©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Spermine Gates Inward-rectifying Muscarinic but Not ATP-sensitive KChannels in Rabbit Atrial Myocytes
INTRACELLULAR SUBSTANCE-MEDIATED MECHANISM OF INWARD RECTIFICATION (*)

Mitsuhiko Yamada , Yoshihisa Kurachi (§)

From the (1) Department of Pharmacology II, Faculty of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The effect of spermine, a low molecular mass aliphatic amine with positive charges, on the strongly inwardly rectifying muscarinic K(K) channel was examined in rabbit atrial myocytes. In inside-out patch membranes, the single channel current-voltage relationship of Kchannels 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 Pof outwardly flowing Kchannel currents without affecting the unitary current amplitude at depolarized potentials, but had no effect on inward Kcurrents under hyperpolarization. This voltage dependence of Pof Kchannels 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 Kcurrents which had been lost in the inside-out configuration. The effect of spermine was concentration-dependent with ICof 10 n M at +40 mV. The order of potency of polyamines in reducing Pat +40 mV was spermine spermidine > putrescine > ornithine; arginine had no significant effect. Intracellular Mgantagonized the effect of spermine. Neither the single channel conductance nor Pof the ATP-sensitive Kchannel, 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 Kand Ichannels.


INTRODUCTION

Inwardly rectifying Kchannels permit the flow of Kions more efficiently in the inward direction (1) . This unique property allows these channels to clamp the resting potential of cell membrane near the Kequilibrium 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 Kions by intracellular Mgions (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 Kchannels 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 Kchannel (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 Mgand/or their gating properties (4, 7, 11, 12, 18, 19) .

In the case of the cardiac Ichannel, Mginstantaneously 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) . Mgalso paradoxically permits some outward flow of Kions at positive membrane potentials by hampering closure of the gate (11, 13) . Upon hyperpolarization to potentials more negative than E, the Mgblockade is nearly instantaneously relieved, followed by a time-dependent increase in inward currents through the gating process (13) . The Ichannels in the open cell-attached patch in Mg-free solution gradually lose the time-dependent change in the open probability ( P); as a result, Premains 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 Kchannel, another strong inward rectifier (20) . Thus, such a substance might universally contribute to the gating of strong inward rectifiers.

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 Kchannels at potentials positive but not negative to E. In contrast, the weak inward rectifier Kchannel 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.


EXPERIMENTAL PROCEDURES

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 NaHPO, 5.5 glucose, and 5.5 HEPES-NaOH buffer (pH 7.4). The composition of the high K, low Clsolution was (in m M): 10 taurine, 10 oxalic acid, 70 glutamic acid, 25 KCl, 10 KHPO, 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 Mgconcentration 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 Kchannel 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 Clsolution 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 Pof 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.


RESULTS

Fig. 1A shows the effect of spermine (10 µ M) on Kchannels in an inside-out patch membrane of a rabbit atrial myocyte. At -60 mV, GTPS (10 µ M) applied to the intracellular side of the patch membrane in the presence of 1.4 m M free Mgpromptly induced inward Kcurrents (27) : N P( N is the number of the channels in a patch; Pis 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 Mgblockade (10, 12) . On removal of Mg, the unitary amplitude of outward Kchannel 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 Kchannel 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 Kchannels 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 Kcurrents in an inside-out patch membrane of rabbit atrial myocytes. Experiments were conducted in symmetrical 150 m M Ksolutions 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 Kand Kchannels, 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 Kcurrents at +40 mV. Panel a, after Kchannels were irreversibly activated by GTPS, 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 Pof the Kchannels, where N is the number of the channels in a patch, whereas Pis 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 Kchannels 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 ICof 10 n M (Fig. 1 B, panel d). Different from Mgblockade 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).

The effect of spermine (100 n M) on Kchannels was examined at different membrane potentials (Fig. 2). In the cell-attached configuration, Kchannels 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 Kchannels were activated by GTPS 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, Pshowed 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, Psteeply decreased at least severalfold as membrane was depolarized across E(0 mV in this condition). When spermine (100 n M) was applied, the N Pat potentials positive to Ebecame 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 Pcould 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 Pmeasured in the inside-out configuration steeply voltage-dependent in a range of potentials around E. This was also the case when the external Kconcentration was decreased to 70, 35, and 5 m M in the presence of 150 m M intracellular K(in each case, Ecalculated 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 Ksolutions. The intracellular surface of patch membranes was perfused with the Mg-free internal solution containing 1 m M ATP to suppress openings of Kchannels. 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 Pof Kchannels in the presence and absence of 100 n M spermine. ATP (1 m M) was added to the intracellular side of patch membranes. N Pwas measured in the absence ( open circles) and presence ( closed circles) of spermine. N Pis 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 Eis followed by a time-dependent decay of an outward Kchannel 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 Kcurrent (28) . These phenomena occur because the Pof Kchannels 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 Pof Kchannels voltage-dependent (Fig. 2 B). In the absence of spermine (control), the time-dependent change in N Pduring 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 Pof inward currents, which is more clearly seen in the ensemble average currents, therefore indicates a certain rundown of the channel during this experiment.

Restoration of the relaxation required relatively high concentrations of spermine (300 n M to 3 µ M) compared with the ICvalue (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 Kchannel was assumed to occur as follows,

On-line formulae not verified for accuracy

 

On-line formulae not verified for accuracy

Then, the time constant of relaxation () is as follows,

 

On-line formulae not verified for accuracy


Figure 3: Spermine evokes the relaxation of K channels. A, the time-dependent change in Kcurrents in the presence or absence of 300 n M, 1 µ M, and 3 µ M spermine. Kchannels in an inside-out patch membrane were preactivated with 10 µ M GTPS and then Mgwas 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.

 

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 Kcurrents 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 10s, 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 Pof the channel in the presence of spermine (Figs. 1 B, panel d, and 2 B).

The possible interaction between spermine and Mgon Kchannels was next examined (Fig. 4). As in the upper trace and the inset in Fig. 4 A, 204 µ M Mgreduced 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 Pof Kchannels. 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 Pto 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, Mgwas applied to the channels pretreated with spermine (1 µ M). In the presence of spermine, Mgreduced 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 Pwas 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 Mgalone. The effect of spermine was also examined when higher concentration (1.4 m M) of Mgwas present (Fig. 4 C). Although not completely, spermine (10 µ M) suppressed flickery openings of Kchannels 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, Kchannels in the inside-out patch membrane were first irreversibly activated by 10 µ M GTPS 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 Pbut not the unitary current amplitude at depolarized potentials (Fig. 5 A): N Pin 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 Pat +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 Kchannels (Fig. 5 B); N Pin the presence of 10 µ M L-arginine was 102.3 ± 5.6% of the control at +40 mV ( n = 3).

Neither the unitary current amplitude nor Pof Kchannels, a weak inward rectifier, was affected by spermine (Fig. 5 C); the N Pin 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 Kchannels and of spermine (10 µ M) on Kchannels ( 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 Kchannel openings, whereas the smaller spikes (1.2 pA) correspond to occasional openings of Kchannels. The very large spikes seen on changing membrane potentials are the artificial capacitative currents introduced by rapid voltage jumps.




DISCUSSION

Spermine makes the Pof Kchannels 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 Kchannels (Fig. 1 B, panel d). Thus, Kchannels 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 Kchannel 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 Kcurrent through Kchannels at the onset of the action potential.

Spermine and Mgsuppress the outward Kcurrents 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 Ichannels also indicates that the closed state of the intrinsic gating and the state of the Mgblockade 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 Mgblockade (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 Kchannel (GIRK1), also has aspartate at position 173 (35) ; cardiac Kchannels, a weak inward rectifier, have asparagine at the equivalent position (36) . The difference in the sensitivity to spermine between Kand Kchannels (Figs. 1 and 5 C) suggests that polyamines gate Kchannels probably through this site as a unidirectional voltage-dependent channel blocker.

It is likely that the polyamine-bound state is an independent state distinguished from the Mg-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.

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 Mregion. Their data are basically similar to ours and support our inference concerning the site of action of polyamines.


FOOTNOTES

*
This work was supported by a grant from the Ministry of Education, Science and Culture of Japan Terumo Life Science Foundation; the Naito Foundation; the Mochida Memorial Foundation for Medical and Pharmaceutical Research; the Ichiro Kanehara Foundation; and the Yamanouchi Foundation for Research on Metabolic Disorders (to Y. K.). 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.

§
To whom correspondence should be addressed: Dept. of Pharmacology II, Faculty of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565, Japan.

The abbreviations used are: Mg, intracellular Mg; Kchannel, the muscarinic Kchannel; Ichannel, the background inward-rectifying Kchannel; Kchannel, the ATP-sensitive Kchannel; GTPS, guanosine 5`-3- O-(thio)triphosphate.


ACKNOWLEDGEMENTS

We thank Dr. Ian Findlay (University of Tours, Tours, France) for his suggestions during the work and critical reading of this manuscript.


REFERENCES
  1. Hille, B. (1991) Ionic Channels of Excitable Membranes, pp 127-130, Sinauer Associates Inc., Sunderland, MA
  2. Ciani, S., Krasne, S., Miyazaki, S., and Hagiwara, S. (1978) J. Membr. Biol. 44, 103-134 [Medline] [Order article via Infotrieve]
  3. Gunning, R. (1983) J. Physiol. ( Lond.) 342, 437-451 [Abstract]
  4. Kurachi, Y. (1985) J. Physiol. ( Lond.) 366, 365-385 [Abstract]
  5. Matsuda, H., Saigusa, A., and Irisawa, H. (1987) Nature 325, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  6. Vandenberg, C. A. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2560-2564 [Abstract]
  7. Findlay, I. (1987) J. Physiol. ( Lond.) 391, 611-629 [Abstract]
  8. Findlay, I. (1987) Pflügers Arch. 410, 313-320
  9. Horie, M., Irisawa, H., and Noma, A. (1987) J. Physiol. ( Lond.) 387, 251-272 [Abstract]
  10. Horie, M., and Irisawa, H. (1987) Am. J. Physiol. 253, H210-H214
  11. Matsuda, H. (1988) J. Physiol. ( Lond.) 397, 237-258 [Abstract]
  12. Horie, M., and Irisawa, H. (1989) J. Physiol. ( Lond.) 408, 313-332 [Abstract]
  13. Ishihara, K., Mitsuiye, T., Noma, A., and Takano, M. (1989) J. Physiol. ( Lond.) 419, 297-320 [Abstract]
  14. Matsuda, H. (1991) Annu. Rev. Physiol. 53, 289-298 [CrossRef][Medline] [Order article via Infotrieve]
  15. Sakmann, B., and Trube, G. (1984) J. Physiol. ( Lond.) 347, 641-657 [Abstract]
  16. Sakmann, B., Noma, A., and Trautwein, W. (1983) Nature 303, 250-253 [Medline] [Order article via Infotrieve]
  17. Noma, A. (1983) Nature 305, 147-148 [Medline] [Order article via Infotrieve]
  18. Ho, K., Nichols, C. G., Lederer, W. J., Lytton, J., Vassilev, P. M., Kanazirska, M. V., and Hebert, S. C. (1993) Nature 362, 31-38 [CrossRef][Medline] [Order article via Infotrieve]
  19. Simmons, M. A., and Hartzell, H. C. (1987) Pflügers Arch. 409, 454-461
  20. Ito, H., Tung, R. T., Sugimoto, T., Kobayashi, I., Takahashi, K., Katada, T., Ui, M., and Kurachi, Y. (1992) J. Gen. Physiol. 99, 961-983 [Abstract]
  21. Morgan, D. M. L. (1990) Biochem. Soc. Trans. 18, 1079-1080 [Medline] [Order article via Infotrieve]
  22. Morgan, D. M. L. (1990) Biochem. Soc. Trans. 18, 1080-1084 [Medline] [Order article via Infotrieve]
  23. Fabiato, A., and Fabiato, F. (1979) J. Physiol. ( Paris) 75, 463-505
  24. Kurachi, Y., Nakajima, T., and Sugimoto, T. (1986) Pflügers Arch. 407, 264-274
  25. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pflügers Arch. 391, 85-100
  26. Colquhoun, D., and Sigworth, F. J. (1983) Fitting and Statistical Analysis of Single-Channel Records: Single-Channel Recording, pp. 191-263, Plenum Press, New York
  27. Kurachi, Y., Nakajima, T., and Sugimoto, T. (1986) Am. J. Physiol. 251, H681-H684
  28. Kurachi, Y. (1990) Regulation of Potassium Transport across Biological Membranes: Muscarinic Acetylcholine-gated KChannels in Mammalian Heart, pp. 403-428, University of Texas Press, Austin, TX
  29. Watanabe, S., Kusama-Eguchi, K., Kobayashi, H., and Igarashi, K. (1991) J. Biol. Chem. 266, 20803-20809 [Abstract/Free Full Text]
  30. Scott, R. H., Sutton, K. G., and Dolphin, A. C. (1993) Trends Pharmacol. 16, 153-160
  31. Kubo, Y., Baldwin, T. J., Jan, Y. N., and Jan, L. Y. (1993) Nature 362, 127-133 [CrossRef][Medline] [Order article via Infotrieve]
  32. Stanfield, P. R., Davis, N. W., Shelton, P. A., Sutcliffe, M. J., Khan, I. A., Brammar, W. J., and Conley, E. C. (1994) J. Physiol. ( Lond.) 478, 1-6 [Abstract]
  33. Lu, Z., and MacKinnon, R. (1994) Nature 371, 243-246 [CrossRef][Medline] [Order article via Infotrieve]
  34. Wible, B. A., Taglialatela, M., Ficker, E., and Brown, A. M. (1994) Nature 371, 246-249 [CrossRef][Medline] [Order article via Infotrieve]
  35. Kubo, Y., Reuveny, E., Slesinger, P. A., Jan, Y. N., and Jan, L. Y. (1993) Nature 364, 802-806 [CrossRef][Medline] [Order article via Infotrieve]
  36. Ashford, M. L. J., Bond, C. T., Blair, T. A., and Adelman, J. P. (1994) Nature 370, 456-459 [CrossRef][Medline] [Order article via Infotrieve]

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