 µ
M
Mg
, 100 n
M spermine
reduced the N
P
to 63.1 ± 7.4
and 21.7 ± 5.9% of the control; 1 µ
M, to 26.9
± 11.1 and 3.3 ± 0.4%; 10 µ
M, to 10.6
± 4.5 and 0.3 ± 0.1%, respectively ( n =
3). In Fig. 4 B, Mg
was
applied to the channels pretreated with spermine (1 µ
M).
In the presence of spermine, Mg
reduced the size of unitary current but rather increased the
frequency of the opening ( inset B versus C). The frequency was
further increased after removal of spermine ( inset D). In this
particular patch, N
P
was 0.287 in
the control, 0.012 in the presence of spermine alone, 0.048 in the
presence of both spermine and
Mg
, and 0.110 in the presence of
Mg
alone. The effect of spermine
was also examined when higher concentration (1.4 m
M) of
Mg
was present
(Fig. 4 C). Although not completely, spermine (10
µ
M) suppressed flickery openings of K
channels in the presence of 1.4 m
M
Mg
, indicating that spermine
decreases the frequency of openings of the channels, even in the
presence of physiological concentration of
Mg
.
Figure 4:
Interaction between spermine and
Mg on K
channels. In each of the
traces, K
channels in the inside-out patch membrane were
first irreversibly activated by 10 µ
M GTP
S and then
allowed to permit an outward current by the removal of intracellular
Mg
. A, as indicated in the perfusion
protocol shown above each trace, 100 n
M, 1 µ
M,
and 10 µ
M spermine was applied to the intracellular side
of the patch membrane in the presence ( upper trace) or the
absence ( lower trace) of 204 µ
M of free
intracellular Mg
. Both of the traces were obtained
from the same patch. The change in the holding potentials is indicated
beneath the traces. Arrowheads indicate the zero current
levels. Insets, high speed current traces obtained from the
parts of the original trace designated by letters under the
traces. B, the interaction between 1 µ
M spermine
and 204 µ
M Mg
. Inset, high
speed current traces obtained from the parts of the records indicated
by letters under the original trace. C, effect of 10
µ
M spermine in the presence of 1.4 m
M of free
intracellular Mg
. The time and current scales are
indicated beneath the traces.
Similar to spermine,
spermidine suppressed the outward but not inward Kchannel currents by decreasing N
P
but not the unitary current amplitude at depolarized potentials
(Fig. 5 A): N
P
in the
presence of 10 µ
M spermidine at +40 mV was 14.3
± 6.8% of the control ( n = 3). For other
polyamines, 10 µ
M putrescine and ornithine decreased
N
P
at +40 mV only to 75.7
± 10.3% and 79.8 ± 7.8% of the control, respectively
(data not shown, n = 3 for each case).
L-arginine (10 µ
M), the parent substance of
polyamines, did not significantly affect K
channels (Fig.
5 B); N
P
in the presence of
10 µ
M
L-arginine was 102.3 ± 5.6% of the
control at +40 mV ( n = 3).
of K
channels, a weak inward rectifier, was affected by spermine
(Fig. 5 C); the N
P
in
the presence of 10 µ
M spermine was 98.6 ± 5.7% of
the control at +40 mV ( n = 3).
Figure 5:
Effect of spermidine and
L-arginine on K channels and effect of spermine
on K
channels. With a protocol similar to that in Fig.
1 A, the effect of 10 µ
M spermidine ( A)
and
L-arginine ( B) on K
channels and of
spermine (10 µ
M) on K
channels ( C)
was examined. The perfusion protocol and the holding membrane
potentials are indicated above and below the current traces,
respectively. The time and current scales shown below the trace
( C) are also applicable to the other recordings. In A and B, larger spikes (
3 pA) observed in the presence
of Mg
, spermine, or spermidine are K
channel openings, whereas the smaller spikes (
1.2 pA)
correspond to occasional openings of K
channels. The very
large spikes seen on changing membrane potentials are the artificial
capacitative currents introduced by rapid voltage
jumps.
of K
channels steeply voltage-dependent by reducing the frequency of
openings selectively at depolarized potentials (Figs. 1 and 2). Another
physiologically existing polyamine spermidine had a similar effect
(Fig. 5). It has been reported that the cytosolic concentration
of the polyamines in the free form is tens of micromolar
(29) ,
much higher than their effective concentration range against K
channels (Fig. 1 B, panel d). Thus, K
channels may be gated by polyamines in the physiological
condition even in the presence of
Mg
. It was previously reported
that the natural gating substance was quite slowly washed out
(11) compared with the relatively rapid washout of the actions
of spermine and spermidine observed in this study (Figs. 1 A,
4 A, and 5 A). The former study was, however, done in
the open cell-attached patches where the washout of intracellular
polyamines would be strongly impeded by less efficient internal
perfusion and the binding of polyamines to intracellular components
(21, 22, 29) . Thus, the apparent difference in
the washout rate between the polyamines and the intrinsic gating
substance does not necessarily indicate the inconsistency between the
actions of these substances. The function of polyamines as the gating
substance is further supported by the fact that the K
channel lacking voltage-dependent gating
(17) did not
respond to spermine (Fig. 5 C). The restoration of the
relaxation by spermine in a cell-free patch is additional evidence for
this notion (Fig. 3 A). The rate constant of the decay of
outward currents at +40 mV was 180 ms in the presence of 3
µ
M spermine. This value is still greater than observed at
this potential in a physiological condition (
50 ms)
(28) .
The rate may be further accelerated and approach the physiological
value when spermine concentration is raised to the physiological range.
That the effective concentration of polyamines is much lower than the
physiological concentration may be the mechanism responsible for the
rapid inhibition of outward K
current through
K
channels at the onset of the action potential.
suppress the
outward K
currents in a different way (Figs. 1, 2, and
4). Either of these substances can act in the presence of the other
(Fig. 4). Mg
, however,
apparently antagonized the effect of spermine (Fig. 4). Previous
kinetic analysis of cardiac I
channels also indicates that
the closed state of the intrinsic gating and the state of the
Mg
blockade are mutually exclusive
(13) .
Thus, there may be some relationship between the sites of action of
these substances. Polyamines are known to interact with
Mg
-binding sites of various enzymes as well as ion
channels through their positively charged residues
(22, 30) . Recent findings show that a single amino acid
residue in the putative second transmembrane segment (M2) of inward
rectifiers is responsible for both the gating and the Mg
blockade
(31, 32, 33, 34) . A
negatively charged aspartate at a position 172 of murine inward
rectifier (IRK1) causes strong inward rectification, whereas a polar,
but uncharged, asparagine at position 171 of ROMK1 leads to weak
rectification. Interestingly, another strong inward rectifier, the
K
channel (GIRK1), also has aspartate at position 173
(35) ; cardiac K
channels, a weak inward
rectifier, have asparagine at the equivalent position
(36) . The
difference in the sensitivity to spermine between K
and
K
channels (Figs. 1 and 5 C) suggests that
polyamines gate K
channels probably through this site as
a unidirectional voltage-dependent channel blocker.
-bound one, because the kinetics of the
action of spermine are substantially slower than those of
Mg
(10, 12) . Our analysis of the
kinetics of relaxation is, however, clearly oversimplified. The fitting
of the relaxation with a single exponential may not necessarily
indicate a single site of action. Further investigations are necessary
with mutated inward rectifiers and on the mechanism by which polyamines
confer the different kinetics of inward rectification to distinct types
of inward rectifiers.
region. Their data are basically
similar to ours and support our inference concerning the site of action
of polyamines.
, intracellular Mg
;
K
channel, the muscarinic K
channel;
I
channel, the background inward-rectifying K
channel; K
channel, the ATP-sensitive K
channel; GTP
S, guanosine 5`-3- O-(thio)triphosphate.
Channels in Mammalian Heart, pp. 403-428, University of Texas Press, Austin, TX
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.