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Journal of Physiology (2001), 535.2, pp. 359-370
© Copyright 2001 The Physiological Society

Ammonium ions induce inactivation of Kir2.1 potassium channels expressed in Xenopus oocytes

R.-C. Shieh and Y.-L. Lee

	ABSTRACT

Top Abstract Introduction Methods Results Discussion References

1. The decay of inward currents was studied using the giant patch-clamp technique and a cloned inward rectifier K⁺ channel, Kir2.1, expressed in Xenopus oocytes.
2. In inside-out patches, inward currents carried by NH₄⁺ or Tl⁺ decayed over time. When the voltage was more negative, the degree and rate of decay were greater. The rate of NH₄⁺-induced decay saturated at a symmetrical [NH₄⁺] of ~100 mM. The decay rate was slow (2.6 10³ M^-1 s^-1) at -140 mV with 10 mM [NH₄⁺].
3. Upon a 10 °C increase in temperature, the single-channel NH₄⁺ current amplitude increased by a factor of 1.57, whereas the NH₄⁺-induced decay rate increased by a factor of 2.76. In the R148Y Kir2.1 mutant (tyrosine 148 is at the external pore mouth), NH₄⁺-induced inactivation was no longer observed.
4. NH₄⁺ single-channel currents revealed one open and one closed state. The entry rate into the closed state was voltage dependent whereas the exit rate from the closed state was not. An increase of internal [NH₄⁺] not only decreased the entry rate into but also elevated the exit rate from the closed state, consistent with the occupancy model modified from the foot-in-the-door model of gating.
5. These results suggest that the decay of NH₄⁺ current is unlikely to be due to a simple bimolecular reaction leading to channel block. We propose that NH₄⁺ binding to Kir2.1 channels induces a conformational change followed by channel closure.
6. The decay induced by permeant ions other than K⁺ may serve as a secondary selectivity filter, such that K⁺ is the preferred permeant ion for Kir2.1 channels.

	INTRODUCTION

Top Abstract Introduction Methods Results Discussion References

Gating mechanisms in inward rectifier K⁺ channels are usually mediated by permeant or blocking ions, whereas voltage-dependent ion channels are gated directly by membrane voltage (V_m). For example, the inward rectification of inward rectifier K⁺ channels is attributed to the V_m-dependent block of outward currents by internal Mg²⁺ (Matsuda et al. 1987; Vandenberg, 1987) or polyamines (Ficker et al. 1994; Lopatin et al. 1994). Furthermore, gating of inward rectifier K⁺ channels is influenced by the species of permeant ion, as illustrated by studies using Tl⁺ (Ashcroft & Stanfield, 1983) and Rb⁺ (Reuveny et al. 1996) in which ion currents were inactivated in an exponential manner during hyperpolarization. In addition, the kinetics of Tl⁺ and K⁺ single-channel currents are different in cloned ROMK2 channels (Chepilko et al. 1995), where it has been suggested that K⁺ ions bring about a brief closed state that exhibits a biphasic V_m dependence (Choe et al. 1998). These previous studies suggest two mechanisms that may underlie the inactivation of inward rectifier K⁺ channels. One states that channels are blocked by permeant ions (permeable block hypothesis) while the other states that the binding of permeant ions induces a conformational change in the channel. Using the cloned inward rectifier K⁺ channel Kir2.1 (Kubo et al. 1993), we recently demonstrated the inactivation of inward currents, due presumably to a V_m-dependent conformational change in Kir2.1. We also showed that external K⁺ can protect Kir2.1 channels from such inactivation (Shieh, 2000). This work provided another line of evidence that V_m-dependent processes are involved in gating inward rectifier K⁺ channels.

The protective effect of external K⁺ on hyperpolarization-induced inactivation is reminiscent of C-type inactivation in voltage-gated K⁺ channels. It has been shown that external permeant ions and blockers slow C-type inactivation (Hoshi et al. 1991; Demo & Yellen, 1992) and that the binding site for slowing C-type inactivation is selective for K⁺ ions (Lopez-Barneo et al. 1993). However, it remains unclear whether such selectivity exists for the binding site involved in the prevention of hyperpolarization-induced inactivation in Kir2.1 channels.

In this study, we tested whether the two permeant ions NH₄⁺ and Tl⁺ could protect Kir2.1 channels from hyperpolarization-induced inactivation. Our results show that there are apparently two types of inactivation in Kir2.1 channels in the presence of NH₄⁺ or Tl⁺. One is similar to the K⁺-protected inactivation in that increases in [NH₄⁺] or [Tl⁺] protect Kir2.1 channels from inactivation (permeant ion-protected inactivation). The other type of inactivation is enhanced as the concentration of NH₄⁺ or Tl⁺ is increased, and it dominates at [NH₄⁺] or [Tl⁺] 10 mM (such that permeant ion-protected inactivation is no longer observed). The on-rate for NH₄⁺-induced inactivation is very slow compared to the effective rate coefficient for ion conduction. Furthermore, a strong hyperpolarizing V_m does not affect the exit rate during inactivation. These results are inconsistent with a model in which NH₄⁺ acts as a permeable blocker. We therefore propose that NH₄⁺ binding to Kir2.1 channels induces a conformational change that promotes channel closure. Inactivation of inward currents through inward rectifier K⁺ channels may lead to membrane hyperpolarization, thereby influencing cell excitability. Our study provides new insights into Kir2.1 channel gating as manifested by direct interactions between NH₄⁺ and Kir2.1.

	METHODS

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Molecular biology and preparation of Xenopus oocytes

Purification of mouse macrophage Kir2.1 DNA and in vitro T7 transcription reactions (mMessage mMachine, Ambion, Dallas, TX, USA) were performed as previously described (Shieh et al. 1998). Xenopus oocytes were isolated by partial ovariectomy from frogs anaesthetized with 0.1 % tricaine (3-aminobenzoic acid ethyl ester). Following the last oocyte collection, frogs were anaesthetized as above and killed by decapitation. All surgical and anaesthetic procedures followed the institutional guidelines for animal use. Oocytes were pressure injected with RNA 24 h after defolliculation and used 1-3 days after RNA injection. Oocytes were maintained at 18 °C in Barth's solution containing (mM): NaCl 88, KCl 1, NaHCO₃ 2.4, Ca(NO₃)₂ 0.3, CaCl₂ 0.41, MgSO₄ 0.82 and Hepes 15, with gentamicin (20 µg ml^-1) at pH 7.6.

Electrophysiological techniques

Currents were recorded at room temperature (21-24 °C) unless otherwise noted, using the giant and single-channel patch clamp techniques (Hamill et al. 1981; 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 for giant patch recordings, and from 1 to 3 M for single-channel recordings when filled with the electrode solutions. For K⁺ current recordings, the electrode (external) and internal solutions contained (mM): KCl 3-100, N-methyl-D-glucamine (NMG) 0-100, EDTA 5 and Hepes 5, at pH 7.4 (titrated with HCl). For NH₄⁺ current recordings, the external solution contained (mM): NH₄Cl 3-100, NMG 0-100, EDTA 5 and Hepes 5, at pH 7.4 (titrated with HCl). The internal solution contained (mM): NH₄Cl 0-500, NMG 0-100, EDTA 5 and Hepes 5, at pH 7.4 (titrated with HCl). For Tl⁺ current recordings, the external and internal solutions contained (mM): TlNO₃ 3-100, NMG 0-100, EDTA 5 and Hepes 5, at pH 7.4 (titrated with HNO₃). For all experiments, the bath grounding was achieved by connecting the bath and grounding Ag-AgCl pellet immersed in 3 M KCl with a U-shape salt bridge filled with KCl (3 M)-agar (3 %) mixture. To stabilize the electrode potential in solutions containing TlNO₃, the Ag-AgCl wire in the pipette was inserted into a piece of tubing filled with KCl (100 mM)-agar (3 %) mixture. The electrode potential was stable as the drift in zero-current potential was < 1 mV in 10 min. No visible TlCl precipitation was observed in the electrode pipettes during experiments. Rundown of channel activity was delayed by treating inside-out patches with L--phosphatidylinositol-4,5-bisphosphate (Sigma Chemical Co., St Louis, MO, USA) (Huang et al. 1998; Shieh et al. 1998).

The command V_m and data acquisition functions were processed using a Pentium computer, a DigiData board and pCLAMP 6 software (Axon Instruments). For K⁺ and NH₄⁺ currents, data sampling rates were 2.5-5 kHz and the data were filtered at 0.5-1 kHz with an 8-pole low-pass filter (Frequency Devices, Rochester, NY, USA). For Tl⁺ currents, data sampling rates were 25 kHz and the data were filtered at 5 kHz. Because single-channel patches did not survive well at V_m values as negative as -200 mV, most of the single-channel currents were recorded using step voltages from a holding potential of 0 mV. The event recorded immediately after the voltage change from 0 to -140 mV as well as the event right before the voltage change from -140 to 0 mV were not included in the single-channel analysis. Because the single-channel openings are uncorrelated (there is only one open state), all the open and closed events obtained in voltage steps have exactly the same distributions as in the steady state (Colquhoun & Hawkes, 1995). Furthermore, the distributions of open and closed times obtained from a continuous holding potential of -140 mV were not different from those obtained using the voltage step from 0 to -140 mV (data not shown).

Capacitive currents were corrected using the built-in capacitance neutralization in the Axopatch 200A amplifier. The residual uncorrected capacitive currents at 2 ms following voltage changes were much smaller than the Kir2.1 currents. In some experiments, the capacitive and leak currents were corrected by subtracting the currents recorded after complete channel rundown from those measured during channel activity. The results using these two methods were the same (data not shown).

Data analysis

Instantaneous NH₄⁺ currents were measured 2 ms after the voltage steps to test V_m. Histograms of the duration of time that channels remained open and closed were constructed with square root-log ordinates (Sigworth & Sine, 1987). The histograms were fitted to monoexponential functions with the maximum log likelihood method, and, in general, biexponential functions did not provide significantly better fits (P > 0.05) than monoexponential functions, as judged by the maximal likelihood ratio test (Horn & Lange, 1983). Substates were observed, but they did not occur frequently. Transitions between the open state and substate as well as between the closed state and substate were not included in the data analysis. Results are presented as means ± S.E.M.

	RESULTS

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Decay of inward currents in Kir2.1 channels

Figure 1A shows the voltage protocol used to measure the steady-state decay of currents through Kir2.1 channels. Figure 1B demonstrates that inward currents decayed over time under physiological ionic conditions (3 mM external [K⁺] ([K⁺]_o) and 100 mM internal [K⁺] ([K⁺]_i)]. However, no decay was observed when [K⁺]_o was raised to 100 mM. Previously, we referred to the decay of currents during hyperpolarization as 'K⁺-protected inactivation' (Shieh, 2000). To test whether NH₄⁺ or Tl⁺ can substitute for K⁺ in this respect, we evaluated the effect of 100 mM [NH₄⁺] or [Tl⁺] on both sides of the membrane (symmetrical [NH₄⁺] or [Tl⁺]) on currents. Figure 1C shows that both inward NH₄⁺ and Tl⁺ currents decayed over time, and that the decay time course fitted a monoexponential function (which differs from the biexponential time course of K⁺-protected inactivation).

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Figure 1. Inward currents carried by K+, NH4+ or Tl+ through Kir2.1 channels A, voltage protocol used to record the steady-state decay of currents through Kir2.1 channels. The holding potential was 0 mV, prepulses ranged from -200 to +100 mV, and the test Vm was -120 mV. B, current traces obtained from two different inside-out patches exposed to 3 mM [K+]o/100 mM [K+]i and 100 mM symmetrical [K+] as indicated. The continuous horizontal lines indicate zero current levels throughout this study. Iinst, instantaneous current. C, current traces obtained from two different inside-out patches exposed to 100 mM symmetrical [NH4+] or [Tl+] as indicated.

We next examined the decay of inward NH₄⁺ and Tl⁺ currents through Kir2.1 channels with respect to the effect of low symmetrical [NH₄⁺] and [Tl⁺]. Figure 2A shows that with 10 mM symmetrical [NH₄⁺], the inward current at V_m = -140 mV still decayed in a monoexponential manner. However, when [NH₄⁺] was decreased to 3 mM, the decay of the inward current exhibited one fast and one slow component. The kinetics of the fast component were similar to those of the 10 mM NH₄⁺ experiment, although the degree was smaller (the slow component was not observed in the 10 mM experiment). In other words, upon increasing [NH₄⁺] from 3 to 10 mM, the magnitude of the fast kinetic component was increased while the slow component was no longer observed. Similar results were observed with Tl⁺ currents (Fig. 2B). These results suggest that Kir2.1 channels exhibit two types of current decay in the presence of NH₄⁺ and Tl⁺. One is a permeant ion-protected inactivation in which increased [NH₄⁺] or [Tl⁺] protects Kir2.1 channels from inactivation. The other type of decay is enhanced by increased [NH₄⁺] or [Tl⁺], and dominates at [NH₄⁺] and [Tl⁺] 10 mM where permeant ion-protected inactivation is not observed. Since NH₄⁺- and Tl⁺-induced decay of inward currents was qualitatively similar, our subsequent experiments focused exclusively on NH₄⁺-induced decay.

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Figure 2. Current traces in low symmetrical [NH4+] or [Tl+] A, inward currents recorded at -140 mV in 3 mM (continuous line) and 10 mM symmetrical [NH4+] (dotted line). B, inward currents recorded at -140 mV in 3 mM (continuous line) and 10 mM symmetrical [Tl+] (dotted line). Current amplitudes were normalized to 1, and the zero current level was offset for comparison and clarity.

We examined NH₄⁺-induced decay using 10 mM symmetrical [NH₄⁺] and the voltage protocol described in Fig. 1A. Instantaneous current was recorded at the test V_m after a specific prepulse, and the degree of decay was quantified by normalizing this current to that obtained with a prepulse of 0 mV (normalized I). Figure 3A shows that normalized I was smaller at more negative V_m. Changes in [NH₄⁺] did not affect the relationship between normalized I and V_m. Since the time course of decay could be fitted to a monoexponential function, the rate of decay was calculated as the reciprocal of the time constant (). The rate of decay depended on V_m (Fig. 3B). Also, the rate of decay increased as a function of [NH₄⁺], and plateaued at ~100 mM (Fig. 3C). These results further indicate that NH₄⁺ promotes a type of V_m-dependent decay in Kir2.1 channels that is distinguishable from the K⁺-dependent inactivation.

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Figure 3. NH4+-induced decay of inward currents through Kir2.1 channels A, relationship between normalized I and Vm. Symbols indicate mean (± S.E.M.) values for [NH4+]: , 10 mM (n = 3); , 30 mM (n = 4); , 100 mM (n = 5); , 200 mM (n = 7); and , 300 mM (n = 7). B, Vm dependence of 1/ in various symmetrical [NH4+]. Symbols are the same as in A. C, dose dependence of 1/. Symbols indicate Vm: , -200 mV; , -180 mV; , -160 mV; , -140 mV; , -120 mV; and , -100 mV. The dotted line is the best linear fit between 0 and 10 mM [NH4+] at -140 mV. The slope is 2.6 103 M-1 s-1.

In the experiments presented in Fig. 3, the solutions containing 10 and 30 mM [NH₄⁺] also contained 100 mM [NMG]. Therefore, we tested whether NMG had any effect on decay. We recorded currents in 100 mM symmetrical [NH₄⁺] plus [NMG] at 0, 18 and 100 mM, and found that both the normalized I-V_m and the 1/-V_m relationship were independent of [NMG] (data not shown), demonstrating that the decay of inward NH₄⁺ currents was not due to NMG.

It is also possible that the observed decay of currents was due to contaminants in the experimental solutions. Na⁺ and Cs⁺ have been shown to block Kir2.1 channels (Kubo et al. 1993). However, Na⁺ blocks the channel with low affinity, and the kinetics of both Na⁺- and Cs⁺-mediated channel block (Kubo et al. 1993) differ from those shown in Fig. 1C. Also, Fig. 3C demonstrates that the rate of decay becomes saturated at ~100 mM [NH₄⁺], and the rate is slow (2.6 10³ M^-1 s^-1 at -140 mV) at a limiting low concentration compared to the diffusion limit (~10¹⁰ s^-1). These two features are inconsistent with a simple bimolecular reaction leading to channel block (Yellen et al. 1994). To rule out the possibility that the NH₄⁺-induced decay is due to contamination by other ions, we also carried out experiments with NH₄NO₃ and NH₄Cl that were manufactured by different companies. The normalized I-V_m and 1/-V_m relationships were similar regardless of the source of NH₄⁺ (data not shown). These results strongly argue that the decay described in this study was due to NH₄⁺, and not chemical contaminants.

What is the mechanism for the NH₄⁺-induced decay of inward currents? There are two possible direct explanations for the time-dependent decay of channel activity. First, NH₄⁺ acts as a permeable blocker of Kir2.1 channels. Second, NH₄⁺ binds directly to Kir2.1 channels and somehow destabilizes the open state, thereby inducing channel inactivation.

Figure 3C shows that the rate of decay was extremely slow (2.6 10³ M^-1 s^-1 at -140 mV) with 10 mM NH₄⁺ compared to the effective rate coefficient for the overall ion transfer (~10⁸ M^-1 s^-1) derived from the single-channel current (2 pA at -140 mV in 100 mM symmetrical [NH₄⁺]). Thus, it is difficult to envisage that the current decay is due to a block of Kir2.1 channels by NH₄⁺ (with multiple ions occupying the pore in single-file). We therefore postulated that direct NH₄⁺ binding promotes a conformational change in Kir2.1 channels, thereby effecting channel closure (see Discussion). The decay of inward NH₄⁺ currents will be referred to as 'NH₄⁺-induced inactivation'.

Temperature dependence of NH₄⁺-induced inactivation

Diffusion-limited processes tend to be less sensitive to temperature than processes such as protein conformational changes (van Lunteren et al. 1993). To determine whether the NH₄⁺-induced channel inactivation is due to a conformational change or NH₄⁺ block, we next studied the effects of temperature on single-channel current amplitude and NH₄⁺-induced inactivation. Figure 4 shows the absolute values of single-channel currents and the rate of NH₄⁺-induced inactivation at V_m = -140 mV over a temperature range of 22-32 °C. Both current amplitude and inactivation rate increased with temperature. The factor by which the current and rate increased over the 10 °C increase (Q₁₀) was 1.57 for the single-channel current and 2.76 for the inactivation rate. The fact that Q₁₀ was higher for the inactivation rate argues that NH₄⁺-induced inactivation probably results from a conformational change rather than from a simple bimolecular reaction resulting in channel block (diffusion limited).

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Figure 4. Effects of temperature on current amplitude and inactivation rate A and B, absolute values of single-channel current and inactivation rate obtained at -140 mV at temperatures ranging from 22 to 32 °C. Continuous lines represent the best fit to the data (in the form of log(1/) = log A - Ea/RT, where A is a temperature-independent constant, Ea is the activation energy, R is the gas constant, and T is the absolute temperature). Q10 = exp[Ea/R(10 K/T1T2)]. n = 4-5.

Effects of external and internal NH₄⁺ on NH₄⁺-induced inactivation

The results in Fig. 3 were obtained using varying symmetrical [NH₄⁺]. However, it was necessary to determine whether the external or internal [NH₄⁺], or both, are critical for inactivation. When the internal [NH₄⁺] ([NH₄⁺]_i) was kept constant, the degree of inactivation increased with increasing external [NH₄⁺] ([NH₄⁺]_o; Fig. 5A). Increased [NH₄⁺]_o also enhanced the inactivation rate in a V_m-dependent manner (Fig. 5B). Figure 5C illustrates the relationship between inactivation rate and driving force (V_m - E). At the same driving force, the rates of inactivation of Kir2.1 channels exposed to four different [NH₄⁺]_o were not superimposible. These results suggest that NH₄⁺ flux is not essential for inactivation. Thus, it is unlikely that channel inactivation is due to NH₄⁺ ions exiting the pore and accumulating near the internal vestibule.

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Figure 5. Effects of [NH4+]o and [NH4+]i on NH4+-induced inactivation A, normalized I-Vm relationships in 100 mM [NH4+]i. Symbols indicate [NH4+]o: , 10 mM (n = 4); , 30 mM (n = 4); , 100 mM (n = 5); and , 200 mM (n = 2). B, Vm dependence of 1/ in various [NH4+]o and 100 mM [NH4+]i. Symbols are the same as in A. C, relationship between 1/ and Vm - E. D, normalized I-Vm relationships in 10 mM [NH4+]o. Symbols indicate [NH4+]i: , 10 mM (n = 3); , 30 mM (n = 4); and , 100 mM (n = 5). E, Vm dependence of 1/. Symbols are the same as in D.

Figure 5D shows the normalized I-prepulse V_m relationships recorded from inside-out patches exposed to 10 mM [NH₄⁺]_o and various [NH₄⁺]_i. A higher [NH₄⁺]_i resulted in a larger normalized I. Figure 5E shows that the rate of inactivation was V_m dependent. At a constant hyperpolarizing potential, changes in [NH₄⁺]_i did not affect the rate of inactivation.

In summary, the results from Fig. 5 suggest that the inactivation rate at a given V_m is predominantly affected by external rather than internal [NH₄⁺].

R148Y mutants do not support NH₄⁺-induced inactivation

The dominant role that external NH₄⁺ plays during inactivation indicates that NH₄⁺ binding may only occur on the external side of the membrane. Previously, we demonstrated that the binding of two K⁺ ions at the external pore mouth of Kir2.1 channels is enhanced in R148Y mutants (arginine at position 148 is replaced by tyrosine), yet the interaction between the two K⁺ ions is reduced (Shieh et al. 1999). We also found that K⁺ protects R148Y mutants from hyperpolarization-induced inactivation with a higher affinity relative to wild-type Kir2.1 channels (Shieh, 2000). Thus, we examined NH₄⁺-induced inactivation in R148Y mutants. Figure 6A (left panel) shows that inward currents in R148Y mutants were inactivated in inside-out patches exposed to 10 mM symmetrical [NH₄⁺], although the kinetics were slower than those for wild-type channels (see Fig. 3B). Intriguingly, both the degree and the rate of inactivation of currents in R148Y mutants were decreased in the presence of 100 mM symmetrical [NH₄⁺] (Fig. 6A, right panel). Figure 6B summaries the effects of various symmetrical [NH₄⁺] on the relationship between normalized I and V_m. These results demonstrate that only NH₄⁺-protected (but not NH₄⁺-induced) inactivation can be observed in the R148Y mutants. Amino acid 148 is located outside the electrical field across the membrane, and two K⁺-binding sites are located near that region. When the positively charged arginine 148 is replaced by an electrically neutral amino acid, the physical distance between the two binding sites becomes larger, thereby affecting ion permeation (Shieh et al. 1999). The two binding sites at the external pore mouth are likely to be accessible to the permeant NH₄⁺ ions, and the NH₄⁺-binding site involved in NH₄⁺-induced inactivation may, in fact, be one of these two binding sites. The lack of NH₄⁺-induced inactivation in the R148Y mutants (as shown in Fig. 6) may be due to a subtle physical change in the binding site. Such a change may, in turn, interfere with the effect that NH₄⁺ binding has on the V_m-dependent process that eventually closes the channel.

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Figure 6. R148Y mutants do not support NH4+-induced inactivation A, current traces obtained from two different inside-out patches (expressing mutant R148Y Kir2.1 channels) exposed to 10 and 100 mM symmetrical [NH4+]. B, normalized I-Vm relationship. Symbols indicate symmetrical [NH4+]: , 10 mM (n = 7); , 100 mM (n = 7); and , 200 mM (n = 2).

Kinetics of NH₄⁺-induced inactivation

We further explored the mechanisms underlying NH₄⁺-induced inactivation by examining the kinetics of single-channel currents. Figure 7A presents single-channel traces recorded at -140 mV for 10 and 100 mM symmetrical [NH₄⁺]. Both open and closed events were clearly identifiable. The distributions of open and closed dwell times fitted well to monoexponential functions. An increase in symmetrical [NH₄⁺] decreased both the mean open time (_O) and mean closed time (_C). These results suggest that, in the presence of NH₄⁺, Kir2.1 channels transit between an open state and a non-conducting state (inactivated state), and thus are consistent with the simple scheme given below (Scheme 1).

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Figure 7. Dose-dependent effects of symmetrical [NH4+] on the kinetics of single-channel currents A, sample sweeps at -140 mV, and corresponding histograms using 10 and 100 mM symmetrical [NH4+] as indicated. The downward arrows identify the beginning of the voltage step and the upward arrows indicate the end of the step from a holding potential of 0 mV. Capacitive transients and leak currents were subtracted off-line using null traces. Histograms in A are plotted with square root (sqrt)-log ordinates. The distributions of the open and closed times were fitted to monoexponential functions (continuous curves). Mean open and closed times obtained from the fitted curve are given above the histograms. B-D, dose dependence of kon, koff and PO on symmetrical [NH4+]. n = 3-4.

In Scheme 1, K_A is the equilibrium constant for transitions between the vacant (O) and occupied (O.NH₄⁺) states, is the entry rate into the inactivated state (I), and is the exit rate from the inactivated state. We assume that the O and O.NH₄⁺ states have the same conductance and that the binding step is very fast compared to any subsequent conformational change. Thus, the O O.NH₄⁺ transition reaction will be maintained at near equilibrium at all times. Therefore, Scheme 1 can be reduced to one open state and one inactivated state (Colquhoun & Hawkes, 1995) as shown in Scheme 2.

Here k_on = /(1 + K_A/[NH₄⁺]) and k_off = . Note that the activation step (voltage-dependent relief of the block by internal polyamines and Mg²⁺) is ignored because it is very fast compared to the inactivation step (Lopatin et al. 1995).

The values of k_on and k_off were calculated as the reciprocals of _O and _C, respectively. Figure 7B and C demonstrates that both k_on and k_off obtained at V_m = -140 mV increased progressively with increasing [NH₄⁺] until plateaus were reached. Since k_on increases with increasing symmetrical [NH₄⁺], this indicates that NH₄⁺ binding is involved in the transition from the conducting to the inactivated state. However, we were unable to quantify K_A because NH₄⁺ is also a permeant ion, and thus the gating of this channel is not at equilibrium. Figure 7D shows the effects of changes in [NH₄⁺] on open probability (P_O = _O/(_O + _C)) at V_m = -140 mV. As with normalized I, P_O values were similar at various symmetrical [NH₄⁺] because _O and _C were affected by [NH₄⁺] to a similar degree.

Regulation of the kinetics of NH₄⁺-induced inactivation

It is interesting that not only k_on, but also k_off, was affected by changes in symmetrical [NH₄⁺]. To further explore whether k_off is affected by external or internal NH₄⁺, or both, we examined the effects of [NH₄⁺]_o on k_on and k_off. Figure 8A presents k_on and k_off values obtained with 0 [NH₄⁺]_i and 30 or 100 mM [NH₄⁺]_o. Both k_on and k_off were significantly higher with 100 mM [NH₄⁺]_o than with 30 mM [NH₄⁺]_o.

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Figure 8. Effects of [NH4+]o and [NH4+]i on kon and koff A, summary of the effects of [NH4+]o on kon and koff at -140 mV with 0 [NH4+]i. B, summary of the effect of [NH4+]i on kon and koff obtained at -140 mV with 100 mM [NH4+]o. Asterisks indicate that groups were significantly different; * P < 0.05 and *** P < 0.001. Student's independent t test was used to assess statistical significance.

Figure 5D and E showed that an increase of [NH₄⁺]_i resulted in a slight decrease in the degree but not the rate of inactivation. To better understand how NH₄⁺-induced inactivation is regulated by internal NH₄⁺, we examined the effects of [NH₄⁺]_i on k_on and k_off. Figure 8B shows that an increase of [NH₄⁺]_i from 10 to 100 mM significantly reduced k_on, but elevated k_off (when [NH₄⁺]_o was 100 mM).

Voltage dependence of k_on and k_off

Figure 3B, and Figure 5B and E showed that the inactivation rates of macroscopic currents were V_m dependent. We next investigated the V_m dependence of k_on and k_off using various combinations of [NH₄⁺]_o and [NH₄⁺]_i. Regardless of the [NH₄⁺] combination, increases in [NH₄⁺]_o or decreases in [NH₄⁺]_i resulted in elevated k_on values without affecting V_m dependence (Fig. 9A). In contrast, k_off values were not affected by V_m values ranging from -200 to -80 mV (Fig. 9B). These results argue against the permeable block hypothesis, which would predict that a more negative V_m would enhance k_off (see Discussion).

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Figure 9. Vm dependence of kon and koff A, Vm dependence of kon. Continuous lines are best fits to data in the form kon = kon(0) exp(F/RT Vm), where kon(0) is the kon at 0 mV and is the apparent electrical distance from the outer membrane. kon(0) and were: 6.2 s-1 and 0.41 in 100 mM symmetrical [NH4+] (); 3.0 s-1 and 0.41 in 100 mM [NH4+]o/500 mM [NH4+]i (); and 2.3 s-1 and 0.43 in 30 mM [NH4+]o/100 mM [NH4+]i (). n = 2-6. B, Vm dependence of koff. Symbols are the same as in A.

Our results are consistent with the hypothesis that a permeant NH₄⁺ ion binds to a site within the Kir2.1 channel, thereby inducing a conformational change such that the channel becomes inactivated and impermeable to NH₄⁺.

	DISCUSSION

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Gating mechanism mediated by NH₄⁺ in Kir2.1 channels

Previously, we showed that inactivation of inward K⁺ currents through Kir2.1 channels is prevented when a K⁺ ion binds to a site at or near the external pore mouth of the channel (Shieh, 2000). In this study, we set out to investigate whether the 'protective effect' is selective for K⁺ ions using either NH₄⁺ or Tl⁺ as the permeant ions. We found that two types of inactivation occur in the presence of 3 mM [NH₄⁺] or [Tl⁺]. One is similar to the K⁺-protected inactivation, in that increases in [NH₄⁺] or [Tl⁺] protect Kir2.1 channels from inactivation (Fig. 2). With the other type, inactivation is actually enhanced by increasing [NH₄⁺] or [Tl⁺], and when these ions were present at 10 mM, only the NH₄⁺- (or Tl⁺-) induced inactivation was observed. Given the similarity in the results obtained using NH₄⁺ and Tl⁺ in our initial experiments, we focused the remaining portion of our study on NH₄⁺-induced inactivation.

What is the mechanism underlying NH₄⁺-induced inactivation? First, channel block by permeant ions has been demonstrated in several types of channel. For example, it has been demonstrated that Ba²⁺ blocks Ca²⁺-activated K⁺ channels, and is transported to the other side of the membrane during the process (Neyton & Miller, 1988a,b). Furthermore, changes in internal or external [Ba²⁺] can either enhance the dissociation of the Ba²⁺ ion or 'lock' the pore-blocking Ba²⁺ within the pore (Neyton & Miller, 1988a,b). These features may be applicable to NH₄⁺-induced inactivation as described in this study. However, detailed examination of our results suggests that the NH₄⁺-induced inactivation is inconsistent with the permeable block hypothesis for the following reasons. First, in this study we demonstrated that the rate of inactivation at low [NH₄⁺] (10 mM) was much slower than the effective rate coefficient for ion conduction. If NH₄⁺ acts as a permeable blocker, then the rate of its association with the binding site located along the permeation pathway has to be extremely low (2.6 10³ M^-1 s^-1). Accordingly, only 1 out of 40 000 NH₄⁺ ions entering the pore would produce a block, a scenario that predicts an open probability close to 1 ((39 999/40 000) 1 + (1/40 000) [k_off/(k_off + k_on)]). This is inconsistent with our experimentally obtained open probability of ~0.4 (see Fig. 7D). Second, it was previously shown that arginine 148 is located outside the electrical field and is crucial for the interaction of K⁺ with Kir2.1 channels (Shieh et al. 1999). The fact that NH₄⁺-induced inactivation is not observed in R148Y mutants indicates that the NH₄⁺-binding site involved in inactivation may be located in the external pore mouth. If NH₄⁺-induced inactivation (which is V_m dependent) is due to NH₄⁺ block, then the NH₄⁺ binding site would have to be located within the electrical field. Third, decreases in V_m did not enhance k_off over a broad voltage range (-200 to -40 mV). Since a permeant blocker can dissociate into either the internal or external space, one would expect that strong hyperpolarization would increase the k_off value. Although it is possible that a voltage of -200 mV is not sufficiently negative to observe an enhancement in k_off, we consider this possibility unlikely for the following reason. While Ba²⁺ is apparently a much better blocker than NH₄⁺ (K_d 0.5 µM for Ba²⁺ versus > 10 mM for NH₄⁺, calculated from data in Fig. 3), it exhibits much poorer permeability than NH₄⁺. However, external Ba²⁺ dissociates into the internal space at V_m < -120 mV (Shieh et al. 1998). Therefore, it is difficult to believe that a voltage of -200 mV is not sufficient to observe an enhancement in k_off for NH₄⁺ block. Together, these features suggest that NH₄⁺-induced inactivation is not due to channel block by NH₄⁺.

It was previously demonstrated that inward Rb⁺ currents inactivate Kir2.1 channels, and two possible mechanisms for this inactivation were proposed (Reuveny et al. 1996). First, once Rb⁺ exits a Kir2.1 channel pore, it may accumulate in or near the internal vestibule. Second, Rb⁺ may induce a conformational change that promotes inactivation. In our studies with NH₄⁺, single-channel conductance did not decrease over time, and the inactivation rate was not dependent on the driving force per se (see Fig. 5C). Therefore, it is unlikely that NH₄⁺-induced inactivation is due to channel inhibition resulting from the back flow of NH₄⁺ accumulating at the internal pore mouth.

If the NH₄⁺-induced inactivation is not due to open channel block by NH₄⁺, what then is the mechanism? Another possibility is that NH₄⁺ binds to a site on the Kir2.1 channel, thereby inducing a conformational change that promotes inactivation. Our findings are consistent with this hypothesis. First, the concentration dependence of the inactivation rate exhibits saturation with respect to NH₄⁺ (Fig. 3C). Second, Q₁₀ for the NH₄⁺-induced inactivation is high (2.76; see Fig. 4). Third, a single amino acid mutation at the external pore mouth of the Kir2.1 channel abolishes NH₄⁺-induced inactivation (Fig. 6). This suggests that a V_m-independent event (the binding of NH₄⁺ to the external pore mouth) is linked to a V_m-dependent process, thereby inducing the V_m-dependent closure of the channel (via a conformational change).

The idea that a conformational change may underlie the hyperpolarization-induced inactivation of inward rectifier K⁺ channels has not commanded much attention, although such a supposition is not unprecedented. Nichols & Lopatin (1997) hypothesized that the V_m-dependent behaviour of both the voltage-gated and inward rectifier K⁺ channels might arise from similar processes if channels share the inner core of the inward rectifier K⁺ channels (Nichols, 1993). They further proposed that the M0 region of the Kir1.1 channel may play a similar role to the S4 region in voltage-gated K⁺ channels. Miller & Aldrich (1996) demonstrated that an outwardly rectifying Shaker channel can be converted to an inward rectifier K⁺ channel by making triple mutations. This conversion is not dependent on a reverse of the charge movement of the voltage sensor, but is instead due to the difference in the gates controlling these two types of channel. According to their kinetic scheme, hyperpolarization induces inward rectifier K⁺ channels to move from a blocked state (Mg²⁺ and polyamine channel block) through the open state, resulting in inward currents, and then to the closed state. If these two types of channel indeed have parallel gating machinery, then the inactivation described in this study is equivalent to deactivation (transition between the open and closed state) that is characteristic of voltage-gated K⁺ channels.

In conjunction with our previous results (Shieh, 2000), the present study provides evidence for the involvement of a V_m-dependent process in gating during inactivation of Kir2.1 channels. It remains to be determined which part(s) of the channel participates in our proposed conformational change during hyperpolarization.

Regulation of NH₄⁺-induced inactivation

External and internal NH₄⁺ affect both k_on and k_off. k_on was determined from the distribution of open times. Since in the conducting channels, NH₄⁺ can enter from both sides of the membrane, the effects of external and internal NH₄⁺ on k_on are due to several rate processes in the multi-ion pore of a Kir2.1 channel and thereby are very complicated (Neyton & Miller, 1988b). On the other hand, k_off was determined from the distribution of closed times. In the non-conducting channels, a regulatory NH₄⁺ ion can bind to its site in a true equilibrium reaction (the closure of a Kir2.1 channel effectively isolates one side of the channel from NH₄⁺ applied on the other side). Thus, the effects of external and internal NH₄⁺ on k_off give a measure of the occupancy of the regulatory NH₄⁺ sites (Neyton & Miller, 1988b). Therefore, we focus only on the regulation of k_off by external and internal NH₄⁺.

The fact that external NH₄⁺ enhances k_off suggests that a regulatory site may be located near the NH₄⁺-binding site involved in inactivation. Two K⁺ binding sites are located near position 148 at the external pore mouth of Kir2.1 channels (Shieh et al. 1999). These binding sites may be involved in NH₄⁺-induced inactivation and k_off enhancement (via ion-ion interactions). It is also possible that k_off enhancement is due to an allosteric effect induced by the binding of another NH₄⁺ to the channel (ion-channel interaction).

Increases in [NH₄⁺]_i not only reduce k_on but also increase k_off. Similar effects of permeant and pore-blocking ions on channel gating have been described. Swenson & Armstrong (1981) proposed that channels cannot close with an ion in the pore (the foot-in-the-door model). However, this model predicts that k_off should not be affected by [NH₄⁺]_i and thus is inconsistent with our data. Our results are consistent with the occupancy model in which the channel can close while occupied by an internal NH₄⁺, but this occupancy destabilizes the inactivated state (Demo & Yellen, 1992).

Conclusions

Inactivation of inward currents through inward rectifier K⁺ channels may result in hyperpolarization of cell membranes and thus affect membrane excitability. This study demonstrates that NH₄⁺ and Tl⁺ inward currents become inactivated in Kir2.1 channels. This inactivation may serve as a second selectivity filter for Kir2.1 channels such that K⁺ permeation is favoured. Our results suggest that inactivation is due to the binding of NH₄⁺ within the Kir2.1 channel, driving the channel into an inactivated state. Inactivation can be regulated by ion-channel and/or ion-ion interactions in the pore. Therefore, the interaction of permeant ions with Kir2.1 channels is important not only for channel permeability, but also for modulation of the gating machinery.

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Acknowledgements

We thank Dr Lily Jan for kindly providing the Kir2.1 clone. We are grateful to Dr Tsung-Yu Chen for his valuable comments and BiomEditor, International Bioscience Consultants, for providing professional English editing services. This work was supported by Academia Sinica and Taiwan (R.O.C.) National Science Council grant 89-2320-B-001-025.

Corresponding author

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}ibms.sinica.edu.tw

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