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Department of Physiology, Saga Medical School, Saga 849-8501, Japan
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Abstract |
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20mV more positive than the reversal potential, with a negative slope at more positive voltages. The relationships exhibited a plateau or a double-hump shape with 1µM spermine/spermidine or 0.1µM spermine, respectively. In the chord conductance–voltage relationships, there were extra conductances in the positive voltage range, which could not be described by the Boltzmann relations fitting the major part of the relationships. The extra conductances, which generated most of the outward currents in the presence of 5–10µM spermine or 10–100µM spermidine, were quantitatively explained by a model that considered two populations of Kir2.1 channels, which were blocked by polyamines in either a high-affinity mode (Mode 1 channel) or a low-affinity mode (Mode 2 channel). Analysis of the inward tail currents following test pulses indicated that the relief from the spermine block of Kir2.1 consisted of an exponential component and a virtually instantaneous component. The fractions of the two components nearly agreed with the fractions of the blockages in Mode 1 and Mode 2 calculated by the model. The estimated proportion of Mode 1 channels to total channels was 0.9 with 0.1–10µM spermine, 0.75 with 1–100µM spermidine, and between 0.75 and 0.9 when spermine and spermidine coexisted. An interaction of spermine/spermidine with the channel at an intracellular site appeared to modify the equilibrium of the two conformational channel states that allow different modes of blockage. Our results suggest that the outward IK1 is primarily generated by channels with lower affinities for polyamines. Polyamines may regulate the amplitude of the outward IK1, not only by blocking the channels but also by modifying the proportion of channels that show different sensitivities to the polyamine block.
(Received 19 September 2003;
accepted after revision 6 January 2004;
first published online 14 January 2004)
Corresponding author K. Ishihara: Department of Physiology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan. Email: keiko{at}post.saga-med.ac.jp
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Introduction |
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Recent progress in understanding the mechanism of the inward rectification (i.e. the property of the channel to open when the direction of the K+ flux is inward and close when the direction of the K+ flux is outward) of IK1 has been achieved from extensive studies on the cloned strong inward rectifier K+ channels in the Kir2 subfamily (for a review, see Lopatin & Nichols, 2001; Stanfield et al. 2002). It is generally accepted that strong rectification is the result of a voltage-dependent blockage of the channel by cytoplasmic spermine and spermidine, which are ubiquitous polyamines in animal cells (Ficker et al. 1994; Lopatin et al. 1994; Fakler et al. 1995). Currently, the blockage of the channel by cytoplasmic Mg2+ (Matsuda et al. 1987; Vandenberg, 1987) is considered to play a role in increasing the amplitude of the outward IK1 during repolarization by inducing an outward transient of IK1 (Ishihara & Ehara, 1998; Ishihara et al. 2002).
Despite the above information, the flow of the outward IK1 during cardiac action potentials has not been quantitatively accounted for by the blockage of the channel by the cytoplasmic blockers. This is chiefly because the mechanism that generates the sustained outward current of IK1 in the voltage range between Vrev and a voltage that is 60mV more positive than Vrev remains unclear. The inward currents of the strong inward rectifier K+ currents show a time-dependent activation phase, following an instantaneous current jump upon hyperpolarization (Hagiwara et al. 1976; Leech & Stanfield, 1981). The parameter of the time-dependent (activation) gating of IK1, which is determined by analysing the amplitude of the activation phase, shows full ‘deactivation’ at a voltage that is 30mV more positive than Vrev; thus, the flow of the outward IK1 is not accounted for by this gating (Ishihara et al. 1989; Oliva et al. 1990). The time-dependent gating of Kir2.1, which strikingly resembles that of IK1, is caused by the blockage of the channel by cytoplasmic spermine (Ishihara et al. 1996). Thus, it has been suggested that intracellular blockers of IK1, other than spermine, play an important role in generating the outward currents. We previously demonstrated that the blockage of IK1 by cytoplasmic Mg2+ and its relief are practically instantaneous (Ishihara et al. 1989). Since the Mg2+ block traps the IK1 channel in subconductance states (Matsuda, 1988), the contribution of the Mg2+ block to the outward IK1 has been considered (Matsuda, 1988; Ishihara et al. 1989; Oliva et al. 1990). However, the steady-state outward current–voltage (I–V) relationship of the strong inward rectifiers is not significantly affected by varying the cytoplasmic free Mg2+ concentration (Silver & DeCoursey, 1990). The steady-state outward I–V relationships of the whole-cell IK1 measured in the previous studies, with different concentrations of cytoplasmic Mg2+, were all similar to each other, i.e. the relationships showed a peak current at a voltage that is 20mV more positive than Vrev and a negative slope at more positive voltages, whether the currents were measured using pipette solutions containing very low concentrations of Mg2+ (Matsuda & Noma, 1984; Tourneur et al. 1987; Giles & Imaizumi, 1988; Oliva et al. 1990), using those containing 1mM Mg2+ (Ishihara & Ehara, 1998), or using the perforated-patch method (Ishihara et al. 2002). Thus, these observations contradict the notion that the Mg2+ block contributes significantly to the mechanism generating the sustained outward IK1. The time courses of the relief from the blockages of the Kir2 channels by spermidine and putrescine (the immediate precursor of spermine and spermidine) have been demonstrated to be much faster than the relief from the spermine block (Kir2.3, Lopatin et al. 1995; Kir2.1, Ishihara et al. 1996). Previously, we demonstrated that putrescine can induce an outward transient of Kir2.1, as can Mg2+ (Ishihara, 1997). However, the low putrescine content in animal cells (see, for example, He et al. 1993) (with the exception of stage V–VI Xenopus oocytes that contain a high level of putrescine, but only a background level of spermine; Shinga et al. 1996) implies that putrescine does not contribute significantly to the inward rectification of IK1. Thus, spermidine was the possible candidate as the molecule that plays an important role in generating the outward IK1.
The Kir2.1 channel (IRK1; Kubo et al. 1993) is considered to provide the major fraction of IK1 in cardiac ventricular cells (Nakamura et al. 1998; Zaritsky et al. 2001), and the properties of Kir2.1 channels, examined by the whole-cell recordings, are very similar to those of IK1 (Stanfield et al. 1994a; Ishihara et al. 1996). In this study, we expressed the Kir2.1 channel in HEK 293T cells and analysed the macroscopic currents recorded from excised inside-out patches in the presence of various concentrations of cytoplasmic polyamines. Contrary to our expectation, the outward I–V relationship and the time-dependent gating of the Kir2.1 channel, in the presence of 5µM spermine alone, are similar to those of IK1. The analysis of the chord conductance–voltage (G–V) relationships in the present study strongly suggests that a small population of channels is less sensitive to the polyamine block than the other channels. Our results suggest that the steady-state outward I–V relationship of IK1 is chiefly determined by the spermine block of the channels that exhibit lower affinities to cytoplasmic polyamines.
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Methods |
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The mouse IRK1 gene (a gift from Professor L.Y. Jan, University of California, San Francisco, CA, USA), previously subcloned into the mammalian expression vector pCXN2 (Ishihara et al. 1996), was used for the present study. HEK 293T cells (derived from the human embryonic kidney cell line 293 and containing the SV 40 large T antigen) were grown in Dulbecco's modified Eagle's medium (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 10% heat-inactivated fetal bovine serum (Gibco-BRL), 0.15% sodium bicarbonate, penicillin (200Uml–1), and streptomycin (200µgml–1). Cells were plated at 1.5 x 105 cells per 30mm dish, 1 day prior to transfection. The cells were transfected with 0.4µg of pCXN2-IRK1 and 0.04µg of plasmid DNA encoding the green fluorescent protein (pEGFP-N1; Clontech, Palo Alto, CA, USA) using an Effectene transfection reagent (Qiagen Inc., CA, USA). Autofluorescence was visualized using an inverted fluorescence microscope to identify the cells expressing exogenous genes.
Solutions
The pipette (extracellular) solution contained (mM): 145 KCl, 1 CaCl2 and 5 Hepes (pH 7.4 with 2mM KOH). The bath solution used as the control Mg2+-free, polyamine-free cytoplasmic solution contained (mM): 125 KCl, 4 EDTA (2K), 7.2 K2HPO4 and 2.8 KH2PO4 (pH 7.2 with 3mM KOH). The free Mg2+ and Ca2+ concentrations in this solution were calculated to be at submicromolar levels (Fabiato & Fabiato, 1979), assuming that the amounts of Ca2+ and Mg2+ contained in the solution were 10µM each. The stock solutions, containing 10mM of spermine/spermidine, were prepared by dissolving spermine-4Cl (Nacalai Tesque, Kyoto, Japan) or spermidine-3Cl (Nacalai Tesque) in distilled water and were stored in small aliquots at –20°C. Before the experiment the cytoplasmic solutions, containing various concentrations of spermine/spermidine, were prepared by diluting these stock solutions with the control Mg2+-free, polyamine-free cytoplasmic solution to the desired concentrations.
Current recordings from HEK 293T cells expressing Kir2.1
On the day of transfection or on the day of recording the data, the cells were trypsinized using 0.25% trypsin–EDTA solution (Sigma) and seeded onto small pieces of collagen-coated coverglass (Asahi Techno Glass Corporation, Tokyo, Japan), such that they could be placed in the recording chamber (area: 5mmx20mm; volume: 0.5ml), which was mounted on the stage of an inverted fluorescence microscope (TMD300; Nikon, Tokyo, Japan).
At 24–56 h after the transfection, currents were recorded from inside-out patch membranes by the conventional patch-clamp technique (Hamill et al. 1981) using a patch-clamp amplifier (Axopatch 200B, Axon Instruments, Union City, CA, USA). Before the experiment, pipettes made from hard borosilicate glass capillaries (1.5mm o.d., 1.0mm i.d.; Narishige, Tokyo, Japan) using a horizontal puller (Sutter Instruments, Novato, CA, USA) were coated with silicone (Shin-Etsu Chemical, Tokyo, Japan) and then heat-polished. The resistance of the electrodes filled with the pipette solution was 2–2.5M
. The recording chamber was continuously perfused with a fresh bath solution at the rate of 3mlmin–1, and the excised patch membrane was located near the site where the bath solution flowed into the recording chamber. The effects of the cytoplasmic polyamines were examined after the native inward rectification had been removed to as great an extent as possible in the control Mg2+-free, polyamine-free cytoplasmic solution. Voltage stimulations and data acquisitions were performed using pCLAMP8 software (Axon Instruments) on a Pentium PC through a 12 bit AD converter (Digidata 1200A, Axon Instruments). Currents were filtered at 5–10kHz and sampled at 25–50kHz. Capacitive currents were compensated for by using the patch-clamp amplifier. The holding potential was 0mV. The horizontal dotted lines superimposed on the current traces indicate the zero-current level. To measure I–V relationships, test pulses were usually applied between –60 and +90mV in 5mV steps. A hyperpolarizing prepulse to –40mV was used before applying a test pulse, to relieve most of the channels from blockage by cytoplasmic Kir2.1 blockers. All experiments were conducted at room temperature (24–26°C).
Analysis of Kir2.1 currents
To evaluate the blockage of Kir2.1 currents by polyamines, we used the chord conductance values calculated from the I–V relationships using the equation G=I/(V – Vrev). Current amplitudes were usually measured at 200ms after the onset of the step pulses. Vrev values were determined using a ramp pulse (0.4–4 V s–1) and were always near 0mV (0–2mV), in agreement with the K+ equilibrium potential of 1mV predicted from the experimental conditions. The small discrepancy could be partially attributed to the junction potential between the pipette and the cytoplasmic solutions, which was not corrected for in this study. For the results, conductance values were normalized with respect to the maximum value obtained using large negative pulses (G/Gmax). In the cases where the application of relatively high concentrations of polyamines reduced the amplitude of the inward currents in the entire negative voltage range measured (Fig. 2), the conductance values were normalized with the maximum conductance value that had been obtained in the absence of or with lower concentrations of polyamines.
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Least-squares fits were performed using the algorithms incorporated in Microcal Origin (ver. 6, Microcal Software, Northampton, MA, USA) and pCLAMP software. The statistical values are given as the means ±S.E.M. (n, number of experiments).
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Results |
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We first tried to identify the spermine and spermidine concentrations at which the Kir2.1 channel exhibits outward I–V relationships similar to the steady-state outward I–V relationship of IK1. Figure 1A shows Kir2.1 currents recorded from inside-out patch membranes by applying different amounts of spermine or spermidine to the cytoplasmic solution after the native inward rectification had been removed to as great an extent as possible in the control Mg2+-free, polyamine-free cytoplasmic solution. In the presence of 0.1–10µM spermine (Fig. 1Aa) or 1–100µM spermidine (Fig. 1Ab), the outward currents were rapidly suppressed during the depolarizing steps. Figure 1Ba and b show the outward I–V relationships obtained with 0.1–10µM spermine and 1–100µM spermidine, respectively. The current amplitudes in these plots are normalized with respect to the inward current amplitude at –40mV measured with 0.1µM spermine or 1µM spermidine that did not notably block the currents at –40mV. The amplitudes of the outward currents observed with these concentrations of spermine were smaller than those obtained with approximately 10-fold concentrations of spermidine. The steady-state I–V relationship of the outward currents obtained with 0.1µM spermine showed two distinct peaks (see also Guo & Lu, 2000a) and that with 1µM spermine or spermidine showed a plateau. These I–V relationships were different in shape from the steady-state I–V relationship of IK1. All the outward I–V relationships obtained with 5–10µM spermine and with 10–100µM spermidine showed a peak at a voltage near Vrev+20mV and a negative slope at more positive voltages, similar to the steady-state outward I–V relationship of IK1 (e.g. Ishihara et al. 2002).
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To examine the voltage dependence of the polyamine block of the Kir2.1 channel, we analysed the G–V relationships of the Kir2.1 currents recorded in the presence of 0.1–10µM spermine and 1–100µM spermidine (Fig. 2). The G–V relationships shifted towards the left as the polyamine concentration increased, in agreement with the voltage-dependent blockage of the channel by these molecules. The half-blocking voltages (V1/2) of spermine were +12.5mV (0.1µM), +1mV (1µM), –8mV (5µM), and –13mV (10µM), and those of spermidine were +16.5mV (1µM), +4.5mV (10µM), –3mV (30µM), and –14.5mV (100µM).
Further, two other features were noted in the G–V relationships. First, with relatively high concentrations of the polyamines (10µM spermine, 30µM spermidine, and 100µM spermidine), conductances at the negative voltages (<–20mV) became small and the relationships in this voltage range exhibited a weak voltage dependence. These findings resulted from the apparent shift of the inward I–V relationships to the negative side (data not shown; see Xie et al. 2002). G/Gmax values at –60mV were 0.92 ± 0.007 (n= 7), 0.95 ± 0.005 (n= 4), and 0.85 ± 0.01 (n= 4) with 10µM spermine, 30µM spermidine, and 100µM spermidine, respectively.
Second, as the polyamine concentration decreased, conductances at voltages more positive than V1/2+10mV increased, which was observed even more clearly by plotting the G–V relationships on a semilogarithmic scale (Fig. 2, lower panels). As shown by the continuous lines, the strong voltage dependence of the conductances observed around V1/2 can be described by the Boltzmann relation:
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| (1) |
10mV, there were ‘extra conductances’ that could not be described by the fitted Boltzmann relations. These extra conductances corresponded to the majority of the outward currents that flowed in the presence of 5–10µM spermine/10–100µM spermidine (Fig. 1B). In the case of the G–V relationships obtained with spermine, it appeared that the extra conductances could be fitted with the Boltzmann relation, in which the maximum level was 0.1, and the Vh value increasingly became more positive than the V1/2 value as the concentration decreased (fits not shown, but see the top panel of Fig. 3F). This finding was essentially the same in the case of spermidine, although the maximum level of extra conductances seemed to be larger, i.e. 0.2–0.3. These observations led us to hypothesize that polyamines may block the Kir2.1 channel in two distinct modes, a high-affinity mode and a low-affinity mode. The steep voltage dependence of the G–V relationships (fitted with Boltzmann relations in lower panels of Fig. 2) may reflect the polyamine block of the major fraction of channels in the high-affinity mode. On the other hand, extra conductances may be generated by a fraction of channels that were blocked by polyamines in the low-affinity mode. The ratio of the channels accepting respective modes of blockage did not appear to be significantly affected by the concentration of polyamines, whereas the ratio was affected by the species of polyamine present in the cytoplasmic solution.
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We assumed the following simplified model for the high-affinity mode (Mode 1) and low-affinity mode (Mode 2) of the polyamine block of the Kir2.1 channel:
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| (2) |
are the voltage-dependent block and unblock rate constants, respectively. According to our hypothesis, the steady-state G–V relationship is calculated by the following equation:
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| (3) |
is the maximum value of the fractional conductance generated by the Mode 1 channels (defined as the channels accepting only the Mode 1 block), and Kd1(PA)(V) and Kd2(PA)(V) are the voltage-dependent dissociation constants (Kd(V)) of the spermine/spermidine block in Mode 1 and Mode 2, respectively. The value is equivalent to the proportion of Mode 1 channels to total channels if the unit conductances of the Mode 1 channels and Mode 2 channels (the channels accepting only the Mode 2 block) are the same.
To examine whether or not the above hypothesis is feasible, we obtained Kd1(PA)(V) and Kd2(PA)(V) values that would be able to explain the data. To gain estimates of the Kd1(PA)(V) values of the spermine (spm) and spermidine (spd) block (Kd1(spm)(V) and Kd1(spd)(V)), dose–response relationships were analysed in the voltage range around 0mV, where the G–V relationships showed a strong voltage dependence (Fig. 3A). The Kd2(PA)(V) values of the spermine and spermidine block (Kd2(spm)(V) and Kd2(spd)(V)) were estimated from dose–response relationships at voltages between +40 and +70mV, normalized with respect to the maximum conductance value in this voltage range (Fig. 3B), since conductances in this voltage range appeared to be generated mostly by the Mode 2 channels at the examined concentrations (Fig. 2, lower panels). The Kd values obtained from the dose–response relationships decreased exponentially as the voltage became more positive (Fig. 3C, symbols). Based on these values, we determined the voltage dependences of Kd1(spm)(V), Kd1(spd)(V), Kd2(spm)(V), and Kd2(spd)(V) (Fig. 3C, lines) and the values of 0.9 for the spermine block and 0.75 for the spermidine block, which resulted in good fits of the G–V relationships with eqn (3), except for the decrease in the conductances at the negative voltages observed with 10µM spermine or 30–100µM spermidine (Fig. 3F). The Kd1(spm)(V), Kd1(spd)(V), Kd2(spm)(V) and Kd2(spd)(V) values could be expressed using the following equation (Woodhull, 1973):
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| (4) |
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| (5) |
value of 0.9, the fractional blockage of the currents in each mode in the presence of 5µM spermine is calculated as shown in Fig. 3E. The fractional block in Mode 1 saturates at voltages more positive than 20mV, whereas the fractional block in Mode 2 increases at voltages more positive than 0mV, causing the complete blockage of currents at voltages more positive than 60mV. Figure 4A and B shows the I–V relationships at various spermine/spermidine concentrations, reconstructed using calculated G/Gmax values. The model reproduced the outward I–V relationships obtained in the experiments (Fig. 1B). With either 5–10µM spermine or 10–100µM spermidine, the outward I–V relationships of Kir2.1 were similar to the steady-state outward I–V relationship of IK1. At these concentrations, the majority of the outward currents were reconstructed by the Mode 2 channels (e.g. Fig. 4C).
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The inward currents of Kir2.1 observed by the whole-cell recordings exhibited an exponential phase following a virtually instantaneous increase upon hyperpolarization, similar to that obtained for IK1, and the exponential phase was considered to reflect the relief from the spermine block of the channel (Ishihara et al. 1996). However, if two modes of blockage are indeed involved in the polyamine block of Kir2.1, the time course of the inward currents reflecting the relief from the polyamine block may contain two components. In the experiment shown in Fig. 5, we analysed the inward tail currents observed following test pulses in the presence of 5µM spermine. This analysis was not performed with spermidine, since the relief from the spermidine block of the Kir2.1 channel was rapid, thus rendering it difficult to analyse its time dependence (Ishihara et al. 1996; Xie et al. 2002). Following test pulses within a voltage range more negative than +20mV (–15, –10, –5 and +15mV in Fig. 5A, left column), the time-dependent fraction of the inward tail currents at –30mV increased as the voltage during the test pulse became more positive (top panel). The majority of the time-dependent components could be fitted by a single exponential function with the same time constant of 0.74ms (middle panel), and the amplitude of this exponential component (ITime) increased, as demonstrated by the theoretical curves (bottom panel). Following test pulses at voltages more positive than +20mV (+20, +40, +60 and +80mV in Fig. 5A, right column), the amplitude of the exponential component saturated (top and middle panels) and failed to explain the whole inward current amplitude at –30mV (IMax; bottom panel). It should be noted that there were no outward currents corresponding to the instantaneous currents at –30mV during the preceding test pulses (top right panel).
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20mV. The voltage dependence of the exponential fraction could be approximated by a single Boltzmann relation (continuous line in Fig. 5B; Vh=–9.8mV, s=–5.7mV, maximum level = 0.84), which was similar to the Boltzmann relation describing the blockage of the Mode 1 channel by 5µM spermine (eqn (5)). On the other hand, in the positive voltage range, the fractional block of currents at the end of the test pulses, estimated from the conductance measurements (1 –G/Gmax; 
in Fig. 5B), was evidently larger than the fraction of the blockage that was relieved with an exponential time course (•). This finding clearly indicated that the time course of the relief from the spermine block consisted of not only an exponential component but also a much faster component (
in Fig. 5B). The virtually instantaneous fraction () increased in the voltage range between 0mV and 60mV, which was analogous to the voltage dependence of the fractional block in Mode2, calculated by the model (Fig. 3E). These findings suggested that the exponential component and the instantaneous component reflect the time courses of the relief from the spermine block in Mode1 and Mode2, respectively. Essentially the same findings were obtained in analyses of the currents obtained from different patches in the presence of 1 or 5µM spermine (data not shown). These results supported the model we employed to explain the G–V relationships of Kir2.1, obtained in the presence of polyamines. Kir2.1 currents under conditions in which spermine and spermidine coexist
Although the outward I–V relationships of Kir2.1 currents obtained in the presence of either 5µM spermine or 30µM spermidine alone (Fig. 1B) were similar to that of IK1 in the steady state, both species of polyamine coexist in the cytoplasm. Next, we examined the outward I–V relationships of Kir2.1 obtained under conditions in which spermidine coexisted with spermine (Fig. 6A). When 10 or 30µM spermidine coexisted with 5µM spermine, the amplitude of the outward currents decreased slightly throughout the entire voltage range examined (left panel). However, when 2, 5, or 30µM spermidine coexisted with 1µM spermine, the amplitude of the outward currents increased at certain voltages (around +20mV), which caused the crossover of the outward I–V relationships obtained in the presence and absence of spermidine (centre and right panels). Moreover, the peak amplitude of the outward currents observed with 1µM spermine + 5µM spermidine was larger than that with 1µM spermine + 2µM spermidine.
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| (6) |
value (the fraction of Mode 1 channels) of 0.9 or 0.75, respectively (Figs 3F and 4). When spermidine coexisted with spermine, the outward I–V relationships were found to be well reconstructed with the same Kd(V) values by reducing the value, to lie below 0.9 and closer to 0.75, as the concentration of spermidine increased (Fig. 6B and D). For example, using a value of 0.89 for 1µM spermine + 2µM spermidine, 0.87 for 1µM spermine + 5µM spermidine, and 0.81 for 1µM spermine + 30µM spermidine, the increase in the amplitude of the outward currents observed at around +20mV could be well reproduced by the increase in the amplitude of the outward currents through the Mode 2 channels (Fig. 6C). This finding indicated that an increase in the spermidine concentration in the copresence of spermine might have two opposing effects on the outward currents: a decrease in the amplitude of the outward current due to an increase in the blockage of the Mode 1 and Mode 2 channels, and an increase in the amplitude of the outward current due to an increase in the fraction of the Mode 2 channels.
The above findings suggest that the probability of the Kir2.1 channel existing in either of the two conformational states that allow different modes of blockage (Mode1 state and Mode 2 state) is not fixed, but is altered by the interaction of spermine/spermidine with the channel at an intracellular site (Fig. 6E). The finding that the equilibrium of the two states did not notably change in the concentration range between 0.1 and 10µM for spermine and between 1 and 100µM for spermidine may be explained if the Kd values of the interaction were much smaller than 0.1µM with spermine and 1µM with spermidine. If we assume that the probabilities of the spermine- and spermidine-interacting channels to exist in the Mode 1 state are 0.9 and 0.75, respectively, and that spermidine and spermine compete for interaction with the channel, then the fractions of the spermine- and spermidine-interacting channels can be calculated from the values (Fig. 6D). The fractions of the spermine-interacting channels thus obtained could be explained by assuming that the Kd value of the spermidine–channel interaction is approximately 25-fold larger than that of the spermine–channel interaction. For example, the continuous lines in Fig. 6D show the fraction of the spermine-interacting channels, calculated using the Kd values of 0.002µM and 0.05µM for the spermine–channel and spermidine–channel interactions, respectively. By provisionally using these Kd values, the value is calculated as follows:
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| (7) |
The G–V relationships obtained in the copresence of spermine and spermidine (Fig. 7A) could be also reproduced by using the values that were able to reconstruct the outward I–V relationships, except for the decrease in the conductances in the negative voltages observed with 30µM spermidine (Fig. 7B). Figure 7C illustrates the fractional blockages of Mode 1 channels (upper panels) and Mode 2 channels (lower panels) caused by spermine and spermidine, calculated for 5µM spermine + 10µM spermidine (left) and 1µM spermine + 30µM spermidine (right). Under both conditions, the voltage dependence of the fraction blocked by spermine of the Mode 1 channels agreed well with the experimental data showing the relationship between the voltage of test pulses and the exponential fraction of the inward tail current at –30mV (Fig. 7C, ). These findings further justified the use of the model.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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) was 0.9 with 0.1–10µM spermine alone, 0.75 with 10–100µM spermidine alone, and between 0.75 and 0.9 when spermine and spermidine coexisted. This finding implied that an interaction of cytoplasmic spermine/spermidine with the channel at an intracellular site affects the equilibrium of the two conformational states of the channel that allow different modes of blockage. If we assume that spermine and spermidine compete for this interaction, the Kd value for the spermidine–channel interaction was calculated to be approximately 25-fold larger than that for the spermine–channel interaction. Our results suggest that the outward IK1 may largely flow through a small population of channels (Mode 2 channels) that are less sensitive to the polyamine block. Polyamines may regulate the amplitude of the outward IK1 by not only blocking the channel currents but also by changing the ratio of the Mode 1 and Mode 2 channels. Experimental conditions
The expression vector pCXN2 (Niwa et al. 1991) allowed such a high level of Kir2.1 channel expression in HEK 293T cells that the macroscopic currents could be recorded from patch membranes using pipette electrodes with the usual tip diameter (Kubo & Murata, 2001). The G–V relationship of the Kir2.1 channel expressed in L cells, obtained with 1µM spermine (Ishihara, 1997), and that expressed in Xenopus oocytes, obtained with 100µM spermidine (Yang et al. 1995), were nearly identical to the results in this study. Thus, a high expression of the channel did not influence the sensitivity of the channel to cytoplasmic polyamines.
Even long after exposing the patch membranes to the Mg2+-free, polyamine-free cytoplasmic solution, a slow decline in the outward currents was generally observed during long depolarizing pulses (Fig. 1Aa), as previously reported (Lopatin et al. 1995; Shieh et al. 1996). Recent studies, using the Xenopus oocyte expression system, have shown that this ‘spontaneous gating’ can be largely eliminated by using a phosphate buffer instead of Hepes and replacing EGTA with EDTA (Guo & Lu, 2000b, 2002). Although we could confirm that this spontaneous gating was significantly slower and less pronounced when a phosphate buffer was used, its complete removal was still unusual, as has been recently shown in the single-channel recordings of Kir2.1 (Matsuda et al. 2003). Since the blockage of Kir2.1 currents by polyamines at the examined concentrations was much more rapid and greater than the above-mentioned spontaneous gating, we considered that the gating scarcely interfered with the analysis of the currents in the present study.
Polyamine block of the strong inward rectifier K+ channels, Kir2, generating two phases in G–V relationships
The polyamine block of the strong inward rectifier K+ channels is not well understood because it shows complex features that are different in distinct concentration ranges (Lopatin et al. 1995; Guo & Lu, 2000a; Xie et al. 2002). In an analysis of the blockage of the inward currents of the Kir2.3 channel using high concentrations of polyamines (25–500µM spermine), Lopatin et al. (1995) were the first to demonstrate that the polyamine block has two components: a type of blockage showing a weak voltage dependence and an instantaneous time course (‘a shallow component’) and another type of blockage exhibiting a strong voltage dependence with time-dependent kinetics. The former component, which we also observed in the inward currents with relatively high concentrations of polyamines (Fig. 2), was recently attributed to a decrease in unit-channel conductance caused by the binding of polyamines to surface charges at the inner vestibule of the channel (Xie et al. 2002). Although this shallow component of the polyamine block may also affect the outward currents that flow in the presence of lower concentrations of polyamines, it does not readily account for the mechanism that generates extra conductances. Therefore, this effect was not considered in the present study.
Yang et al. (1995) demonstrated that the dose–response relationships of the polyamine block of Kir2.1 analysed at +40mV with symmetrical 140mM[K+] were fitted by the sum of two Hill equations (eqn (3) in this study). The Kd1 and Kd2 values at +40mV and the maximum fractional conductances of the two channel populations we obtained (0.16nM (0.9) and 0.49µM (0.1) for spermine, and 11nM (0.75) and 3.9µM (0.25) for spermidine; maximum fractional conductance in parentheses) were similar to those reported by Yang et al. (0.9nM (0.86) and 0.61µM (0.14) for spermine, 8nM (0.7) and 2.87µM (0.3) for spermidine). Thus, when we reconstructed the dose–response relationship of the spermine block at +40mV (Fig. 8), a hump of conductances appeared in the higher concentration range, as demonstrated by Yang et al. Since the channels expressed from a cloned gene are expected to be homogeneous, Yang et al. instead speculated that the Kd value for a single type of channel may have increased with higher concentrations of the blockers. They suggested that the channel might accommodate more than one blocking particle, which may in turn lead to negative cooperativity due to mechanisms such as electrostatic repulsion between the blockers. Indeed, they later provided evidence that the Kir2.1 channel can simultaneously accommodate three polyamines or Mg2+ in the wide inner pore of the channel (Lu et al. 1999). However, we found that the extra conductances, which may be generated by the repulsion between the blockers inside a channel, were larger with lower concentrations of polyamines (Fig. 2, lower panels). Further, the dose–response relationship obtained at 0mV could be approximated by a single Hill equation (Fig. 3A; see also Xie et al. 2002), which differs as regards the observation of the dose–response relationship obtained at +40mV (Fig. 8). These findings cannot be explained by the repulsion hypothesis unless we further hypothesize, for example, that the channel undergoes a voltage-dependent conformational change, which would then allow it to accommodate more than one blocking particle in the positive voltage range. On the other hand, if we hypothesize two types of channels that are blocked by polyamines in distinct modes (with different voltage dependencies and potencies), various shapes of G(I)–V relationships observed with different concentrations of polyamines could be quantitatively accounted for (Figs 3, 4, 6 and 7). The dose–response relationship at 0mV was also reconstructed (Fig. 8).
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| (8) |
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| (9) |
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| (10) |
is the voltage-dependent rate constant. The apparent dissociation constant Kd1(PA)(V) is (1+)/ß1, and it approximates to /ß1 if is large compared with 1. When the extra conductances in the G–V relationships were simulated using eqn (9) with this assumption (Fig. 9Ab), Kd1(spm) values in the positive voltage range, which may represent /ß1, significantly varied at different spermine concentrations (Fig. 9Aa). When the same set of Kd1(spm)(V) values was used to calculate the relationships at different spermine concentrations, the amplitude of the extra conductances greatly varied (Fig. 9B), which was obviously different from our finding that the maximum values of the extra conductances were similar in the concentration range between 0.1 and 10µM (Fig. 2, lower panels). Furthermore, the blockage at the low-affinity site, calculated by model (8), significantly increased at the expense of that at the high-affinity site in the positive voltage range (Fig. 9C). When we analysed the relationship between the voltage of the test pulses and the exponential fraction of the inward tail currents (Fig. 5B), which may indicate the fractional block at the high-affinity site in model (8), the exponential component always approached a limit at a submaximal level in the positive voltage range. This finding was consistent with model (2), which considers two types of channels that are blocked by polyamines in distinct modes (Fig. 3E).
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The voltage dependences of the spermine/spermidine block in Mode 1 were steeper than those in Mode2 (Fig. 3C). Thus, polyamines may move deeper into the pore in the case of the Mode 1 block. It has been demonstrated that polyamines plug the Kir2.1 channel pore at several distinct sites: at D172 in the M2 transmembrane domain (Stanfield et al. 1994b; Wible et al. 1994) and at glutamate residues E224 and E299 in the C-terminal region (Taglialatela et al. 1995; Yang et al. 1995; Kubo & Murata, 2001). It has been shown that the D172N mutant channel not only lacks the time-dependent gating mechanism (Stanfield et al. 1994b; Wible et al. 1994), but also shows G–V relationships fitted with a single Boltzmann relation in the presence of polyamines (Yang et al. 1995). Furthermore, the polyamine sensitivity of D172N channel is comparable to that of the Mode 2 channel obtained in this study or that of the wild-type channel at the low-affinity site (Yang et al. 1995). These findings may suggest that D172 forms the spermine-binding site for the Mode 1 block, and that the polyamine-binding site in the D172N channel (which is not fully explained by E224 and E299; Kubo & Murata, 2001) is nearly the same as the site for the Mode 2 block.
There may be two types of Kir channels that exhibit different sensitivities to the polyamine block. Indeed, it has been demonstrated by the single-channel analysis that there are Kir2.1 channels showing different sensitivities to the external Cs+ block (Picones et al. 2001). The pore structure of the Kir channel may be flexible to some extent, such that the channel undergoes transitions between the two conformational states, which allows the blockers to plug the pore at different sites. Our results suggest that the equilibrium between the two conformations is affected by the interaction of polyamines with the channel at an intracellular site (Fig. 6E). Thus, polyamines themselves may change the sensitivity of the channel to the polyamine block. The mechanisms underlying the two types of blockage need to be further examined in the future.
Suggested mechanisms of the outward IK1
In this study, we focused on the mechanism that generates the sustained flow of the outward IK1 in the voltage range near Vrev (below Vrev+60mV). It is known that the strong inward rectification of IK1 is primarily caused by a time-dependent gating mechanism operating at around Vrev. According to the present study, this time-dependent gating is presumably caused by the spermine block of the Mode 1 channels and its relief. When the I–V relationship of IK1 was reconstructed using the parameter of the time-dependent gating, the voltage range in which the outward currents flowed was much narrower (below Vrev+30mV; Kurachi, 1985; Ishihara et al. 1989; Oliva et al. 1990) than the range in which the outward IK1 actually took place, as in the case of the outward I–V relationship of Mode 1 channels reconstructed at 5µM spermine (Fig. 4C). We concluded that there is another population of channels (Mode 2 channels) that generate most of the outward IK1. Our finding that the fraction of Mode 2 channels is changeable might be of physiological importance in regulating the amplitude of the outward IK1.
The present results indicate that the concentration of spermidine near the IK1 channel may not be considerably higher than that of spermine, since the time-dependent gating of the Kir2.1 channel lost its steep voltage dependence with an excess amount of spermidine (Fig. 7C). Accordingly, it is strongly suggested that the blockage of channels by spermine primarily determines the outward I–V relationships of both Mode 1 and Mode 2 channels of IK1. Assuming that the IK1 and Kir2.1 channels show similar sensitivities to the polyamine block, the free spermine concentration
), 1µM (•), 5µM (
), 10µM (
). Right, spermidine (spd) 1µM (
), +50 (
). The values of G/Gmax' shown were obtained by normalizing the G/Gmax values with the maximum value in the voltage range between +40 and +70mV after subtracting a minute background conductance. Similar analyses were performed for the spermidine block. C, voltage dependences of the Kd(V) values of the spermine block (thick lines) and the spermidine block (thin lines) in Mode 1 (Kd1(spm)(V) and Kd1(spd)(V); continuous lines) and Mode 2 (Kd2(spm)(V) and Kd2(spd)(V); dotted lines). The Kd values obtained from dose–response relationships are shown by symbols (filled symbols, spermine; open symbols, spermidine). See text for equations reconstructing Kd1(spm)(V), Kd1(spd)(V), Kd2(spm)(V) and Kd2(spd)(V) values. D, fractional blockages of Mode 1 (upper panel) and Mode 2 (lower panel) channels by spermine at different voltages, calculated using Kd1(spm)(V) and Kd2(spm)(V) values, respectively (dotted line, 0.1µM; dashed line, 1µM; continuous line, 10µM). E, fractional blockage of currents in Mode 1 (dotted line) and Mode 2 (dashed line) at 5µM spermine calculated by the model. The proportion of Mode 1 channels to total channels (
