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The Role of Weak Blocking Molecules
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0.4 mM) to the intracellular milieu where endogenous polyamines remained, and then examined outward IRK1 currents using the whole-cell patch-clamp method at 5.4 mM external K+. Without internal Mg2+, small outward currents flowed only at potentials between –80 (the reversal potential) and –40 mV during voltage steps applied from –110 mV. The strong inward rectification was mainly caused by the closed state of the activation gating, which was recently reinterpreted as the endogenous-spermine blocked state. With internal Mg2+, small outward currents flowed over a wider range of potentials during the voltage steps. The outward currents at potentials between –40 and 0 mV were concurrent with the contribution of Mg2+ to blocking channels at these potentials, judging from instantaneous inward currents in the following hyperpolarization. Furthermore, when the membrane was repolarized to –50 mV after short depolarizing steps (>0 mV), a transient increase appeared in outward currents at –50 mV. Since the peak amplitude depended on the fraction of Mg2+-blocked channels in the preceding depolarization, the transient increase was attributed to the relief of Mg2+ block, followed by a re-block of channels by spermine. Shift in the holding potential (–110 to –80 mV), or prolongation of depolarization, increased the number of spermine-blocked channels and decreased that of Mg2+-blocked channels in depolarization, which in turn decreased outward currents in the subsequent repolarization. Putrescine caused the same effects as Mg2+. When both spermine (1 µM, an estimated free spermine level during whole-cell recordings) and putrescine (300 µM) were applied to the inside-out patch membrane, the findings in whole-cell IRK1 were reproduced. Our study indicates that blockage of IRK1 by molecules with distinct affinities, spermine and Mg2+ (putrescine), elicits a transient increase in the outward IRK1, which may contribute to repolarization of the cardiac action potential.
Key Words: inward rectification • Mg2+ • spermine • repolarization • putrescine
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; Noma et al., 1984; Sakmann and Trube, 1984; Hume and Uehara, 1985). The property of this channel that strongly impedes the flow of outward currents (rectification) is important for the long plateau phase of the cardiac action potential (Hutter and Noble, 1960; Sakmann and Trube, 1984). It has also been suggested that the small outward iK1 currents that flow at potentials near the reversal potential (Erev)1 contribute to the final repolarization of the action potential (Giles and Imaizumi, 1988; Ibarra et al., 1991; Shimoni et al., 1992). Interferences by other currents, however, have hampered the analysis of outward currents through iK1 channels (Shimoni et al., 1992).
Mechanisms underlying the strong inward rectification of iK1 have been studied extensively. A voltage-dependent gating has been shown to cause the rectification of iK1 currents at potentials around Erev (for review, see Vandenberg, 1994). With physiological concentrations of internal free Mg2+ (0.5–1.2 mM in cardiac myocytes, Murphy et al., 1991), blockage of the channel by Mg2+ (Matsuda et al., 1987; Vandenberg, 1987) also contributes to the rectification mainly at depolarized levels far from Erev (Ishihara et al., 1989). Recently, studies on channels exogenously expressed from cloned inward rectifier K+ channel genes, IRK1 (Kir 2.1, Kubo et al., 1993) and HRK1 (Kir 2.3, Makhina et al., 1994), have revealed that the channels are also blocked by internal cationic polyamines, spermine (Spm), spermidine (Spd), and putrescine (Put) (Fakler et al., 1994; Ficker et al., 1994; Lopatin et al., 1994; Fakler et al., 1995). Furthermore, these studies have strongly suggested that the gating of the strong inward rectifiers is caused by the blockage of the channels by endogenous Spm and Spd (Ficker et al., 1994; Lopatin et al., 1994, 1995). Among cations known to block strong inward rectifiers, Spm, which can possess four protonated sites at a physiological pH, shows the highest affinity with the channels, being about a 10-fold more potent blocker than Spd (Lopatin et al., 1994; Yang et al., 1995). The potency of Mg2+ for blocking IRK1 and HRK1 was shown to be similar to that for blocking iK1 channels (Matsuda, 1988; Lopatin et al., 1994; Taglialatela et al., 1994). However, Spm is more potent than Mg2+ or Put (diamine with two protonation sites) in blocking IRK1 and HRK1, by a factor of about 10,000 and 100, respectively (Lopatin et al., 1994; Yang et al., 1995).
When the IRK1 gene is expressed in murine fibroblast cells (L strain), the macroscopic currents well reconstitute the gating properties of iK1, including the slowing of the gating observed at depolarized levels in the presence of intracellular Mg2+, due to competitive blocking of the channel by Mg2+ (Ishihara et al., 1989; Stanfield et al., 1994; Ishihara et al., 1996). Therefore, it was worthwhile to use this channel to investigate in detail the kinetics of the outward component of the inward rectifier K+ current at a physiological low concentration of extracellular K+ without any necessity to isolate it from other ionic current systems. In this study, we show a novel time-dependent change of outward currents through the inward rectifier K+ channel observed in the presence of internal Mg2+ or Put. When the membrane potential is repolarized from depolarized levels to a level near Erev where currents still flow in the outward direction, we find that outward currents show a transient increase, which can be attributed to relief of Mg2+ (Put) block followed by re-block of channels by Spm. The blockage of the channels by Mg2+ at membrane potentials in the plateau range is thus suggested to be important for generating outward currents that repolarize the membrane during the cardiac action potential.
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). IRK1-expressing L cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% heat-inactivated fetal bovine serum. Before experiments, cells were dispersed from the culture dish by treating them with a PBS containing 0.5 mM EDTA. The spherical cells in suspension were then stored in Dulbecco's modified Eagle's medium at room temperature until they were used within 3 h.
Measurements of Currents from IRK1-expressing L Cells
Macroscopic currents were measured from whole-cell and inside-out patch membranes using the patch-clamp technique (Hamill et al., 1981) using an EPC-7 amplifier (List Electronics, Darmstadt, Germany). Patch electrodes were pulled from a Pyrex glass tube (o.d. 1.5 mm, i.d. 1.0 mm; Narishige, Tokyo, Japan) on a horizontal puller (Sutter Instruments Co., Novato, CA). The resistance of pipettes used for whole-cell recordings was 1.8–2.5 M
when filled with pipette solutions (see below). For recording currents from patch membranes, pipettes with large-diameter tip openings (7–10 µm) were prepared (Hilgeman, 1995), and the open-cell attached inside-out patch technique was used (Horie et al., 1987). Briefly, after a gigaohm seal was established using the large patch pipette, the cell membrane was torn using another thin glass pipette in order to expose the intracellular side of the patch membrane to the bath solution. Usually, the tip of the thin pipette was broken by crushing it against the bottom of the recording chamber, and then a large opening of cell membranes (5 µm in distance) was achieved by scratching the cell membrane with the broken pipette. Voltage stimulation and data acquisition were performed using pCLAMP software (ver. 6.02; Axon Instruments, Foster City, CA) on a 486 DOS/V computer (Compaq, Prolinea 4/33) through Digidata 1200A AD converter (Axon Instruments). Membrane potentials indicate transmembrane potentials at the inside of the cell membrane. In whole-cell current measurements, the liquid junction potential in pipettes was measured to be –10 mV relative to the extracellular solution, and all membrane potentials were corrected for this value. All experiments were conducted at room temperature (20–22°C).
Solutions
In whole-cell experiments, the extracellular solution perfused in the bath contained (in mM), 140.0 NaCl, 5.4 KCl, 1.8 CaCl2, 0.33 NaH2PO4, 5.0 HEPES (pH 7.4 with NaOH). When extracellular K+ concentration (Ko) was increased to 15.4 mM, 10.0 mM NaCl was replaced with KCl. Unless otherwise stated, whole-cell currents were recorded at 5.4 mM Ko. The Mg2+-free pipette solution contained (in mM): 20.0 KCl, 90.0 K-aspartate, 10.0 KH2PO4, 5.0 EDTA, 1.9 K2ATP, 5.0 HEPES (pH 7.2 with KOH) Using this solution containing EDTA, intracellular free Mg2+ concentration (Mgi) is expected to be less than 10–8 M, even if the deionized water contained 10 µM of Mg2+ (Fabiato and Fabiato, 1979). To prepare pipette solutions containing either 1.1 mM or 440 µM free Mg2+, 7.9 or 7 mM MgCl2 were added to the Mg2+-free pipette solution, respectively (Fabiato and Fabiato, 1979). Put (Sigma Chemical Co., St. Louis, MO) was added to the Mg2+-free pipette solution containing 1.9 mM ATP, at 500 µM. The concentration of free Put buffered by ATP at a physiological concentration (2–3 mM, Watanabe et al., 1991) is estimated to be 330–470 µM using an apparent binding constant of 0.205–0.037 mM–1 (Miyamoto et al., 1993). The total K+ concentration in pipette solutions was 150 mM.
For the open-cell attached inside-out patch experiments, the pipette solution facing the extracellular side of patch membranes contained (in mM), 145.0 KCl, 1.0 CaCl2, 5.0 HEPES (pH 7.4 with KOH). To increase the current amplitude, Ko was 150 mM. The Mg2+-free bath solution contained (in mM) 120.0 KCl, 10.0 KH2PO4, 4.0 EDTA, 5.0 HEPES (pH 7.2 with KOH). Spm (Sigma Chemical Co.) and Put were added to this solution before use.
Data Analysis
Currents were plotted and analyzed using pCLAMP software. The horizontal dashed lines superimposed on current traces indicate the zero current level. In the present study, distribution of channels in the open state, the Spm-blocked state and the Mg2+- or the Put-blocked state was estimated based on macroscopic-current changes. Since the chord conductance of whole-cell and patch-membrane (with 1 or 10 µM internal Spm) currents reached a maximum value at a membrane potential of about 40 mV negative to Erev (Erev – 40 mV), and since the maximum conductance did not notably change subsequent to making the whole-cell patch using pipette solutions containing either Mg2+or Put, the proportions of the channels in the individual states were estimated as a value relative to the amount of the channels maximally opened at Erev – 40 mV (–120 and –90 mV at 5.4 and 15.4 mM Ko, respectively).
The proportion of the channels in the open state (PO) was estimated from the chord conductance by assuming that the unitary conductance of the channel is independent of the membrane potential. The chord conductance g was calculated by dividing the current level I with the deviation of the membrane potential V from Erev, and then it was normalized with its maximum value gmax obtained at Erev – 40 mV.
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To obtain the proportion of channels blocked by Spm (PSpm), Mg2+ (PMg), and Put (PPut), inward currents were recorded by hyperpolarizing the membrane potential from various levels to Erev – 40 mV (for patch-currents Erev – 30 mV was also used). The single exponential increase of inward currents was attributed to the relief of Spm block, based on the previous study (Ishihara et al., 1996) and the results shown in Fig. 2. The theoretical curve –A exp{–(t – k) / 
+ C was fitted to time-dependent currents using the Simplex least squares fitting method, and the current levels at the onset of the voltage change, I(0), was obtained from the curve. The amplitude of the current component showing a single exponential increase, C – I(0), was used to obtain PSpm at the membrane potential that preceded hyperpolarization,
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When we recorded the single-channel currents of IRK1 from L cells in the cell-attached mode, the open probability of the channels generally showed a value larger than 0.9 at 30–40 mV negative to Erev. We therefore consider that PO, PSpm, PMg, and PPut, which were all obtained as above, are close to the probability of the channel in each state.
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). The input capacitance of the cells employed for whole-cell recordings was 26.9 ± 6.0 pF (mean ± SD, n = 27), and the steady-state current density, measured at –120 mV, 5.4 mM Ko, was 43.2 ± 14.9 pA · pF–1 (n = 27). The currents shown in Fig. 1 A were recorded using the Mg2+-free pipette solution containing 5 mM EDTA, at a time 32 min after making the whole-cell patch. When the membrane was depolarized from –110 mV where an inward current flowed, outward currents were rapidly suppressed both at –60 and 10 mV, indicating the strong inward rectification of the current without Mg2+. Subsequent hyperpolarization to –120 mV activated an inward current with a single exponential time course. This time-dependent increase of inward currents has been attributed to the activation kinetics of the inward rectifier K+ currents (Hagiwara et al., 1976; Leech and Stanfield 1981). Recently, internal polyamines were shown to block outward currents through strong inward rectifiers, and the activation of inward currents was reinterpreted as the relief of polyamine block from the channels (Ficker et al., 1994; Lopatin et al., 1994; Lopatin et al., 1995). We found that an increase in the internal Spm concentration changes the activation of whole-cell IRK1 currents in L cells, which was explicable by an increase in the closing rate of the activation gating, and this observation was considered to be evidence indicating that the "closed state" of the activation gating is the blocked state of the channel by endogenous Spm, a tetravalent polyamine (Ishihara et al., 1996). The theoretical curves superimposed on the current traces in Fig. 1 A show that the increase in inward currents started from approximately a 25% level of the maximum inward current level. According to the above notion, this observation infers that 75% of the channels that opened at –120 mV were in the Spm-blocked state, both at –60 and 10 mV. When the relationship between the membrane potential and PSpm was estimated from currents recorded at 8 and 32 min after starting the experiment (Fig. 1 B), the relation at 32 min was shifted in a depolarizing direction compared to that at 8 min, which most likely reflects the decrease in the internal Spm level due to washout of endogenous Spm from the cell. Even after the intracellular milieu had been exposed to the pipette solution for >30 min, however, a large fraction of channels were still blocked by Spm at potentials positive to Erev (–80 mV). In I-V relationship, outward currents are in evidence at potentials positive to Erev, with a negative slope at potentials positive to –50 mV, thus showing only a hump of outward currents (Fig. 1 C). The inward rectification of whole-cell IRK1 currents mainly caused by Spm block was generally so strong that no measurable outward currents flowed at potentials positive to –30 mV.
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; Lopatin et al., 1994), the time course of inward currents did not change (Figs. 2 Ab and B). When 1 µM Spm was added to the bath solution after the decrease in outward currents had obviously slowed, the decrease became rapid without affecting the time course of inward currents (Fig. 2 Ac and B). These observations show that the "closing process" of the activation gating slows after opening the cell, and that the increase in the internal Spm concentration restores the speed of the closing process, supporting that Spm block "closes" the activation gate, and that the time-dependent increase of inward currents under the whole-cell voltage clamp condition (Fig. 1 A) reflects the relief of Spm block (see DISCUSSION). With 1 µM internal Spm, PSpm, estimated from the amplitude of time-dependent inward currents, steeply increased at potentials around Erev (Fig. 2 C). With 10 µM Spm, the increase in PSpm occurred at more negative membrane potentials (Fig. 2 C). When the voltage dependence of PSpm obtained from whole-cell currents (Fig. 1 B) was plotted against the deviation of the membrane potential from Erev, these relationships were found to be similar to those of the patch currents (Fig. 2 C). We thus speculate the concentration of endogenous free Spm left in cells during whole-cell recordings to be around 1–10 µM.
Inward Rectification of Whole-cell IRK1 with Internal Divalent Cations
In the following sections, we show the results obtained from whole-cell currents recorded in the presence of internal Mg2+ or Put. When the membrane was depolarized in the presence of 1.1 mM Mgi, outward currents were rapidly suppressed, and remaining current components further decayed with a slow time course (Fig. 3 A, top). Although the outward-current levels at the end of 20-ms depolarizing steps to –60 and 10 mV were small (as they were in the absence of Mgi, Fig. 1 A), currents in the subsequent hyperpolarization to –120 mV were different: exponential increase in the inward current started from 24% of the maximum inward current on hyperpolarization from –60 mV, whereas the current increased exponentially from 67% of the maximum with the same time constant on hyperpolarization from 10 mV. As an increase in Mgi increases the fraction of nonconductive channels which instantaneously activates on hyperpolarization, these observations infer that the number of Spm-blocked channels decreased, while the number of Mg2+-blocked channels increased, by depolarizing to 10 mV, rather than to –60 mV (Ishihara et al., 1989, 1996). The lower panel of Fig. 3 A shows a similar effect caused by 500 µM internal Put. During the steps to both –60 and 10 mV, sizable outward currents remained after the rapid suppression of the currents, and then decayed slowly. At the end of the steps, the outward current at 10 mV was even smaller than that at –60 mV. However, the current observed in the subsequent step from –60 to –120 mV showed a time-dependent activation whereas that from 10 to –120 mV showed an instantaneous activation. These observations indicate that internal Put blocked the channels at the end of the step at 10 mV and that the relief of Put block is also virtually instantaneous (Lopatin et al., 1995; Ishihara et al., 1996).
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). These findings indicate that the outward currents flow through the inward rectifier IRK1. The presence of either internal Mg2+ or Put appeared to increase the flow of outward currents.
Distribution of Channels in the Mg2+-blocked State Increases Amplitude of Outward Currents
From I-V relations obtained with Mg2+ (Fig. 3 B, top), the voltage dependence of PO at the end of 20-ms voltage steps was obtained (Fig. 4 A). PSpm and PMg at the end of the voltage steps, estimated based on the currents in the following hyperpolarization, are shown in Fig. 4, B and C, respectively. PSpm steeply increased at potentials around Erev (–82 and –54 mV at 5.4 and 15.4 mM Ko, respectively), but decreased at more depolarized potentials. PMg increased in a voltage-dependent manner at potentials positive to Erev + 20 mV at both Ko, as denoted by continuous curves. These plots indicate that distribution of channels in the individual states apparently depends on Ko. From these plots, it is suggested that the increase in the fraction of channels blocked by Mg2+ (Fig. 4 C) increases the amplitude of outward currents at positive membrane potentials (Fig. 3 B).
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), by replacing the closed state of iK1 channel with the Spm-blocked state:
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where β and µ are the block rates (M–1 · S–1) that are the function of the concentration of Spm and Mg2+, respectively, and 
and 
are the unblock rates (S–1). It is inferred from the plots that channels were distributed from the open state to the Mg2+-blocked state at potentials far positive to Erev by the larger µ compared with β. We previously showed, however, that Mg2+ block and Put block are replaced by Spm block during extended depolarization (Ishihara et al., 1996). The simulation of the same phenomenon, i.e., redistribution of the Mg2+ blocked state to the closed state of iK1 channel (Ishihara et al., 1989) suggests that the channels transiting frequently between the Mg2+-blocked state and the open state are gradually trapped in the Spm-blocked state because of the small that gives Spm a high affinity with the channel. Fig. 4 D shows PSpm obtained at the end of 500-ms voltage steps. The amount of PSpm that increased at each membrane potential by extending voltage steps from 20 to 500 ms is plotted in Fig. 4 E. For example, at 15.4 mM Ko, the increase in PSpm is conspicuous at potentials between –20 and 20 mV. Concomitantly, outward currents at corresponding potentials decreased by extending voltage steps from 20 to 500 ms (Fig. 5 A), which resulted in a disappearance of the second hump in the outward I-V relation. These observations show that replacement of Mg2+ block by Spm block during extended depolarization decreases the amplitude of outward currents, and indicate that blockage of channels by Mg2+ facilitates the flow of outward currents. In the experiment performed with internal Put, outward currents generating the second hump in the outward I-V (Fig. 3 B, bottom) were also decreased by extending depolarizing steps (Fig. 5 B) due to the replacement of Put block by Spm block (data not shown).
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, 1996). Therefore, PMg remains large during depolarization at potentials positive to 0 mV, near the action potential plateau (e.g., Fig. 4). We next show the influence of Mg2+-blocked channels at depolarized potentials on outward currents which flow upon repolarization. Fig. 7 A shows a representative current observed in response to repolarization at 440 µM Mgi. When the membrane potential was stepped from 10 to –50 mV, a transient outward current was elicited after the inward capacitive current: the outward current increased rapidly, and then decreased with a slower time course. Before repolarization, the membrane was briefly depolarized from –120 to 10 mV, and PMg estimated at the end of the step at 10 mV was 0.7. Since the relative potency of Spm block versus Mg2+ block is stronger at –50 mV than at 10 mV (Fig. 4), Mg2+ block is expected to be substituted with Spm block during the repolarizing step at –50 mV. If this kinetic occurs through the open state, then the larger the number of Mg2+-blocked channels are, the more channels will reside in the open state before passing into the Spm-blocked state, thereby transiently increasing the outward-current amplitude.
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Factors that Change the Amplitude of Transient Outward Currents
Transient increase in outward currents also appeared when the pipette solution contained 500 µM Put. Fig. 8 illustrates the factors that affected the amplitude of transient outward currents. In Fig. 8 A, currents were recorded by depolarizing the membrane from –120 to 0 mV for various durations (100–500 ms), followed by repolarization to –50 mV. Upon repolarization to –50 mV, outward currents increased rapidly after inward capacitive transients, and then decreased with a slower time course to levels which are larger than those at 0 mV due to the negative slope conductance of the current. Prolongation of the depolarizing step at 0 mV reduced the amplitude of the transient outward current during the following repolarizing step at –50 mV. Elevation of the holding potential from –120 to –80 mV also decreased the size of transient outward currents (Fig. 8 B). When neither Mg2+ nor Put was added to the pipette solution, there was no transient increase in outward currents on repolarization (Fig. 8 C).
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). It is noticed from these relations that PO with 300 µM Put was significantly larger than that with 1 µM Spm at potentials between 0 and 40 mV.
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I-V Relationships Obtained with a Repolarizing Ramp in the Presence of Mgi
In the repolarizing phase of the action potential, the membrane potential successively changes while the net current flows in the outward direction. Thus, we obtained the outward I-V relation from whole cells using a repolarizing ramp pulse. As shown in Fig. 11 A, the outward current measured with 440 µM Mgi was significantly larger than that measured at Mgi free. With Mg2+, the outward current was prominent at potentials around –50 mV. Without Mg2+, only a small outward current flowed at potentials negative to –40 mV, which was comparable to the levels measured using depolarizing steps (Fig. 1). At 440 µM Mgi, a shift in the holding potential from –120 to –80 mV reduced the outward current at potentials around –50 mV (Fig. 11 B), and the prolongation of the preceding depolarization further reduced it (Fig. 11 C).
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400 µM in free form), used within physiological Mgi (Murphy et al., 1991), contributed to blocking IRK1 during large depolarizing steps. Subsequent repolarizing steps to a level where currents still flow in the outward direction elicited a transient increase in outward currents, which was attributed to the relief of Mg2+ or Put block followed by a re-block of channels by Spm. When 300 µM Put was applied to the intracellular side of the patch membrane with 1 µM Spm (an estimated endogenous free Spm level during whole-cell recordings), the results obtained from whole-cell currents were reproduced. The findings in the present study indicate that blockage of the inward rectifier K+ channel by molecules with distinct affinities, i.e., Spm and Mg2+ (or Put), can elicit a time-dependent change in outward currents.
Inward Rectification of Whole-cell IRK1 Caused by Endogenous Polyamines
Without Mg2+, the strong inward rectification of whole-cell IRK1 in L cells was mainly determined by the closed state that shows an exponential activation on hyperpolarization (Fig. 1), similar to native strong inward rectifiers (Ishihara et al., 1989; Silver and DeCoursey, 1990). In the previous study (Ishihara et al., 1996), we attributed this closed state to the state of the channel blocked by endogenous Spm according to the findings that (a) an increase in the internal Spm concentration shifted the voltage dependence of the activation curve in a hyperpolarizing direction and changed the time constant of the activation process, which were both explained by an increase in the closing rate of the activation gating, and (b) an increase in the concentration of other naturally occurring polyamines (Spd and Put) increased the fraction of nonconductive channels which open faster than the activation process on hyperpolarization. In this study, the results obtained from inside-out patch-currents (Fig. 2, A and B) were also compatible with the idea that closed state is caused by Spm block of the channel. That is, when we consider the following simple kinetic model, 
where k (M–1 · S–1) and k–1 (S–1) are the voltage-dependent block and unblock rates of molecule X, the slowing of relaxation, 1 / (k + k–1), at potentials positive to Erev after opening the cell (Fig. 2 Ab) can be explained by a decrease in the concentration-dependent k due to the washout of molecule X. In this case, relaxation is not necessarily affected at potentials negative to Erev where k–1 is significantly larger than k (Ishihara et al., 1996). The currents recorded with 1 µM Spm (Fig. 2Ac) infer that unblock rate from the Spm-blocked state is the same as k–1, strongly suggesting that Spm is the molecule X that is acting as a gating charge to cause the closed state of the activation gating (Lopatin et al., 1995).
Even when the pipette solution contained EDTA to eliminate Mgi, 20% of the channels apparently showed an instantaneous activation on hyperpolarization (e.g., Fig. 1), similar to the observation in native inward rectifier channels (e.g., Silver and DeCoursey, 1990). Since the concentration of total Put has been reported to be much lower than that of Spm or Spd in mammalian tissues (Pegg and McCann, 1982), this component may reflect rapid relief of endogenous-Spd block (with much higher affinity for IRK1 than Mg2+ or Put; Yang et al., 1995) rather than that of endogenous-Put block. In fact, it was shown for the HRK1 channel expressed in Xenopus oocytes (stages V-VI) that Spd contributes to blocking the channel together with Spm (Lopatin et al., 1995). In experiments performed with either internal Mg2+ or Put, we obtained PMg or PPut from the fraction of channels that open instantaneously on hyperpolarization. For the above reason, however, it is possible that the fraction of channels blocked by endogenous Spd contaminated the values PMg and PPut, by less than 0.2. However, outward currents at potentials >–40 mV (Fig. 3 B), and transient outward currents during repolarizing steps (Figs. 7 and 8), appeared only when PMg or PPut substantially increased at depolarized levels by adding Mg2+ or Put to pipette solutions, respectively (Figs. 4, 7, and 9), indicating that these current changes were caused by the effects of internal Mg2+ and Put (cf., Figs. 1 and 8 C).
Blockage of IRK1 by Mg2+ or Put in the Presence of Spm
During prolonged whole-cell experiments, the current changes caused by the effects of internal Mg2+ and Put became more prominent as PMg or PPut gradually increased (data not shown). Since we always analyzed currents recorded within a short period of time (1–3 min), the influence of this time course on our findings appeared to be minimal. As a decrease in the endogenous Spm level was implied from the shift in the voltage dependence of PSpm during experiments (Fig. 1 B), the above observation is consistent with the kinetic scheme that Spm and Mg2+ (Put) compete to block IRK1. The currents recorded from cell-attached inside-out patch membranes (Fig. 2 C) suggested that the concentration of endogenous free Spm decreased from 10 µM to 1 µM during whole-cell recordings. When both 1 µM Spm and 300 µM Put (an amount close to what we used in whole-cell experiments) were added to the solution facing the intracellular side of patch membranes, Put contributed to blocking channels at positive potentials, and the findings in whole-cell currents were reproduced (Fig. 10). When a combination of 10 µM Spm and 300 µM Put was tested, outward currents were negligible due to Spm block (Fig. 2 C), and the contribution of Put block was insignificant (data not shown), which is also compatible with the competitive access of Spm and Put to IRK1.
Although the internal free Spm levels at 1–10 µM is similar to those reported in mammalian cells (Watanabe et al., 1991), our whole-cell data were obtained after part of the endogenous Spm had been washed out from L cells. However, we speculate such a Spm level might be close to that found in cardiac myocytes, since the outward iK1 currents are usually prominent (e.g., Shimoni et al., 1992). Polyamine levels vary among different cell types since it is related to cell growth (Pegg and McCann, 1982). Thus, it may be necessary to examine the content of polyamines in the cells expressing the inward rectifiers to determine the function of the channel. In the following sections we discuss the effects of Mg2+ block of the inward rectifier K+ channel on the cardiac action potential based on our results, as they were observed at physiological Mgi.
Paradoxical Increase in the Outward-current Amplitude due to Mg2+ Block
Mgi at 0.44-1.1 mM blocked IRK1 in the presence of endogenous Spm, when large depolarizing steps were applied from a hyperpolarized level where most of the channels are in the open state (Fig. 4), same as the observation in iK1 currents (Ishihara et al., 1989). We showed in this paper that the contribution of Mg2+ to blocking IRK1 facilitate the flow of outward currents (Figs. 3–5). This is an interesting phenomenon since endogenous-Spm block can cause a stronger rectification of currents when there is less contribution of Mg2+ block (Figs. 1 and 5). The increase in open probability caused by Mg2+ block might be explained by the finding that iK1 channels can be trapped frequently in partially conducting states by Mg2+ block, which interferes with the long closure of the channel (Matsuda, 1988). The outward-current component with two humps (Fig. 3 B) was also noticed in I-V relations of iK1 in guinea-pig ventricular cells at 0.5–3 mM Mgi (unpublished observation), and this kind of I-V relation is predicted by a kinetic model with partially conducting states (Ishihara et al., 1989; Oliva et al., 1990). We still do not know whether Mg2+ block induces subconductance levels in IRK1 or not, and this point needs to be further clarified. The outward currents increased by Mg2+ block may be important not only for the repolarization phase but also in respect to the influence on the plateau phase of the cardiac action potential. For example, the increase in Ko to 15.4 mM, which shifts I-V relation in a depolarizing direction by about 30 mV, readily increases outward currents during short voltage steps to >0 mV (Fig. 3 B). Outward iK1 currents mediated by Mg2+ block may contribute to shortening the action potential at an elevated Ko (Noble, 1965).
Outward Currents during Repolarization Increased by Relief of Mg2+ Block
We found that transient increase appears in outward IRK1 currents during a repolarizing step pulse in the presence of Mgi (Fig. 7). This current change was attributed to the rapid relief of Mg2+ block, followed by a re-block of channels by Spm. The relatively slow decrease of outward currents infers that the remaining Mg2+ block interfered with the time course of Spm block at the repolarized level, the same as the currents in depolarization (Fig. 3 A). The current change is similar to the rapidly activating delayed rectifier K+ current, iKr, in pace-maker and ventricular cells, as fast recovery from inactivation instantaneously increases outward iKr on repolarization, and then proceeds to time-dependent deactivation (Shibasaki, 1987; Sanguinetti and Jurkiewicz, 1990; Ito and Ono, 1995; Smith et al., 1996). Outward iK1 currents increased by the relief of Mg2+ block (Fig. 11) may contribute to repolarization of the cardiac action potential together with iKr.
The replacement of Mg2+ block by Spm block, which gradually occurs during depolarization (Ishihara et al., 1989, 1996), decreased the flow of outward IRK1 currents during repolarization (Figs. 8 and 11). An increase in the number of Spm-blocked channels at the holding level also decreased the number of Mg2+-blocked channels in depolarization (Fig. 6), which in turn decreased the outward IRK1 during repolarization (Figs. 8 and 11). These results suggest that a prolongation of the action potential or a small depolarization in the resting potential may affect the repolarization phase of the cardiac action potential by reducing the fraction of the Mg2+-blocked inward rectifier K+ channels during the action potential plateau. As repolarization of the cardiac action potential occurs by a small net outward current, the relevance of this phenomenon to the early after-depolarization needs to be further investigated.
Our study suggests that a change in the concentration intracellular polyamines and Mg2+ affects the cardiac action potential by changing iK1 currents (see also Nichols et al., 1996). Therefore, regulating mechanisms of intracellular Mg2+ (Murphy et al., 1991) and polyamines (Pegg and McCann, 1982) may play an important role in cardiac function.
1 Abbreviations used in this paper: Erev, the reversal potential; Ko, extracellular K+ concentration; Mgi, intracellular Mg2+ concentration; PO, the proportion of the channels in the open state; PPut, the proportion of the channels blocked by putrescine; PMg, the proportion of the channels blocked by Mg2+; PSpm, the proportion of the channels blocked by spermine; Put, putrescine; Spd, spermidine; Spm, spermine.
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ACKNOWLEDGMENTS |
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Submitted: 9 May 1996
Accepted: 21 October 1996
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, open cell 3 min; 
, open cell 6 min; 
, 1 µM Spm). (C) Voltage dependence of PSpm with 1 (•) or 10 µM (
) internal Spm. PSpm was obtained at the end of 15-ms voltage steps applied from –40 mV. Mean values from two to three experiments are plotted against the deviation of the membrane potential from Erev. For comparison, PSpm estimated at 8 (
) are different. The time constant of the exponential curves superimposed on the time-dependent inward currents was 1.5 and 1.7 ms in the upper and the lower panel, respectively. (B) Isochronal I-V relations obtained at the end of 20-ms voltage steps applied from –110 and –80 mV at 5.4 mM (•) and 15.4 mM Ko (
PSpm) while extending voltage steps from 20 to 500 ms. Values were obtained by subtracting PSpm in B from that in D at each membrane potential. All negative values (>–0.04) are omitted. The increase in PSpm is designated by curves.
