Biophysical Journal - Interaction of Ba2+ with the Pores of the Cloned Inward Rectifier K+ Channels Kir2.1 Expressed in Xenopus Oocytes

Article Outline

Abstract
Introduction
Materials and methods
Results
Discussion
Acknowledgement
References
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Copyright © 1998 The Biophysical Society. All rights reserved.
Biophysical Journal, Volume 75, Issue 5, 2313-2322, 1 November 1998

doi:10.1016/S0006-3495(98)77675-1

Channels, Receptors, and Transporters

Interaction of Ba²⁺ with the Pores of the Cloned Inward Rectifier K⁺ Channels Kir2.1 Expressed in Xenopus Oocytes

Ru-Chi Shieh^*^,^, , Jui-Chu Chang^* and Jorge Arreola^#

^* Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, R.O.C.
^# Instituto de Física, Universidad Autónoma de San Luis Potosí, San Luis Potosí, S.L.P. 78000, Mexico

Address reprint requests to Dr. Ru-Chi Shieh, Institute of Biomedical Sciences, Academia Sinica, 128 Yen-Chiu Yuan Road, Section 2, Taipei 11529, Taiwan, R.O.C. Tel.: 02-2789-9024; Fax: 02-2785-3569.

Abstract

Interactions of Ba²⁺ with K⁺ and molecules contributing to inward rectification were studied in the cloned inward rectifier K⁺ channels, Kir2.1. Extracellular Ba²⁺ blocked Kir2.1 channels with first-order kinetics in a V_m-dependent manner. At V_m more negative than −120mV, the K_d-V_m relationship became less steep and the dissociation rate constants were larger, suggesting Ba²⁺ dissociation into the extracellular space. Both depolarization and increasing [K⁺]_i accelerated the recovery from extracellular Ba²⁺ blockade. Intracellular K⁺ appears to relieve Ba²⁺ blockade by competitively slowing the Ba²⁺ entrance rate, instead of increasing its exit rate by knocking off action. Intracellular spermine (100μM) reduced, whereas 1mM [Mg²⁺]_i only slightly reduced, the ability of intracellular K⁺ to repulse Ba²⁺ from the channel pore. Intracellular Ba²⁺ also blocked outward I_Kir2.1 in a voltage-dependent fashion. At V_m≥+40mV, where intrinsic inactivation is prominent, intracellular Ba²⁺ accelerated the inactivation rate of the outward I_Kir2.1 in a V_m-independent manner, suggesting interaction of Ba²⁺ with the intrinsic gate of Kir2.1 channels.

Introduction

Inward rectifier K⁺ channels conduct inward currents at potentials more negative than the K⁺ reversal potential but permit much smaller currents at potentials positive to the reversal potential (Hille, 1992). Thus inward rectifier K⁺ channels set the resting membrane potential and control the excitability of many cell types. Two mechanisms have been proposed to account for inward rectification of K⁺ channels. The first involves the voltage-dependent blockade of outward K⁺ currents by both intracellular Mg²⁺ (Matsuda et al,Vandenberg, 1987) and polyamines (Fickler et al,Lopatin et al). These observations are consistent with the hypothesis that inward rectification is caused by intracellular pore blocking particle(s) (Hille and Schwarz, 1978). The second mechanism is a novel pH_i-dependent gating mechanism that is involved in the inactivation of the outward currents and is independent of intracellular Mg²⁺ and polyamines (Shieh et al).

Ba²⁺ is known to inhibit several types of K⁺ channels expressed in different tissues, including delayed rectifier K⁺ channels of squid giant axon (Armstrong and Taylor, 1980,Eaton and Brodwick, 1980,Armstrong et al), native inward rectifier K⁺ channels of starfish eggs and frog skeletal muscle (Hagiwara et al,Standen and Stanfield, 1978), and Ca²⁺-activated K⁺ channels (Vergara and Latorre, 1983,Neyton and Miller, 1988a,b). The inhibitory effects of extracellular Ba²⁺ have also been demonstrated in different cloned inward rectifier K⁺ channels, including those cloned from mouse macrophage (Kubo et al), human and mouse brain (Makhina et al,Morishige et al,Perier et al), rat kidney (Zhou et al), and human heart (Ashen et al). Application of extracellular Ba²⁺ to either native or cloned inward rectifier K⁺ channels resulted in voltage- and time-dependent inhibitions of the K⁺ currents. In contrast, the application of extracellular Ba²⁺ to the delayed rectifier K⁺ channels from squid axon reduced the magnitude of K⁺ current without changing its kinetics, whereas intracellular Ba²⁺ induced time-dependent inhibition of the channels (Armstrong and Taylor, 1980,Eaton and Brodwick, 1980,Armstrong et al). However, detailed characterization of Ba²⁺ blockade in the cloned inward rectifier K⁺ channels has not been carried out.

Because Ba²⁺ and K⁺ have similar crystal radii, it has been hypothesized that Ba²⁺ ions inhibit K⁺ channels by entering the pores when the channels are opened by changes in membrane potential, as evidenced by the findings that extracellular K⁺ could either knock off the Ba²⁺ (Eaton and Brodwick, 1980,Armstrong and Taylor, 1980) or compete for the Ba²⁺ binding site (Vergara and Latorre, 1983) in K⁺ channels. These previous studies suggest that the voltage-dependent inhibition of K⁺ channels by Ba²⁺ is due to the interaction of Ba²⁺ ions with the amino acid residues lining the pores of the channels, which are presumably located within the electrical field. Thus Ba²⁺ has been used as a probe to learn about the permeability of the K⁺ channels. For example, from the studies of K⁺ and Ba²⁺ interaction in the pores of the high-conductance Ca²⁺-activated K⁺ channels, it has been shown that each channel has at least three binding sites for the permeant K⁺ ions (Neyton and Miller, 1988b).

In this study we examined the interactions of Ba²⁺ with the permeant K⁺ ion in Kir2.1 channels and found that extracellular Ba²⁺ blockade was relieved by intracellular K⁺ through competition for the same binding site. Ba²⁺ thus is a useful probe for studying K⁺ binding in Kir2.1 channels. In addition, we identified the interactions of Ba²⁺ with the rectifying factors, including intracellular Mg²⁺ and spermine and the “intrinsic” gate. The results demonstrate that although both Mg²⁺ and spermine induce inward rectification by blocking the Kir2.1 channel, their binding sites are not identical. Ba²⁺ applied intracellularly appears to accelerate the inactivation of outward current by interfering with the “intrinsic” gating mechanism and thus is a potential tool for unraveling the essence of the intrinsic gating property of the Kir2.1 channel. A preliminary report of these findings has been presented to the Biophysical Society (Shieh et al).

Materials and methods

Molecular biology and preparation of Xenopus oocytes

Mouse macrophage Kir2.1 DNA (the original clone in pCDNPAI/Amp was generously provided by Dr. Lily Jan, UCSF)) subcloned into Bluescript II SK+ was a generous gift from Drs. Scott A. John and James N. Weiss (UCLA). Purified linear Kir2.1 DNA and in vitro transcription were carried out as previously described (Shieh et al).

Xenopus oocytes were obtained by partial ovariectomy from frogs fully anesthetized with 0.1% tricaine and then defolliculated using 2% collagenase as previously described (Shieh et al). Oocytes were pressure injected 24h after defolliculation with 10–100 pg of Kir2.1 cRNAs for whole-cell recordings and 1–10ng for giant patch recordings. Oocytes were maintained at 18°C in Barth’s solution containing (in mM) 88 NaCl, 1 KCl, 2.4 NaHCO₃, 0.3 Ca(NO₃)₂, 0.41 CaCl₂, 0.82 MgSO₄, 15 HEPES, and 20μg gentamicin/ml at pH 7.6 and were used 1–3 days after RNA injection.

Electrophysiology techniques

Extracellular Ba²⁺ blockade of whole-cell I_Kir2.1 was examined at room temperature (21–24°C) using a two-electrode voltage-clamp amplifier (Ca-1 clamp; Dagan, Minneapolis, MN). Oocytes were bathed in a solution containing (in mM) 98 KCl, 2 KOH, 1.8 CaCl₂, and 5 HEPES at pH 7.4. Both the voltage-sensing and current-injecting electrodes were filled with 3M KCl (resistance 0.2–1 MΩ). Voltage steps were applied from a holding potential of 0mV to various test voltages ranging from −160 to +40mV in 10-mV increments. To ensure that Ba²⁺ blockade reached steady state, the effects of [Ba²⁺]≤30μM were studied using 10-s depolarizing pulses, whereas those of [Ba²⁺]≥100μM were examined using 2-s pulses. Ba²⁺ was added directly to the bath solution until the desired concentration was reached, and the pH was adjusted to 7.4.

To avoid inadequate voltage clamping in whole cells caused by large currents, we used oocytes expressing an absolute value of less than −10μA at −160mV. The time constants for the blockade (τ_block) of the I_Kir2.1 recorded at −120mV by 10μM [Ba²⁺] in whole cells (123.6±8.8ms, n=13) were not significantly different (p=0.19) from those recorded in cell-attached patches (104.3±10.4ms, n=8). Furthermore, the recovery time constants in cell-attached patches (τ_recov=412.3±33.6ms, n=8) were not significantly different (p=0.49) from those obtained in whole cells (τ_recov=385.9±9.8ms, n=7) at V_r=+100mV. These data suggest that the voltage clamping in whole cells was as adequate as that in patches.

In experiments where intracellular ionic conditions needed to be changed, I_Kir2.1 was recorded using the inside-out giant patch-clamp technique (Hilgemann, 1995,Shieh et al) and an Axopatch 1-D amplifier (Axon Instruments, Foster City, CA). Patch electrode solution contained (in mM) 98 KCl, 2 KOH, 1.8 CaCl₂ (or 5 MgCl₂), and 5 HEPES at pH 7.4. Bath solution (control intracellular solution) contained (in mM) 82 KCl, 18 KOH, 5 EDTA, 2K₂ATP, and 5 HEPES at pH 7.2. Concentrations of K⁺ from 200mM down to 20mM were obtained by changing the concentrations of KCl while the concentrations of HEPES, EDTA, and KOH were kept constant. The rundown of channel activity was avoided by treating the patches with 25μM l-α- phosphatidylinositol-4,5-bisphosphate (PIP₂) (Sigma Chemical Co., St. Louis, MO) (Huang et al) for 20–60s. PIP₂ stock solution (1mM) was prepared by dissolving PIP₂ in chloroform. On the day of experiments, 125μl of the stock solution was N₂-dried and resuspended in 5ml of control intracellular solution by sonication for 15s three times, separated by 30-s intervals. Although the amplitude of outward I_Kir2.1 was increased by 25μM PIP₂, the recovery from the extracellular Ba²⁺ blockade and the intrinsic inactivation of the outward currents were not significantly different (p>0.1) between patches treated with and without PIP₂ (data not shown). To wash out the residual intracellular Mg²⁺ and polyamines, experiments were carried out after at least 5min of continuous perfusion of Mg²⁺- and polyamine-free solution after patch excision. Free [Ba²⁺] and [Mg²⁺] in the intracellular solution were calculated with the MaxC program (Chris Patton, Stanford University) and stability constants by Martell and Smith, 1974.

The command voltage pulses and data acquisition functions were processed using a Pentium 100 computer, a DigiData board, and pClamp6 software (Axon Instruments, Burlingame, CA). Data were filtered at 1kHz by a 8-pole low-pass filter (Frequency Devices, Rochester, NY). Frequencies of stimulation and data sampling are described in the figure legends.

Data analysis

Students’ independent or paired t-test was used to assess statistical significance. Results are presented as mean±SEM.

Results

The blockade of inward I_Kir2.1 by extracellular Ba²⁺

Figure 1A shows current traces recorded at −120mV from a representative oocyte in the absence and in the presence of the indicated [Ba²⁺]_o. The addition of Ba²⁺ to the extracellular side of the membrane induced a time-dependent decrease of the inward currents. The rate of inhibition of I_Kir2.1 was accelerated by increasing [Ba²⁺]_o, and the currents reached steady state at the end of hyperpolarization. I_Kir2.1 at the onset of hyperpolarization was little affected by Ba²⁺. For example, 10μM [Ba²⁺]_o reduced the instantaneous I_Kir2.1 recorded at −120mV by only 5±2% (n=6), despite the fact that at the end of the pulse less than 5% of the current remained. This observation suggests that the Kir2.1 channels are not affected by Ba²⁺ at 0mV, and channels must open before Ba²⁺ inhibition takes place.

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Figure 1

Blockade of inward I_Kir2.1 by extracellular Ba²⁺. (A) Representative currents elicited by voltage steps to −120mV for 2s from a holding potential of 0mV in the presence of the indicated [Ba²⁺]_o (μM). The horizontal line indicates the zero current level. Current traces recorded in the presence of 5mM [Ba²⁺]_o were used to eliminate the linear leak currents. (B) Normalized current-voltage (I-V) relationships for the steady-state inward I_Kir2.1. Data are the averages from 4–12 cells in the presence of 0 (●), 1 (○), 10 (▾), 100 (▿), and 1000μM Ba²⁺ (■). Steady-state currents measured at the end of the 2- or 10-s pulses were normalized to that recorded at −160mV in the absence of Ba²⁺ in each cell and then averaged. (C) Dose-response curves for extracellular Ba²⁺ blockade of the steady-state I_Kir2.1 at −160mV (●), −120mV (○), −80mV (▾), and −40mV (▿). Lines are the best fits to the averaged data (n=4–12 for each point) using the Hill equation (Eq. (1)). (D) K_d values obtained from the concentration-dependent inhibition of the steady-state I_Kir2.1 are plotted against membrane potentials. The solid line is the best fit of the data obtained at V_m ranging from −120mV to −40mV, using the Boltzmann equation (Eq. (2)). The frequencies of stimulation were 0.05–0.083Hz, and the sampling rates of the I_Kir2.1 were 1.43–1.67kHz.

Normalized steady state current-voltage (I-V) relationships in the presence of various [Ba²⁺]_o are summarized in Figure 1B, which shows that the effects of extracellular Ba²⁺ on I_Kir2.1 were voltage dependent. Extracellular Ba²⁺ preferentially affected Kir2.1 channels at more negative potentials. This voltage-dependent effect of [Ba²⁺]_o (1 or 10μM) on Kir2.1 channels resulted in a negative conductance at negative potentials (Figure 1B), as has previously been observed in Kir2.1 channels (Kubo et al).

Figure 1C shows, from left to right, the dose-response curves for the blockade of the steady-state I_Kir2.1 by extracellular Ba²⁺ at V_m=−160, −120, −80, and −40mV. I_Kir2.1 amplitude was normalized to the control amplitude ([Ba²⁺]_o=0) obtained at the same potential and expressed as fractional I_Kir2.1. The dose-response curves also demonstrate the voltage-dependent inhibition of the inward I_Kir2.1 by extracellular Ba²⁺. As the membrane potential became more hyperpolarized, the steady-state I_Kir2.1 was more sensitive to extracellular Ba²⁺, as if the negative electrical field attracted Ba²⁺ inside the membrane. The dose-response curves at each potential were well described by the Hill equation (Eq. (1)):

(1)

where K_d is the apparent dissociation constant and n is the Hill coefficient. The K_d values obtained from the fittings were 0.2μM, V_m=−160mV; 0.5μM, V_m=−120mV; 2.2μM, V_m=−80mV; 12.7μM, V_m=−40mV. The Hill coefficients were 0.84, V_m=−160mV; 0.90, V_m=−120mV; 1.06, V_m=−80mV; 0.99, V_m=−40mV. The corresponding Hill coefficients were close to 1, suggesting one Ba²⁺ interacted with one channel protein.

To quantitatively assay the voltage dependence of the inhibition of I_Kir2.1 by extracellular Ba²⁺, the K_d values obtained from the blockade of the steady-state I_Kir2.1 were plotted against the membrane potential (Figure 1D). The solid line is the fit to the data points from −40 to −120mV, using the Boltzmann equation:

(2)

where K_d(0) is the dissociation constant at 0mV, z is the valence of Ba²⁺=+2, δ is the apparent electrical distance between the Ba²⁺-channel interacting site and the outer pore of the channel, and F, R, T, and V_m have their usual meanings. The estimated K_d(0) was 62μM, and δ was 0.54 for the blockade of steady-state I_Kir2.1. Note that the K_d-V_m relationship became less steep at V_m more negative than −120mV. This may be due to Ba²⁺ dissociation into the intracellular site at very negative V_m, as previously described in the high-conductance Ca²⁺-activated K⁺ channel (Neyton and Miller, 1988b).

Kinetics of the extracellular Ba²⁺-induced blockade of Kir2.1 channels

To quantitatively examine the kinetics of the blockade of Kir2.1 channels by extracellular Ba²⁺, we obtained the association (k_on) and dissociation (k_off) rate constants of Ba²⁺ interacting with the binding site. Assuming the interaction of Ba²⁺ with Kir2.1 channels follows a first-order reaction, as suggested by the dose-response curves (Figure 1C), we can then compute k_on and k_off, using the equations described by Holmgren et al:

(3)

(4)

where f=I_steady-state/I_ctrl.

Figure 2A summarizes the k_on-V_m relationship. The solid line is the fit to the data points from −40 to −120mV, using the Boltzmann equation with a k_on at 0 mV=6.8×10³ s⁻¹M⁻¹ and a δ=0.52. This value of δ was comparable to that (0.54) obtained using stationary analysis of dose-response curves (Figure 1C). These results demonstrate that the voltage-dependent interaction of Ba²⁺ ions with the binding sites in the channels to induce blockade of Kir2.1 channels is mainly due to the voltage dependence of k_on. In contrast, the k_off-V_m relationship (Figure 2B) showed little V_m dependence at V_m between −130 and −50mV. However, k_off increased as V_m became more negative than −130mV. These results are indicative of Ba²⁺ dissociating into the intracellular space, as previously shown in the high-conductance Ca²⁺-activated K⁺ channels (Neyton and Miller, 1988b) and Shaker K⁺ channels (Harris et al). This is also consistent with the observation that the K_d-V_m relationship became less steep at V_m more negative than −120mV.

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Figure 2

Kinetics analysis of inward I_Kir2.1 blockade by extracellular Ba²⁺. (A) k_on values obtained from Eq. (3) are plotted at the corresponding membrane potentials (n=6). The solid line was the fit of the data using the Boltzmann equation. (B) The voltage dependence of the k_off values computed from Eq. (4) (●, n=6) and those obtained from recovery from extracellular Ba²⁺ blockade experiments (○). k_on and k_off values were estimated by using f and τ_block obtained at [Ba²⁺]_o=3μM. Similar k_on-V_m and k_off-V_m relationships were also observed with [Ba²⁺]_o=1, 10, and 30μM.

Recovery of Kir2.1 channels from extracellular Ba²⁺ blockade

Figure 2B shows that k_off tends to increase at V_m>−50mV. Unfortunately, currents recorded at V_m>−40mV were too small to allow us to estimate k_off at more positive V_m. To obtain experimental estimates of k_off over the positive range of potentials, the rate of recovery from 10μM [Ba²⁺]_o blockade was examined using a two-pulse protocol. The fraction of channels blocked by 10μM [Ba²⁺]_o at −120mV was tested by the first pulse, whereas the fraction of channels recovered from blockade after a given time interval at various recovery voltages (V_r) was recorded by the second pulse. Figure 3AB, shows the voltage protocol (A, top panel) with various recovery time intervals and the corresponding current traces recorded with V_r=0 (A, lower panel) and V_r=+60mV (B). I_Kir2.1 activated by the first hyperpolarizing pulse displayed a monoexponential blockade until only 5% of the instantaneous current amplitude remained at the end of the pulse. The instantaneous current recorded with the second hyperpolarizing pulse increased as the interval between the two pulses increased. The recovery from the blockade followed a monoexponential time course and was faster with V_r=+60mV (time constant=867ms) than that with V_r=0mV (time constant=1506ms). The steady-state I_Kir2.1 at the end of the −120-mV pulse remained constant, independent of the recovery time interval, the recovery voltage, or the fraction of channels recovered at the beginning of the second hyperpolarizing pulse.

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Figure 3

Time course of recovery from extracellular Ba²⁺ blockade. (A) Upper panel shows the two-pulse voltage protocol. The time interval between the two identical pulses (each for 1s) was increased by 0.6s between sweeps (starting at 0.05s) to assess recovery from blockade induced by extracellular Ba²⁺ (10μM) during the first pulse. The corresponding current traces recorded with V_r=0 are shown in the lower panel. (B) Current traces recorded with V_r=+60mV, using the same protocol as described in A. Currents recorded in the presence of 5mM [Ba²⁺]_o were subtracted from the currents recorded in 10μM [Ba²⁺]_o to eliminate the linear leak currents. The horizontal lines indicate the zero current level. (C) Time courses for recovery from extracellular Ba²⁺ blockade with V_r=−40 (▿), 0 (●), 60 (○), and 100 (▾) mV. The recovery time constants (τ_recov) were 1160±53ms, V_r=−40mV (n=5); 1315±85ms, V_r=0mV (n=6); 775±48ms, V_r=+60mV (n=6); 386±10ms, V_r=+100mV (n=7). Continuous lines are single-exponential function fits to the data. The estimated maximum fractional recoveries obtained from fittings were 0.43±0.03, V_r=−40mV (n=5); 0.94±0.03, V_r=0mV (n=6); 1.01±0.01, V_r=+60mV (n=6); 1.06±0.05, V_r=+100mV (n=7). The frequency of stimulation was 0.04Hz, and the data sampling rate was 0.5kHz.

The recovery time courses for the instantaneous current recorded at the second pulse at different recovery voltages are shown in Figure 3C. The fractional recovery was calculated as I₂/I₁, where I₂ was the current recorded 4ms after the onset of the second pulse minus the steady-state current, and I₁ was the current recorded 4ms after the onset of the first pulse minus the steady-state current. The fraction of Kir2.1 channels recovered from extracellular Ba²⁺ blockade followed an exponential time course, as shown by the fits to monoexponential functions. The τ_recov values calculated from the monoexponential fits, the k_on values described in Figure 2A, and the function 1/τ_recov=k_on×[Ba²⁺]_o+k_off were used to estimate k_off at positive voltages. The k_on values at 0, +60, and +100mV were obtained from the extrapolation of the k_on-V_m relationship shown in Figure 2A. The calculated k_off values were plotted against V_m in Figure 2B. k_off at −40mV is very close to k_off determined from the kinetic analysis of the extracellular Ba²⁺ blockade. k_off obtained from the recovery for the extracellular Ba²⁺ blockade at potentials positive to 0mV increased significantly (p<0.0005) when compared to that calculated from the kinetic analysis of the extracellular Ba²⁺ blockade at −40mV. The voltage dependence of k_off at positive voltages determined from recovery of blockade is probably due to the effects of the electrical field on the dissociation of Ba²⁺ into the extracellular side.

Effects of intracellular K⁺ on the recovery from extracellular Ba²⁺ blockade

To further investigate whether extracellular Ba²⁺ enters the pore of Kir2.1 channels to induce blockade, we examined the effects of intracellular K⁺ concentration ([K⁺]_i) on the recovery from extracellular Ba²⁺ blockade, as previously described for delayed rectifier K⁺ channels of squid giant axon (Armstrong and Taylor, 1980). To control the intracellular ionic compositions, I_Kir2.1 was recorded using a giant patch technique and a voltage protocol similar to that described in Figure 3A. The hyperpolarizing pulses were set at V_m=−120mV, V_r was set at +100mV, and the holding potential was set at V_m=E_K throughout this series of experiments. Figure 4A shows a set of I_Kir2.1 currents recorded from a cell-attached patch, with a pipette solution containing 10μM [Ba²⁺] used to induce blockade. The overall characteristics (τ_block and τ_recov) of I_Kir2.1 recorded in cell-attached patches were similar to those recorded in whole cells (compare to Fig. 3). When the same patch was excised (inside out) into a Mg²⁺-free and polyamine-free control intracellular solution, both the rate of the blockade for the inward I_Kir2.1 and the rate of the recovery from extracellular Ba²⁺ blockade were increased (Figure 4B), and outward currents were observed during the recovery interval. τ_block decreased from 106ms to 47ms, and τ_recov decreased from 452ms to 42ms. When the same patch was perfused with an intracellular solution containing only 20mM [K⁺], the outward current decreased at V_r=+100mV, τ_recov increased from 42 to 62ms, and τ_block decreased from 47 to 22ms (Figure 4C).

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Figure 4

Effects of [K⁺]_i on the time course of recovery from extracellular Ba²⁺ blockade of the inward I_Kir2.1. (A) Current traces were recorded at V_m=−120mV from a holding potential of 0mV and V_r=+100mV in a cell-attached patch. (B and C) Currents were recorded from the same patch shown in A after excising into the Mg²⁺- and polyamine-free solution containing 100mM [K⁺]_i and 20mM [K⁺]_i, respectively. The blockade of I_Kir2.1 was induced by the presence of 10μM [Ba²⁺] in the pipette solution. The insets in B and C display currents recorded during the second pulse in amplified time scales. The horizontal lines indicate the zero current level. The frequencies of stimulation were 0.067Hz. The sampling rates were 1kHz (A) and 2.5kHz (B and C).

It was previously considered that there are two possible mechanisms for relief of the channel blocker by intracellular K⁺ (Yellen, 1984). One is that K⁺ relieves the block by competing with the blocker for the same binding site in the channel. The other is that K⁺ accelerates the exit of the blocker. We can distinguish between these two possibilities by examining the effects of [K⁺]_i on the kinetics of extracellular Ba²⁺ blockade. If K⁺ competes with Ba²⁺ for a binding site, then k_on should be affected but k_off should not be affected by [K⁺]_i. On the other hand, if K⁺ knocks off Ba²⁺, k_off should be affected.

Table 1 summarizes the effects of various [K⁺]_i on the extracellular Ba²⁺ blockade of the Kir2.1 channels with V_r=+100mV. τ_recov was about one order of magnitude faster in the inside-out excised patches perfused with the control intracellular solution compared to cell-attached patches. Furthermore, τ_block was smaller in the inside-out patches than in the cell-attached patches. When [K⁺]_i was increased from 20 to 200mM, τ_recov decreased, whereas τ_block increased. In addition, the fractions of nonblocked channels at the end of the −120-mV pulse increased as intracellular [K] increased. These results indicate that K⁺ ions interfere with extracellular Ba²⁺ blockade of the Kir2.1 channels at V_m=−120mV. To examine how [K⁺]_i affects the kinetic parameters for the extracellular Ba²⁺ blockade of I_Kir2.1 recorded at −120mV, we estimated k_on and k_off using Eqs. (3), respectively. Note that here f=I_ss,Ba/I_inst,Ba; I_ss,Ba and I_inst,Ba are the steady-state and instantaneous currents recorded at −120mV in the presence of 10μM [Ba²⁺]_o. The accurate expression of f should be I_ss,Ba/I_ss,ctrl, where I_ss,ctrl is the steady-state current recorded at −120mV in the absence of extracellular Ba²⁺. Because currents were recorded in inside-out patches, we were not able to obtain both I_ss,Ba and I_ss,ctrl in the same patch. However, we consistently observed that the inward currents recorded in inside-out patches perfused with Mg²⁺-free and polyamine-free solution did not decrease over time when there was no Ba²⁺ in the pipette (Figure 6AB), and that 10μM [Ba²⁺]_o only inhibited 5±2% of the instantaneous inward I_Kir2.1 in whole-cell experiments. These observations support our assumption that I_inst,Ba≈I_ss,ctrl. The averaged k_on and k_off values at different [K]_i are summarized in Table 1. The results show that increasing [K⁺]_i decreased k_on without significantly affecting k_off for the binding of Ba²⁺ to the channels at −120mV. Thus intracellular K⁺ appears to relieve Ba²⁺ blockade by competing for the Ba²⁺ binding site in the channel.

Table 1 Effects of various intracellular cations on the kinetics of the extracellular Ba²⁺ blockade of K_ir2.1 channels


	C-A (n=8)	100mM [K⁺]_i (n=14)	200mM [K⁺]_i (n=4)	20mM [K⁺]_i (n=3)	+1mM [Mg²⁺]_i (n=4)	+100μM [spm]_i (n=3)
τ_block (ms)	104.3±10.4^§	53.3±2.3	65.0±7.7^*	24.0±1.5^#	70.8±0.8^#	122.7±5.9^#
τ_recov (ms)	412.3±33.6^§	38.1±1.9	32.3±3.4^*	57.1±6.0^*	82.8±5.9^#	2300±503^§
f	0.05±0.00^§	0.12±0.01	0.20±0.03^*	0.08±0.01^*	0.07±0.01^#	0.05±0.03^#
k_on (10⁶×s⁻¹M⁻¹)	1.00±0.15^§	1.71±0.12	1.31±0.23^*	3.85±0.25^*	ND	ND
k_off (s⁻¹)	0.54±0.11^§	2.31±0.11	3.17±0.47	3.49±0.65	ND	ND
			(p=0.24)	(p=0.17)

Paired t-tests were carried out by comparing data in various groups to the control 100mM K_i solution group.^*,#,§The groups were statistically different at p<0.05, p<0.005, and p<0.0005, respectively, unless otherwise indicated. +1mM [Mg²⁺]_i=control 100mM [K⁺]_i solution containing 1mM free [Mg²⁺]; +100μM [spm]_i=control solution containing 100μM spermine. τ_recov was obtained from I_Kir2.1 recorded with V_r=+100mV at V_m=−120mV; τ_block, f, k_on, and k_off were obtained at V_m=−120mV. ND, not determined.

Spermine contributes to the slow recovery from extracellular Ba²⁺ blockade in cell-attached patches

Fig. 4 demonstrates that the rates of recovery from Ba²⁺-induced blockade were faster in inside-out patches exposed to Mg²⁺-free and polyamine-free control solution containing 100mM [K]_i than those observed in cell-attached patches. To examine whether intracellular Mg²⁺ and spermine were responsible for the difference between the recovery rates obtained in the whole cells (or cell-attached patches) and the inside-out patches, we examined the effects of perfusing patches with the control intracellular solution containing Mg²⁺ or spermine on the recovery from the extracellular Ba²⁺ blockade. Figure 5A shows a set of current traces recorded from an inside-out patch exposed to the control intracellular solution, using the two-pulse voltage protocol. Perfusing this patch with the control intracellular solution containing 1mM free [Mg²⁺] induced a slight increase in the recovery time constant from the control value of 45ms (Figure 5A) to 99ms (Figure 5B). Note that outward I_Kir2.1 at V_r=+100mV was blocked. However, when the same patch was exposed to the control solution containing 100μM spermine, the block time constant was increased, the recovery time course was dramatically retarded (Figure 5C, τ_recov=3071ms), and the outward I_Kir2.1 was completely blocked. The effects of 1mM free [Mg²⁺]_i and 100μM spermine on the extracellular Ba²⁺ blockade of the Kir2.1 channels are also summarized in Table 1. These results suggest that spermine, but not Mg²⁺, is one of the major factors that contribute to the slow recovery from extracellular Ba²⁺ blockade observed in whole cells and cell-attached patches, possibly by prohibiting the interaction between the bound Ba²⁺ and K⁺. Becauase intracellular Mg²⁺ and spermine inactivated the inward I_Kir2.1 recorded in the absence of extracellular Ba²⁺ (data not shown), k_on and k_off were not determined in patches treated with Mg²⁺ or spermine.

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Figure 5

Effects of intracellular Mg²⁺ and spermine on the recovery of the inward I_Kir2.1 from blockade by extracellular Ba²⁺. A, B, and C are the current traces recorded in an inside-out patch perfused with the control solution, the control solution containing 1mM free [Mg²⁺], and the control solution containing 100μM spermine, respectively. The same voltage protocol as described in Fig. 4 was applied. The frequencies of stimulation were 0.067Hz in A and B and 0.04Hz in C. Sampling rates were 2.5kHz (A and B) and 0.5kHz (C).

Blockade of Kir2.1 channels by intracellular Ba²⁺

To examine whether Kir2.1 channels are inhibited by intracellular Ba²⁺, we recorded I_Kir2.1 from excised inside-out giant patches. The I_Kir2.1 at positive and negative voltages were recorded in the absence of intracellular Mg²⁺ and polyamines. Fig. 6 shows the effects of 0, 0.1, and 3μM [Ba²⁺]_i on the I_Kir2.1 recorded at +50 and −50mV (Figure 6A) and at +30 and −30mV (Figure 6B) from a holding potential of 0mV and a prepulse voltage=−40mV. As previously described (Shieh et al), control I_Kir2.1 ([Ba²⁺]_i=0) at +50mV showed inactivation. In contrast, the current recorded at −50mV was nearly constant throughout the 1.2-s period. The perfusion of the intracellular side of the membrane with 0.1 and 3μM [Ba²⁺] resulted in reductions of the currents at the end of depolarization to +50 or +30mV and acceleration of the inactivation of the outward I_Kir2.1. In contrast to the effects on outward I_Kir2.1, 0.1 and 3μM [Ba²⁺]_i caused no change in the inward I_Kir2.1 recorded at −50 or −30mV. All of these effects were reversible upon Ba²⁺ removal.

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Figure 6

Blockade of Kir2.1 channels by intracellular Ba²⁺. I_Kir2.1 was recorded at +50 and −50mV (A) and at +30 and −30mV (B) in the presence of 0, 0.1, and 3μM [Ba²⁺]_i. The residual current obtained in the presence of 30mM intracellular TEA was used to subtract leak and capacitative components. The horizontal lines indicate the zero current level. (C) Normalized I-V relationships in the presence of 0 (control, ●, n=5), 0.1 (○, n=5), and 3μM [Ba²⁺]_i (▾, n=5). I_Kir2.1 measured at the end of the pulse was normalized to the values obtained at −100mV in the absence of Ba²⁺. The frequency of stimulation was 0.15Hz, and the sampling rate was 5kHz.

The normalized current-voltage relationships in the presence of 0, 0.1, and 3μM [Ba²⁺] are shown in Figure 6C. The normalized I_Kir2.1 under control condition showed inward rectification at positive potentials, reflecting the presence of intrinsic inactivation. Intracellular Ba²⁺ induced a dose-dependent and V_m-dependent inhibition of the outward currents.

Figure 7A displays from right to left the dose-response curves for the I_Kir2.1 blockade by intracellular Ba²⁺ at +10, +20, +30, +40, +60, and +80mV. The currents recorded at the end of the 1.2-s pulses in the presence of intracellular Ba²⁺ were normalized to the control at each voltage and expressed as fractional I_Kir2.1. Relations of the fractional I_Kir2.1 versus [Ba²⁺]_i were fitted by the Hill equation (Eq. (1)). Figure 7B shows that K_d obtained from fitting in Figure 7A was steeply dependent on V_m between +10 and +40mV, and the K_d-V_m relationship fit well with the Boltzmann equation (Eq. (2)). The K_d(0) was 91μM, and the δ value was 1.79, indicative of the multiionic pore feature of the Kir2.1 channel (Armstrong et al,Cecchi et al). In contrast, K_d showed a slight tendency to increase at V_m≥+40mV.

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Figure 7

Dose-response curves and voltage dependence of Kir2.1 channel blockade by intracellular Ba²⁺. (A) Dose-response curves at +10 (●), +20 (○), +30 (▾), +40 (▿), +60 (■), and +80 (□) mV were constructed for intracellular Ba²⁺ blockade of steady state I_Kir2.1. Data were fitted with the Hill equation (Eq. (1), continuous lines). The fit K_d and n values were 0.32μM, 0.69, V_m=+80mV; 0.10μM, 0.61, V_m=+60mV; 0.19μM, 0.63, V_m=+40mV; 1.09μM, 0.83, V_m=+30mV; 4.91μM, 1.07, V_m +20mV; 22.00μM, 0.87, V_m +10mV. (B) Voltage-dependence of K_d. K_d at V_m between +10 and +40mV were fitted (continuous line) with the Boltzmann equation (Eq. (2)) with K_d (0)=91μM and zδ=3.57.

It was previously shown that the inactivation of outward Kir2.1 currents recorded at V_m≥+40mV followed a double-exponential time course and that this inactivation may be due to a pH_i-sensitive intrinsic gating movement (Shieh et al). To analyze the effects of intracellular Ba²⁺ on the two time constants that describe the inactivation of the outward I_Kir2.1, currents recorded at V_m≥+40mV were fitted with a double-exponential function, and the time constants were extracted at each potential and [Ba²⁺]_i. The time constants τ₁ (fast, Figure 8A) and τ₂ (slow, Figure 8B) calculated from currents recorded in various Ba²⁺ were normalized to the time constants calculated in the absence of Ba²⁺ and are shown as a function of membrane potential. Both τ₁ and τ₂ were decreased by intracellular Ba²⁺ in a dose-dependent but voltage-independent manner. These results suggest that once intracellular Ba²⁺ was bound to the channel, it interfered with the intrinsic gating process of the channel in a manner that accelerated the inactivation process.

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Figure 8

Acceleration of outward I_Kir2.1 inactivation by intracellular Ba²⁺. The τ_1,Ba/τ_1,Ctrl-V_m relationship (A) and τ_2,Ba/τ_2,Ctrl-V_m relationship (B) for 0.03 (●, n=3), 0.1 (○, n=11), 1.0 (▾, n=11), and 10 (▿, n=8) μM [Ba²⁺]_i.

Discussion

Blockade of Kir2.1 channels by extracellular Ba²⁺

The blockade of inward rectifier K⁺ channels by extracellular Ba²⁺ has been extensively studied in starfish eggs (Hagiwara et al) and in frog skeletal muscle fibers (Standen and Stanfield, 1978). These studies showed that extracellular Ba²⁺ blocked the inward currents in a V_m- and time-dependent manner. The K_d(0) values for Ba²⁺ blockade of the inward rectifier K⁺ channels in starfish egg and frog skeletal muscle were 560 and 500μM, respectively (Hagiwara et al,Standen and Stanfield, 1978). The effects of extracellular Ba²⁺ on these tissues were interpreted as the binding of Ba²⁺ to sites located within the pores of the channels where they sensed 64–70% of the electrical field (Hagiwara et al,Standen and Stanfield, 1978). The K_d(0) value obtained for the extracellular Ba²⁺ blockage of the Kir2.1 channels was 62μM, suggesting that cloned Kir2.1 channels are about one order of magnitude more sensitive to extracellular Ba²⁺ than the native inward rectifier K⁺ channels in starfish egg or in frog skeletal muscle. Furthermore, the apparent electrical distance (δ=0.54) in Kir2.1 channels was smaller in the oocyte expressed channels than in the native channels.

Our results demonstrated that extracellular Ba²⁺ entered the pores to induce blockade of Kir2.1 channels as previously described in cloned and native inward rectifier K⁺ channels. In addition, we showed that the K_d-V_m relationship became less steep as V_m<−120mV, and k_off values increased when V_m<−130mV. These observations are similar to those reported in the high-conductance Ca²⁺-activated K⁺ channels (Neyton and Miller, 1988b) and Skaker K⁺ channels (Harris et al) and suggest that extracellularly applied Ba²⁺ can dissociate into the intracellular space at very hyperpolarizing potentials. On the other hand, k_off estimated from recovery of Ba²⁺ blockade increased as V_m became more positive. This suggests that the majority of Ba²⁺ dissociated into the extracellular space in a V_m-dependent manner.

Interactions of intracellular cations with Ba²⁺ applied extracellularly

Consistent with the previous finding that permeant K⁺ can compete with Ba²⁺ for binding within the pores of the Ca²⁺-activated K⁺ channels (Vergara and Latorre, 1983), we demonstrated that in Kir2.1 channels, increasing [K⁺]_i from 20 to 200mM increased the rate of recovery from extracellular Ba²⁺ blockade by decreasing the Ba²⁺ entrance rate (Figure 3C and Table 1). We also found that when an inside-out patch was perfused with Mg²⁺-free and polyamine-free control solution, the recovery from extracellular Ba²⁺ (10μM) blockade was much faster than in whole cells or cell-attached patches. One of the intracellular factors that contributed to the slow recovery from extracellular Ba²⁺ blockade in cell-attached patches was identified as spermine. We found that although spermine carries a +4 charge, it was not as efficient as intracellular K⁺ or Mg²⁺ in facilitating the dissociation of the bound Ba²⁺ from the pore of a Kir2.1 channel at V_r=+100mV. Note that in the presence of extracellular Ba²⁺, spermine (100μM) was still able to completely block the outward I_Kir2.1 at V_r=+100mV, which indicates that spermine bound to its binding site(s) in the pore of the channel. These results provide evidence that a spermine binding site(s) is distinct from that for K⁺ or Mg²⁺.

In summary, the interactions of extracellular applied Ba²⁺ with other cations in the pore of the Kir2.1 channel are very complicated. So far we have identified a common binding site for K⁺ and Ba²⁺ and a distinct site for spermine in the Kir2.1 channel. Further investigation is required to determine whether Kir2.1 channels have Ba²⁺ lock-in and enhancement sites lined up with the Ba²⁺ binding site similar to those described in the high-conductance Ca²⁺-activated K⁺ channels (Neyton and Miller, 1988b).

Blockade of Kir2.1 channels by intracellular Ba²⁺

Intracellular Ba²⁺ at submicromolar concentrations blocked and accelerated the rate of inactivation of the outward I_Kir2.1. [Ba²⁺]_i higher than 10μM resulted in complete inhibition of outward currents with very little blockade of the inward currents activated by hyperpolarization. All of these effects were observed in the absence of intracellular Mg²⁺ and polyamines. Figure 7B shows that K_d values for the inhibition of the outward currents decreased sharply as the depolarization increased until a minimum value of 0.16±0.07μM was reached at +50mV. As depolarization became more positive, K_d for intracellular Ba²⁺ blockade of the outward I_Kir2.1 increased. This may be due to the dissociation of Ba²⁺ into the extracellular space facilitated by depolarization, as previously shown for Na⁺ permeating through the K⁺ channels of the squid giant axon (French and Wells, 1977). Alternatively, this may be a result of the limitation of Ba²⁺ access to its binding site when the intrinsic inactivation gate closed the channel. Our experimental data also demonstrate that the exposure of the intracellular side of the channels to Ba²⁺ results in an acceleration of the inactivation at positive voltages. It is tempting to propose that Ba²⁺ may directly interact with the intrinsic gating mechanism responsible for the inactivation of Kir2.1 channels at positive voltages. The possibility of Ba²⁺ interaction with the intrinsic gate renders it a useful tool for probing the movement of the intrinsic gate of the Kir2.1 channels.

In conclusion, in this study we report the voltage-dependent blocking effects of both intracellular and extracellular Ba²⁺ on the cloned inward rectifier K⁺ channel Kir2.1. Ba²⁺ applied extracellularly can enter the pore of a Kir2.1 channel and dissociate into the intracellular space at very negative membrane potentials. Our study of the effects of intracellular cations on the recovery from Ba²⁺ blockade suggest that various cations entering the pore of a Kir2.1 channel may occupy different binding sites. Whereas Mg²⁺ produced a degree of efficiency similar to that of K⁺ in relieving Ba²⁺ blockade, spermine bound in the channel was much less effective in enhancing the dissociation of Ba²⁺ from the pore. We also showed that intracellular Ba²⁺ closely interacts with the “intrinsic” gate and may be applied to reveal the characteristics of this “intrinsic” gating mechanism in Kir2.1 channels.

Acknowledgement

We thank Dr. Patricia Pérez (UASLP, Mexico) and Dr. Jim Weiss (UCLA, USA) for their critical reading of the manuscript and helpful comments on this study and Mr. Douglas Platt for editing the English of the manuscript.

This work was supported by National Science Council grant 86-2314-B-001-025; Academia Sinica, Taiwan, R.O.C.; and the Fundación Mexicana para la Salud and Fondo de Apoyo a la Investigación, UASLP, Mexico.

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Received: January 21, 1998
Revised: July 28, 1998

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Interaction of Ba2+ with the Pores of the Cloned Inward Rectifier K+ Channels Kir2.1 Expressed in Xenopus Oocytes

Abstract

Introduction

Materials and methods

Molecular biology and preparation of Xenopus oocytes

Electrophysiology techniques

Data analysis

Results

The blockade of inward IKir2.1 by extracellular Ba2+

Kinetics of the extracellular Ba2+-induced blockade of Kir2.1 channels

Recovery of Kir2.1 channels from extracellular Ba2+ blockade

Effects of intracellular K+ on the recovery from extracellular Ba2+ blockade

Spermine contributes to the slow recovery from extracellular Ba2+ blockade in cell-attached patches

Blockade of Kir2.1 channels by intracellular Ba2+

Discussion

Blockade of Kir2.1 channels by extracellular Ba2+

Interactions of intracellular cations with Ba2+ applied extracellularly

Blockade of Kir2.1 channels by intracellular Ba2+

Acknowledgement

References

Publication Information

Article Outline

Interaction of Ba²⁺ with the Pores of the Cloned Inward Rectifier K⁺ Channels Kir2.1 Expressed in Xenopus Oocytes

The blockade of inward I_Kir2.1 by extracellular Ba²⁺

Kinetics of the extracellular Ba²⁺-induced blockade of Kir2.1 channels

Recovery of Kir2.1 channels from extracellular Ba²⁺ blockade

Effects of intracellular K⁺ on the recovery from extracellular Ba²⁺ blockade

Spermine contributes to the slow recovery from extracellular Ba²⁺ blockade in cell-attached patches

Blockade of Kir2.1 channels by intracellular Ba²⁺

Blockade of Kir2.1 channels by extracellular Ba²⁺

Interactions of intracellular cations with Ba²⁺ applied extracellularly

Blockade of Kir2.1 channels by intracellular Ba²⁺