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MS 0606 Received 25 January 2000; accepted after revision 20 April 2000.
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ABSTRACT |
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INTRODUCTION |
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Inward rectifier K+ channels are important in maintaining stable resting membrane potentials and controlling the excitability of many excitable cells such as neurons and cardiac myocytes (Hille, 1992). The physiological functions of these channels are closely related to their unique property of inward rectification which allows much more current to flow inward than outward through the channels. The mechanisms underlying inward rectification have been ascribed to the Vm-dependent block of outward currents by intracellular Mg2+ and polyamines (Matsuda et al. 1987; Vandenberg, 1987; Ficker et al. 1994; Lopatin et al. 1994) and to a gating process dependent on internal pH (Shieh et al. 1996).
In addition to inhibition of currents upon depolarization (inward rectification), time-dependent decay of currents upon hyperpolarization has also been demonstrated in native inward rectifier K+ channels from several preparations. The decay of inward currents through native channels has usually been explained by depletion of K+ ions (Adrian & Freygang, 1962; Adrian et al. 1970; Almers, 1972; Maughan, 1976). However, experiments using voltage-clamp techniques have indicated that decreases in K+ permeability could also contribute to the time-dependent decay (Adrian et al. 1970; Almers, 1972; Kameyama et al. 1983; Sakmann & Trube, 1984; Kurachi, 1985). Decay of currents during hyperpolarization has also been attributed to a block by external cations (Ohmori, 1978; Standen & Stanfield, 1979). In the cloned Kir2.1 channels (Kubo et al. 1993) expressed in Xenopus laevis oocytes, inward currents have also been shown to decline over time in whole-cell and cell-attached recordings (Reuveny et al. 1996; Shieh et al. 1998). Although Rb+ has been demonstrated to induce time-dependent decay of currents through the cloned Kir2.1 channels on hyperpolarization (Reuveny et al. 1996), little is known about the hyperpolarization-induced decay of currents with K+ ions as the only external cations.
In the present study the decay of currents through the Kir2.1 channels was examined during hyperpolarization using the giant patch-clamp technique. The decay of inward currents is attributed to inactivation of the channels and is dependent on external [K+] and membrane potential. Using site-directed mutagenesis, it is demonstrated that this inactivation could be prevented by external K+ ions binding to sites affected by the positive charges of the amino acids located at positions 148. Furthermore, internal Mg2+ and polyamines induce time-dependent inhibition of currents upon hyperpolarization. These new findings present novel mechanisms underlying the decay of inward currents through the Kir2.1 channels during hyperpolarization.
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METHODS |
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Molecular biology and preparation of Xenopus oocytes
Site-directed mutations were generated using 'pAltered sites II: in vitro mutagenesis systems' (Promega, Madison, WI, USA). The correctness of site-directed mutagenesis was confirmed using the ABI Prism dRhodamine terminator cycle sequencing ready reaction kit (PE Applied Biosystems, Foster City, CA, USA). RNA was synthesized with in vitro T7 (for wild-type DNA) and Sp6 (for mutant DNA) transcription reactions (mMessage mMachine, Ambion, Dallas, TX, USA).
Xenopus laevis oocytes were prepared as previously described (Shieh et al. 1998). In brief, Xenopus laevis oocytes were isolated by partial ovariectomy from frogs anaesthetized by immersion in 0·1 % tricaine (3-aminobenzoic acid ethyl ester). The incision was sutured and the animal was monitored during the recovery period before being placed back in its tank. No further collections were made for at least 3 months. Following the last collection frogs were anaesthetized as above and killed by decapitation. The surgical and anaesthetic procedures were approved by the institutional animal use committee. The day after isolation, Xenopus laevis oocytes were pressure injected with 0·1-1 ng wild-type, R148Y, D172N or E224G cRNA. Oocytes were maintained at 18°C in Barth's solution containing (mM): NaCl 88, KCl 1, NaHCO3 2·4, Ca(N2O6) 0·3, CaCl2 0·41, MgSO4 0·82, Hepes 15 and 20 µg ml-1 gentamicin, pH 7·6; and used 1-3 days after RNA injection.
Electrophysiology techniques
Currents were recorded at room temperature (21-24°C) using the giant patch-clamp technique (Hilgemann, 1995) with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA, USA). The resistance of the electrode pipette ranged from 0·15 to 0·25 M
when filled with the electrode solution. The electrode solutions contained (mM): KCl-KOH 1-100, EDTA 5 and Hepes 5, pH 7·4. The internal solutions contained (mM): KCl-KOH 1-100, EDTA 5, K2ATP 2 and Hepes 5, pH 7·2. In order to maintain the ionic strength of solutions, 100 mM NMGCl was added to solutions containing [K+] < 100 mM. Addition of 100 mM NMGCl to either the external or internal solution containing 100 mM [K+] did not produce any quantitative change on the inactivation of inward currents (data not shown). Free [Mg2+], [Ca2+] and [Ba2+] in the 100 mM [K+] internal solution was calculated using the MaxC program (Chris Patton, Stanford University, CA, USA) and stability constants previously reported by Martell & Smith (1974).
The rundown of channel activity was delayed by treating inside-out patches with 25 µM L-
-phosphatidylinositol-4,5-bisphosphate (PIP2, Sigma Chemical Co., St Louis, MO, USA) for 20-60 s (Huang et al. 1998; Shieh et al. 1998). The degree of decay of inward currents was not significantly different between patches treated with and without PIP2 (data not shown).
The command voltage pulses and data acquisition functions were processed using a Pentium-100 computer, a DigiData board and pClamp6 software (Axon Instruments, Foster City, CA, USA). Data sampling rates were 1-2 kHz and pulse stimulation rate was 0·25 Hz. Data were filtered at 1 kHz with an eight-pole low-pass filter (Frequency Devices, Rochester, NY, USA).
Data analysis
Instantaneous current (Iinst) was measured at 2 ms after pulse changes or by placing a cursor at the point of full activation of a current trace when activation was slow. Steady-state current (Iss) was measured at the end of the test pulse. Results are presented as means ± S.E.M.
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RESULTS |
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Decay of inward currents through Kir2.1 channels
Figure 1A shows the voltage protocol used to measure the steady-state decay of the Kir2.1 channels. Figure 1B demonstrates currents recorded in two different cell-attached patches in 3 mM and 100 mM external [K+], respectively. Inward currents recorded at hyperpolarizing prepulses showed time-dependent decreases. The instantaneous current recorded at the test pulse estimated the fraction of channels that remained open during the prepulse. After a hyperpolarizing prepulse, instantaneous current became smaller, and after a depolarizing prepulse, larger. The decay of currents was more prominent in the presence of lower external [K+]. Figure 1C illustrates currents recorded from two inside-out patches exposed to symmetrical 3 and 100 mM [K+]. Currents still showed time-dependent decay at hyperpolarizing prepulses in symmetrical 3 mM [K+]. However, the decay was completely removed in the inside-out patch exposed to symmetrical 100 mM [K+]. Two major findings are disclosed from this set of experiments. First, hyperpolarization-induced decay of currents through the Kir2.1 channels depends on the bulk [K+]. Throughout this paper, this [K+]-dependent decay is referred to as inactivation. When [K+] is higher, the inactivation is less prominent. Second, exposure of inside-out patches to 100 mM internal [K+] solution free of Mg2+ and polyamines alleviates the decay of inward currents. As will be shown later, this effect is due to inhibition of inward currents by internal Mg2+ and polyamines.
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A, voltage protocol used to record steady-state inactivation of currents through the Kir2.1 channels. The holding potential was 0 mV, prepulses ranged from -200 to +100 mV, and test voltage was -120 or -140 mV. B, current traces obtained from two different cell-attached (C-A) patches exposed to 3 and 100 mM external [K+] as indicated. C, current traces obtained from two inside-out patches exposed to symmetrical 3 and 100 mM [K+] as indicated. The horizontal lines indicate the zero current level throughout this study. | ||
Effects of symmetrical variation of [K+] on inactivation of inward currents
In Fig. 1 K+ ions were demonstrated to have a regulatory effect on the hyperpolarization-induced inactivation. The effects of K+ ions were next examined by quantifying the degrees of inactivation at various symmetrical K+ concentrations. The degree of inactivation during a prepulse was quantified by normalizing the instantaneous current recorded at the test voltage to the instantaneous current with a prepulse of 0 mV (normalized I). The larger the degree of the inactivation is, the smaller is the normalized I. Figure 2A shows the normalized I-prepulse Vm relationships recorded from inside-out patches exposed to various K+ concentrations. The degrees of inactivation were larger at more negative potentials and in reduced [K+]. Figure 2B illustrates the dose-response effects of K+ ions on inactivation. K+ ions showed a protective effect against inactivation but the Kd for this effect did not seem to depend on Vm in the range -200 to -100 mV (see Fig. 2 legend). The Hill coefficients (nH) were close to 1 indicating that only one K+ binding site is involved in the K+ protective effect. The estimated maximum degrees of inactivation (i.e. extrapolated value obtained at [K+] = 0 mM, see Fig. 2 legend) increased as the prepulses became more negative. These results suggest that the inactivation is Vm dependent and the occupancy of a K+ ion in the Kir2.1 channel prevents the channel from inactivation at hyperpolarization. The K+ binding site is probably not located in the electrical field as the effects of K+ ions were not Vm dependent.
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A, normalized I-prepulse Vm relationships. Symbols are [K+] = 100 mM ( | ||
Inactivation of inward currents is regulated by external K+ ions
Because the results in Fig. 2 were obtained in symmetrical [K+], it is desirable to further examine whether it is the external K+ or internal K+ ions, or both, that are crucial for inactivation. Figure 3A and B shows the normalized I-prepulse Vm relationships and the dose-response effect of external K+ ions on the inactivation at various prepulse Vm values in the presence of 100 mM internal [K+]. Similar to the effect of symmetrical [K+], the Kd values for the protective effect of external [K+] were not sensitive to Vm and the two sets of Kd values were similar (
5 mM). These results suggest that the K+ binding site involved in protection of the channel from inactivation is probably located externally. The calculated maximum degree of inactivation also increased as the prepulses became more negative. However, the values were less than those obtained in symmetrical [K+] indicating that internal K+ ions may also have a regulatory effect on the inactivation of the Kir2.1 channels.
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A, the normalized I-prepulse Vm relationships. Symbols are [K+] = 100 mM ( | ||
To further explore the properties of the external K+ and Vm-dependent inactivation of inward currents, the kinetics of the inactivation was analysed. Inward currents recorded in the presence of 100 mM internal [K+] free of Mg2+ and polyamines were well fitted by double exponential functions. Figure 4A demonstrates that both the fast and slow time constants showed little external K+ and Vm dependence. The percentage of channels remaining in the fast inactivation component was dependent on both Vm and external [K+] (Fig. 4B) whereas the percentage of channels remaining in the slow inactivation component (Fig. 4C) was not.
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A, the fast (closed symbols) and slow (open symbols) time constants ( | ||
Internal K+ ions also have a mildly protective effect on inactivation
Figure 5A shows the normalized I-prepulse Vm relationships recorded from inside-out patches exposed to various internal K+ concentrations in the presence of 3 and 100 mM external [K+]. In the presence of 100 mM external [K+], no inactivation was observed even when internal [K+] was reduced to 3 mM. These results indicate that once the external K+ binding site is saturated and the hyperpolarization-induced inactivation is thus prevented, decreases of internal [K+] are not sufficient to induce inactivation. However, when external [K+] was kept at 3 mM so that inactivation of inward currents was allowed to occur, increases of internal [K+] also protected the channels against inactivation. For example, an increase of internal [K+] from 3 mM to 100 mM at a prepulse Vm of -200 mV enhanced the normalized I from 0·4 to 0·6 (Fig. 5A). These results suggest that internal K+ ions also regulate the inactivation of inward currents but they are not as effective as external K+ ions.
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A, normalized I-prepulse Vm relationships in various external and internal [K+] combinations. Open symbols are 3 mM external [K+] with internal [K+] = 100 mM ( | ||
Unlike several other known properties of the inward rectifier K+ channels such as inward rectification (Hille, 1992) and the activation of inward currents (Kubo, 1996), the inactivation of inward currents through the Kir2.1 channels does not seem to depend on the driving force for K+ ions. Figure 5B illustrates the normalized I-driving force relationships of four different external and internal [K+] combinations. At the same driving force, the degrees of inactivation of Kir2.1 channels exposed to the four different [K+] combinations were not superimposed.
K+ binding sites in the external pore region are crucial for the external K+-dependent inactivation
The protective effect of external K+ ions on inactivation is not Vm dependent suggesting that external K+ ions may interact with the Kir2.1 channel at the external region and the interaction obstructs the inactivation process. In other words, external K+ ions act as stabilizing ligands such that the K+ occupied channels are more likely to stay open than are the unoccupied channels. Recently, we have identified a set of two K+ binding sites in the external pore mouth of the Kir2.1 channel and shown that interaction of K+ ions around this region decreases K+ binding affinity and thus enhances K+ transportation rate (Shieh et al. 1999). When the positively charged amino acids at positions 148 are neutralized, K+ binding affinity increases. It was therefore hypothesized that these K+ binding sites may be involved in the external K+-dependent inactivation process and that replacement of the positively charged amino acids located at positions 148 by neutral amino acids may thus enhance the protective effect of external K+ ions against inactivation. To test this hypothesis, the inactivation property of the R148Y mutants (arginine at position 148 is replaced by tyrosine) was examined.
Figure 6A shows that in the cell-attached patch no inactivation was observed in the R148Y mutants in 100 mM external [K+] and only slight inactivation was observed in 3 mM external [K+] at hyperpolarization. These results are different from those observed in the wild-type channels (Fig. 1B). In inside-out patches exposed to 3 mM symmetrical [K+] (Fig. 6B), the inactivation was still much less than that in the wild-type channels (cf. Fig. 1C). The effects of K+ ions on the hyperpolarization-induced inactivation in the wild-type channels and R148Y mutants were compared (Fig. 6C). These results indicate that when the external K+ binding affinity is higher, the degree of inactivation is smaller and thus are consistent with the idea that the K+ binding sites at the external pore mouth are involved in the effect of external K+ ions on inactivation.
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A, current traces obtained from two different R148Y cell-attached patches exposed to 3 and 100 mM external [K+]. B, current traces from two inside-out patches exposed to symmetrical 3 and 100 mM [K+] as indicated. C, the normalized I-prepulse Vm relationships for the wild-type channels ( | ||
Internal Mg2+ and polyamines induce time-dependent decay of inward currents
Figure 1 shows that in the presence of 100 mM external [K+], hyperpolarization induced time-dependent decay of currents in a cell-attached patch and the decay disappeared after patch excision (inside out) into the 100 mM internal [K+] solution. Since the internal [K+] is also about 100 mM in cell-attached patches, the phenomenon cannot be explained as a relief of the decay by perfusion of 100 mM [K+] alone. Because internal Mg2+ and polyamines are known to block Kir2.1 channels their possible involvement in the time-dependent decay was explored. Since no [K+]-dependent inactivation occurs at a Vm of 0 mV, the Vm protocol was simplified and inward currents were recorded at Vm values of -150 to -10 mV from a holding potential of 0 mV (Fig. 7). Addition of internal Mg2+ restored the time-dependent decay observed in cell-attached patches in symmetrical 100 mM [K+] and reduced the instantaneous amplitude of inward currents in a dose-dependent manner (Fig. 7B -E). Both effects were reversible upon washout (Fig. 7F).
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A, inward currents recorded in a cell-attached patch. B-F, inward currents recorded in the same patch shown in A in an inside-out configuration perfused with the 100 mM [K+] internal solution containing the indicated free [Mg2+] and after washout. | ||
In the presence of symmetrical 100 mM [K+], spermine (+4 charges) and spermidine (+3 charges) also showed similar effects. Figure 8A-C summarizes the ratio of Iss over Iinst (Iss/Iinst)-Vm relationships obtained in the presence of different concentrations of Mg2+, spermine and spermidine, respectively. The maximum Iss/Iinst was about 10 % for Mg2+, 15 % for spermine, and 30 % for spermidine. Figure 8D shows that the Kd for the decay induced by internal Mg2+, spermine and spermidine did not seem to show Vm dependence at Vm ranging from -150 to -60 mV. Along with producing a larger degree of decay of inward currents, spermidine seemed to have higher affinity for the Kir2.1 channels. The order of efficiency in generating the decay of inward currents was spermidine > spermine 
Mg2+. On the other hand, the order of potency for inducing inward rectification was spermine > spermidine > Mg2+ (data not shown) (Lopatin et al. 1994; Yang et al. 1995). These results suggest that the effects of internal Mg2+ and polyamines on the decay of inward currents are distinct from their inward rectification.
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The Iss/Iinst-Vm relationships in the presence of various concentrations of Mg2+ (A, n = 5), spermine (B, n = 5), and spermidine (C, n = 4). Data obtained from cell-attached patches are shown as | ||
Figure 8E shows the time constants of the current decay at different Vm values in cell-attached and inside-out patches exposed to spermidine ranging from 0·1 to 1 mM. Unlike the external K+- and Vm-dependent inactivation, the decay of inward currents was well fitted with single exponential functions. The time constants were not Vm dependent nor were they dependent on the concentration of internal spermidine. The time constants obtained in cell-attached patches were similar to those obtained in inside-out patches exposed to spermidine.
To further determine whether the decay of inward currents was due to specific interaction of internal Mg2+ and polyamines with the Kir2.1 channels, the effects of internal Ca2+ and Ba2+ on inward currents were examined. Internal free [Ca2+] (1 mM) or [Ba2+] (1 mM) did not induce any decay of inward currents (data not shown).
Different effects of internal Mg2+ on decay of inward currents through D172N and E224G mutant channels
It has been shown that internal Mg2+ and polyamines induce inward rectification with reduced efficiency in D172N and E224G mutants (Wible et al. 1994; Lopatin et al. 1994; Yang et al. 1995). The order of inward rectification is wild-type channel > D172N mutant > E224G mutant. To gain more insight into the molecular mechanism by which internal Mg2+ and polyamines induced the decay of inward currents, the inward currents through the D172N and E224G mutants were studied. Figure 9 demonstrates that, unlike inward currents through the wild-type channels, those through the D172N mutants showed little decay (Fig. 9A), whereas those through the E224G mutants decayed over time (Fig. 9B). Figure 9C and D summarizes the degrees of decay of inward currents through the D172N and E224G mutants under various experimental conditions. Inward currents of the E224G mutants decayed by about 10 % in the cell-attached patches and inside-out patches perfused with 1 mM free [Mg2+], whereas inward currents of the D172N mutants did not show any decay under the same experimental conditions. Thus, the order of Mg2+-induced decay of inward currents is wild-type channel E224G mutant > D172N mutant.
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Inward D172N (A) and E225G (B) currents recorded in a cell-attached patch (upper panel) and the same patch in the inside-out configuration perfused with 100 mM internal [K+] solutions containing the indicated [Mg2+] (middle and lower panels). Iss/Iinst-Vm relationships for D172N (C) and E224G (D) mutants. Data are from cell-attached patches ( | ||
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DISCUSSION |
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Inactivation of the Kir2.1 channels upon hyperpolarization
The decay of inward currents through the inward rectifier K+ channels has been attributed to the depletion in K+ near the channels (Adrian & Freygang, 1962; Adrian et al. 1970; Almers, 1972; Maughan, 1976), a decrease of K+ permeability (Adrian et al. 1970; Almers, 1972; Kameyama et al. 1983; Sakmann & Trube, 1984; Kurachi, 1985) and inhibition of inward currents by external cations such as Na+, Rb+, Mg2+ and Ca2+ (Ohmori, 1978; Standen & Stanfield, 1979; Reuveny et al. 1996). The external K+- and Vm-dependent decay of inward currents described in this study is consistent with decreases of the time-dependent K+ permeability (inactivation). Because the binding of a K+ ion to the regulatory site does not depend on Vm the Vm dependence of the inactivation is probably not due to Vm-dependent K+ binding. Hyperpolarization increases the percentage of channels in the fast inactivation component without affecting the time constants suggesting that stronger hyperpolarization forces the channels into a more stable inactivated state.
It is unlikely that the K+- and Vm-dependent inactivation described in this study is due to the depletion of external K+ ions because no change of reversal potential occurs (data not shown). The inactivation is not due to inhibition of Kir2.1 channels by external divalent cations such as Mg2+, Ba2+ and Ca2+ because experiments were carried out in the presence of 5 mM internal and external EDTA. It is also unlikely that the inactivation is due to contamination of external monovalent cations in the solutions. The time constants for the monovalent cation block of Kir2.1 channels have been demonstrated to be steeply Vm dependent (Kubo et al. 1993) yet the time constants for the inactivation described in this study show little external K+ and Vm dependence (Fig. 4A).
Is it possible that the external K+- and Vm-dependent inactivation is due to block by residual Mg2+ and polyamines? It has been previously suggested that internal Mg2+ and polyamines may not be completely washed out in inside-out patches. The greater degree of inactivation in the presence of reduced external [K+] thus may be due to the enhanced block of inward currents by these cations. However, our data are not consistent with this idea for the following reasons. First of all, the inactivation is Vm dependent whereas the internal Mg2+- and polyamine-induced block is not at the same Vm range tested. Second, even if it is further argued that the internal Mg2+- and polyamine-induced block may become Vm dependent in low external [K+], it is difficult to envisage why the block induced by positive internal cations should be more severe at more negative Vm. Third, the time constants of the inactivation are different from those of internal Mg2+- and polyamine block. Also, the time constants for the internal cation block are not dose dependent (Fig. 8E).
Regulation of Vm-dependent inactivation by K+ ions
Increases in external [K+] decrease the degree of inactivation of inward currents with Kd 5 mM. The protective effect of external K+ ions against channel inactivation is reminiscent of the C-type inactivation of the voltage-gated K+ channels. It has been shown that external permeant ions and blockers can relieve the C-type inactivation (Grissmer & Cahalan, 1989; Hoshi et al. 1991; Lopez-Barneo et al. 1993; Baukrowitz & Yellen, 1995). One possible mechanism to explain the protective effect of K+ ions is the foot-in-the-door model of gating (Swenson & Armstrong, 1981) in which a channel cannot close when occupied by a permeant or blocking ion. This model postulates that a permeant ion (K+ ion in this case) or a blocker binds within a region of the channel pore that becomes obstructed upon a voltage change, thereby preventing inactivation. The protective binding region for K+ ions in Kir2.1 channels is affected by mutation at position 148 in the pore region. This finding is also similar to the C-type inactivation which is influenced by mutations in the pore and S6 regions (Iverson & Rudy, 1990; Hoshi et al. 1991; Lopez-Barneo et al. 1993; Olcese et al. 1997; Yang et al. 1997). Since K+ channels from various families comprise a pore region and two transmembrane domains flanking the pore region, it will be interesting to determine whether slow inactivation is a common feature to all K+ channels baring this architecture.
It has previously been demonstrated that K+ influx following K+ binding to the channel is also involved in the K+ effect for C-type inactivation (Baukrowitz & Yellen, 1995, 1996). However, our results are not consistent with this mechanism for the following reasons. First, the K+-regulated inactivation process is not dependent on the driving force for K+ ions (Fig. 5B). Second, we have previously demonstrated that the K+-K+ interaction in the external pore mouth of a Kir2.1 channel is crucial for a high K+ influx rate (Shieh et al. 1999). When the positively charged amino acids at positions 148 are neutralized this K+-K+ interaction is greatly reduced even in the presence of high external [K+] (
100 mM) and thereby decreasing K+ influx. Therefore, if an increase of K+ influx associated with high external [K+] indeed played a role in the protective effect on inactivation, a greater degree of inactivation would have been observed in the R148Y mutants than the wild-type channels. These results suggest that the binding of K+ ions in the external pore mouth rather than the K+ movements following the binding is directly involved in the regulation of inactivation.
The external K+-dependent decay of inward currents have also been demonstrated in other subtypes of inward rectifier K+ channels such as RBHIK1 (Ishii et al. 1994), RB-IRK2 (Morishige et al. 1994), HIR (Perier et al. 1994), and BIK (Forsyth et al. 1997). All these channels have an arginine at the position equivalent to position 148 in Kir2.1 channels. Furthermore, inward currents through the Kir7.1 channels (with a methionine instead of an arginine at the equivalent position) do not decay in the presence of external [K+] ranging from 2 to 96 mM (Doring et al. 1998). However, when the methionine is replaced by an arginine external [K+]-dependent decay of inward currents is evident. These results suggest that the highly conserved arginine may play an important role for the external K+-dependent decay of inward currents in the inward rectifier K+ channel family.
Internal Mg2+ and polyamines induce decay of inward currents
The decay of inward currents induced by internal Mg2+ and polyamines is most probably due to specific interaction of these cations with the Kir2.1 channels. A non-specific effect such as surface-charge screening cannot explain why internal Ca2+ and Ba2+, both having the same positive charge as Mg2+, do not generate similar degrees of decay of inward currents. Also, a surface-charge screening effect is not consistent with the finding that spermidine (+3) is more effective than spermine (+4) in inducing decay of inward currents (Fig. 8D).
It is puzzling why the decay induced by internal Mg2+ and polyamines is Vm dependent (it is only observed at strong hyperpolarization) yet it does not show much Vm dependence in the Vm range -150 to -60 mV. One possibility is that the Mg2+- and polyamine-induced decay of currents is not intrinsically Vm dependent. The lack of effect of Mg2+ and polyamines at less hyperpolarizing voltages may be due to interaction of the decaying and inward rectifying processes.
Mg2+- and polyamine-induced decay and inward rectification can be differentiated
The evidence that distinguishes the Mg2+- and polyamine-induced decay of inward currents from inward rectification is as follows. First, the order of potency in producing decay of inward currents is spermidine > spermine Mg2+ (Fig. 8D) whereas the order of efficiency for inducing inward rectification is spermine > spermidine > Mg2+. Second, the order of inward rectification is wild-type channel > D172N mutant > E224G mutant (Yang et al. 1995). On the other hand, the order of the decay of inward currents is wild-type E224G > D172N. These results strongly suggest that the two effects of internal Mg2+ and polyamines on Kir2.1 channels involve different binding sites. The physiological significance of the Mg2+- and polyamine-induced decay of the Kir2.1 channels is unknown. However, it is possible that the corresponding low affinity binding site located in the internal part of the channel serves as a reservoir (Lee et al. 1999) for 'catching' Mg2+ and polyamines dissociating from their high affinity sites (responsible for inward rectification).
Conclusions
The major findings in this study are: (1) Kir2.1 channels inactivate at hyperpolarization, (2) external K+ ions protect the channels against the inactivation, (3) neutralization of the positively charged amino acids at positions 148 enhances the protective effect of external K+ ions against the inactivation, (4) internal Mg2+ and polyamines induce decay of inward currents in the presence of 100 mM symmetrical [K+] and (5) the effects of Mg2+ and polyamines on the decay can be differentiated from their inward rectifying effects. Although it remains controversial whether the inward rectifier K+ channels are voltage-gated channels, data from our previous study (Shieh et al. 1996) and the present study provide supportive evidence that Vm-dependent processes may be involved in the 'gating' for the inactivation of the Kir2.1 channels. Despite that the regulations of the inactivation have been characterized to some details, the inactivation processes per se are still not clear. Efforts are required to further determine whether a physical gate or protein conformational change is involved and whether this inactivation interacts with the inactivation gate dependent on internal pH.
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REFERENCES |
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I thank Dr Tsung-Yu Chen for critical reading of the manuscript and helpful comments and Drs Jorge Arreola and Lung-Sen Kao for reading part of the manuscript and discussions. This study was supported by Academia Sinica and National Science Council grants (88-2314-B-001-042 and 89-2320-B-001-025) in Taiwan, Republic of China.
Correspondence
R.-C. Shieh: Institute of Biomedical Sciences, Academia Sinica, 128 Yen-Chiu Yuan Road, section 2, Taipei 11529, Taiwan, Republic of China.
Email: ruchi{at}novell.ibms.sinica.edu.tw
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, n = 5), 30 mM (
, n = 3), 15 mM (
, n = 3), 10 mM (
, n = 7), 3 mM (
, n = 5) and 1 mM (
, n = 4). B, dose-response effects of [K+] on normalized I. Symbols are -200 mV (
f and 
), and 3 mM (
). D, Kd-Vm relationships for Mg2+ (