| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MOLECULAR AND GENOMIC |
1 Department of Physiology, Faculty of Medicine, Saga University, 5-1-1 Nabeshima, Saga 849-8501, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
(Received 11 May 2007;
accepted after revision 13 July 2007;
first published online 19 July 2007)
Corresponding author K. Ishihara: Department of Physiology Faculty of Medicine, Saga University 5-1-1 Nabeshima, Saga 849-8501, Japan. Email: keiko{at}med.saga-u.ac.jp
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
IKir flows through tetrameric channels formed by subunits in the Kir2 family (Kir2.1–2.4). The polyamine block of Kir2 channels has been studied mainly using Kir2.1, which may be the predominant subunit mediating the cardiac IKir (Zaritsky et al. 2001; Miake et al. 2003). Studies have shown that block of the outward currents by internal particles involves both high- and low-affinity block (Yang et al. 1995a; Guo & Lu, 2000), but the molecular mechanisms for two types of block, and the physiological relevance of the low-affinity block, remained unclear. Yang et al. (1995a) showed that their dose–response data contained two distinct components corresponding to high and low affinity binding. Although they had considered the possibility that there are two different populations of channels with distinct affinities for the blockers, they later proposed that the high-affinity block may not abolish the current flow and that the binding of a second blocker to a channel may occur with lower affinity as a result of electrostatic repulsion and steric hindrance between the blockers (Yang et al. 1995b). Guo & Lu (2000) showed that the current–voltage (I–V) relations of the outward currents exhibit multiple phases, which they explained by assuming that, in the high-affinity blocking state, the polyamine traverses the pore to reduce the block at positive voltages. In our previous study (Ishihara & Ehara, 2004), we showed that the different shapes of the I–V relations of the outward currents obtained in the presence of various concentrations of polyamines are nicely explained by the sum of two conductances showing different susceptibilities to polyamine block. In that study, we demonstrated that the model of Guo & Lu (2000), in which two blocked states occur in a mutually exclusive manner (or that two types of blockers compete for block), does not explain the polyamine concentration dependence of the I–V relations. Moreover, internal Mg2+ at physiological intracellular concentrations (0.5–1 mM) appeared to block only the major conductance susceptible to high-affinity block, thereby inducing a time-dependent component in the outward currents that reflected the competition between Mg2+ and SPM for block of the channel (Yan & Ishihara, 2005). Thus, our current hypothesis is that Kir2 channels exist in two different states with differing susceptibilities to internal cationic blockers (Ishihara & Ehara, 2004).
Our earlier studies of Kir2.1 indicated that a large portion of the outward currents of the native IKir is likely carried by a small fraction of the conductance susceptible to the low-affinity mode of block (Ishihara & Ehara, 2004; Yan & Ishihara, 2005). To further examine the physiological relevance of the low-affinity block, here we first compared the SPM block of Kir2.2 with that of Kir2.1. The former shares 70% identity with the latter at the amino-acid level (Takahashi et al. 1994) and, together, Kir2.2 and Kir2.1 likely mediate the cardiac IKir (Yamashita et al. 1996; Zaritsky et al. 2001; Liu et al. 2001). We then studied the SPM block of Kir2.1 under the low external [K+] conditions. To explore the molecular mechanism of the two types of block, we also studied Kir2.1 mutants in which the negatively charged residues D172 and E224 situated on the wall of the pore (Nishida & Mackinnon, 2002; Kuo et al. 2003; Pegan et al. 2005) were replaced with corresponding uncharged residues in the weak inward rectifier Kir1.1. These residues are known to be involved in the high-affinity SPM block (Lu & Mackinnon, 1994; Stanfield et al. 1994; Yang et al. 1995a; Taglialatela et al. 1995), though the site to which polyamines bind to block the K+ permeation remains controversial (Guo & Lu, 2003; Guo et al. 2003; John et al. 2004; Kurata et al. 2004; Kurata et al. 2006). Our results indicate that E224 situated on the wall of the cytoplasmic pore plays a key role in facilitating the high-affinity block and its relief, but is not the binding site for block in the ‘high-affinity channel’, though it is a critical component of the binding site related to block of the ‘low-affinity channel.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
293T cells (derived from the human embryonic kidney cell line 293 and expressing the SV40 large T antigen) were grown in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum, 0.15% sodium bicarbonate, 200 U ml–1 penicillin and 200 µg ml–1 streptomycin. Using Effectene transfection reagent (Qiagen Inc.), the cells were cotransfected with 1 µg of Kir2.2 (mouse; Takahashi et al. 1994), Kir2.1 (mouse; Kubo et al. 1993) or Kir2.1 mutant cDNA, all subcloned into the mammalian expression vector pCXN2 (Niwa et al. 1991), plus 0.1 µg pEGFP-N1 (Clontech) per 60 mm dish. Cells expressing exogenous genes were identified by visualizing the EGFP fluorescence using an inverted fluorescence microscope.
Current recordings from 293T cells expressing Kir2 channels
On the day of transfection, cells were seeded onto small pieces of collagen-coated cover glass (Asahi Techno Glass Corporation, Tokyo Japan). Within 24–56 h after transfection, a piece of cover glass was placed in a recording chamber (area: 4 mm x 23 mm; volume: 
0.5 ml) mounted on the stage of an inverted fluorescence microscope (TMD300 or TE2000, Nikon, Tokyo, Japan), which was continuously perfused with bath solution at a rate of 3 ml min–1. Currents were recorded from inside-out patches with a patch-clamp amplifier (EPC-8, HEKA; or Axopatch 200B, Axon Instruments) using Silicone-coated and fire-polished patch electrodes made from borosilicate glass capillaries (1.65 mm o.d., 0.165 mm wall thickness; Hilgenberg GmbH, Malsfeld, Germany). The pipette (external) solution used to compare the SPM block of Kir2.1 and Kir2.2 (Fig. 1) and to examine the SPM block of Kir2.1 mutants (Figs 7, 8 and 9) contained (mM): 145 KCl, 1 CaCl2, and 5 Hepes (pH 7.4 adjusted with KOH). The pipette solutions used to study the external K+ dependence of the SPM block of Kir2.1 (Fig. 4) were devoid of CaCl2 and were buffered to pH 7.4 using 8 mM K2HPO4 and 2 mM KH2PO4: 150 mM K+, 50 mM K+ and 20 mM K+ solutions contained 132 mM, 32 mM and 2 mM KCl, respectively. The osmolarities of the 50 mM and 20 mM K+ solutions were not adjusted (see Discussion). The 5.4 mM K+ pipette solution (Fig. 6) contained (mM): 5.4 KCl, 139.6 NMDG-Cl, 1 CaCl2 and 5 Hepes (pH 7.4 adjusted with NMDG). The pipette resistance was typically 1.7–2.0 M
when filled with the pipette solution.
|
|
|
|
|
|
4 mM 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 10 µM each. Bath solutions containing various concentrations of SPM were made by adding the appropriate amount of spermine-4HCl (Nacalai Tesque, Kyoto, Japan; as a 10 mM stock solution in distilled water; stored in small aliquots at –20°C) to the control bath solution. Bath solution containing SPM was applied after the native inward rectification had been removed to as great an extent as possible in the control bath solution. All experiments were conducted at room temperature (24–25°C). Data analysis
Steady-state current amplitudes were obtained using appropriate pulse duration. We did not subtract any background currents because of the difficulty in defining those currents. 293T cells show voltage-gated K+ currents activated at depolarized voltages under whole-cell conditions (Yu & Kerchner, 1998). These endogenous currents were usually negligibly small in our inside-out patch experiments using pipettes of 2 M, especially when expression levels of exogenous Kir2 channels were high. Because Kir2 channels show an apparently ohmic open channel conductance (as can be seen from the instantaneous current amplitudes at various test potentials under the SPM free condition in Figs 1A and 4A), we analysed the normalized chord conductance (G/Gmax) to estimate the unblocked fraction of Kir2 channels at different voltages, as before (Ishihara & Ehara, 2004). Unless otherwise noted, the maximum value of the chord conductance (Gmax) obtained at the same SPM concentration was used for normalization to minimize the influence of current rundown. This analytical procedure was chosen because outward Kir2 currents exhibit slow gating, even in the nominal absence of SPM (e.g. Fig. 1A), which may cause a marginal overestimation of the block in this procedure, but may give rise to a significant underestimation of the block if the percentage block is calculated from isochronal current amplitudes in the presence and absence of SPM.
The voltage dependence of G/Gmax (G–V relation) was fitted to the sum of two Boltzmann relations:
|
| (1) |
is the fractional amplitude of the first Boltzmann component, and R, T and F are the usual thermodynamic constants. In the experiments conducted under the symmetrical [K+] condition, G/Gmax approached a constant value at positive voltages where the Kir2 conductance became very small, presumably reflecting a non-specific leak conductance (GL). This value was less than 1% of Gmax in the analysed experiments. In the experiments conducted in low external [K+], the reversal potential of currents (Vrev) did not notably change when outward Kir2.1 currents were reduced by increasing the internal SPM concentration, and I–V relations crossed at the zero current potential (Fig. 4C), indicating that the contribution of the leak current was also insignificant. Still, to minimize interference by GL, eqn (1) was fitted to G–V relations using G/Gmax values larger than 3%. Fitting of the data to theoretical equations was done using Origin (version 6, OriginLab Corp., Northampton, MA, USA).
The junction potentials between the pipette and bath solutions were negligibly small (< 0.4 mV), except for those in the experiments shown in Fig. 6, which were 12 mV. These junction potentials were not corrected for.
All statistical values are given as means ± S.E.M.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
We first examined the SPM-mediated block of Kir2.2 currents in symmetrical 150 mM
[K+]. When SPM was added to the bath (internal) solution to final concentrations ranging from 0.1 µM to 10 µM, it blocked the outward component of Kir2.2 currents in a concentration-dependent manner without significantly affecting the inward component (Fig. 1A and B). The shapes of the I–V relations for the outward currents were distinct at different SPM concentrations: a peak was observed with 5 or 10 µM SPM, a plateau with 1 µM and two peaks with 0.1 µM (Fig. 1Ca). These findings were similar to those obtained with Kir2.1 (Fig. 1Cb in this study; Ishihara & Ehara, 2004). However, the outward-to-inward ratios of Kir2.2 current amplitudes were smaller than those obtained with Kir2.1; the ratio was roughly 2-fold smaller for the largest outward current observed at each SPM concentration (Fig. 1C). In the presence of 5 or 10 µM SPM, the outward components of Kir2.1 currents were similar to those of IKir in native cardiac ventricular myocytes in that they were observed at voltages ranging to at least Vrev
+
50 mV and showed a clear peak at Vrev
+
20 mV (e.g. Ishihara et al. 2002). For Kir2.2, the outward currents flowed in a narrower voltage range, and the peak amplitude was observed at more negative voltages.
We then analysed the G–V relations to estimate the unblocked fraction of Kir2.2 channels at different membrane potentials (Fig. 2A). The conductance values became smaller as the membrane potential become more positive, indicating the voltage-dependent block of the channels by SPM. Since the normalized conductance values for the outward currents were small, the relations were plotted on a semilogarithmic scale in Fig. 2B. As with Kir2.1 (Ishihara & Ehara, 2004), the G–V relations obtained in the presence of various concentrations of SPM could be approximated by the sum of two Boltzmann relations (eqn (1)). Increasing the SPM concentration shifted the half-activation voltages of the two Boltzmann components (dashed lines and dotted lines in Fig. 2B) in the negative direction without changing their fractional amplitudes. This finding strongly suggests that there are two conductances susceptible to different modes of SPM block. Indeed, the apparent dissociation constants for the two blocking modes, inferred from the relationship between the SPM concentration and the half-activation voltages of the Boltzmann components, declined exponentially with increasing membrane potential (Fig. 2C), and the relations could be expressed by the equation (Woodhull, 1973):
|
| (2) |
|
|
It is well known that the G–V relation of the whole-cell IKir shifts along the voltage axis in parallel with the shift in EK when the extracellular [K+] is altered at a constant intracellular [K+] (e.g. Hagiwara & Takahashi, 1974). We would thus expect that low-affinity block of Kir2 channels mediates the outward currents even under low external [K+] conditions. Our next aim was to carefully test that idea for Kir2.1 using inside-out patches. When [K+] in the pipette (external) solution was reduced to 50 mM or 20 mM, the characteristic shapes of the I–V relations of the outward currents, which showed a peak, a plateau (or a slightly positive slope region) and two peaks at 5 µM, 1 µM and 0.1 µM SPM, respectively, were largely unaffected (Fig. 4). The negative shift of the I–V relation of the inward currents seen with Kir2.1 in the presence of relatively high concentrations of SPM (Xie et al. 2002) was also seen at lower external [K+] (Fig. 4C, lower panels). This effect was not observed for Kir2.2 with 10 µM SPM, the highest concentration we used (Fig. 1B).
As was shown previously (Lopatin & Nichols, 1996), the open channel conductance of Kir2.1 was apparently ohmic, even with a [K+] gradient across the membrane, as can be seen from the instantaneous currents at various test potentials in the absence of SPM (Fig. 4A). We therefore simply compared G–V relations obtained at different external [K+] (Fig. 5A). The G–V relations could be fitted by eqn (1) using the same RT/z1F and RT/z2F values (4.7 mV and 9 mV, respectively) for all external [K+] (Fig. 5B). The voltage dependences of the dissociation constants for the high- and low-affinity blocks, inferred from the half-activation voltages of the Boltzmann components (Fig. 5C, symbols), were well approximated by eqn (2) using the aforementioned RT/z1F and RT/z2F values (Fig. 5C, lines), and were almost superimposable when plotted against the difference between the membrane potential and Vrev at each external [K+] (Fig. 5C). When the Kd(0) values obtained from those fittings were plotted against the external [K+] (Fig. 5D), they deviated slightly from the theoretical relation as [K+] was reduced, which may reflect the higher activity of K+ in the low [K+] pipette solutions (see Discussion). The fittings in Fig. 5B also showed that the fractional conductance susceptible to low-affinity block became slightly larger at lower external [K+]: the values were 0.075, 0.08 and 0.09 with 150 mM, 50 mM and 20 mM external K+, respectively.
|
140 mM NMDG was added to the pipette solution to compensate for the reduced osmolarity and to obtain a high seal resistance. In the presence of external NMDG, inward currents during hyperpolarizing steps showed a significant relaxation, which hampered the analysis of G–V relations (see Discussion). Nevertheless, it could be clearly seen that the shape of the I–V relation of the outward currents at each SPM concentration (Fig. 6B) was similar to that observed at higher external [K+] (Fig. 4C), which confirms that the conductance susceptible to low-affinity block mediates the outward currents, even under the physiologically low external [K+] conditions. Block of Kir2.1(D172N) is similar to low-affinity block of the wild-type channel
It has been shown that neutralization of the negatively charged residues situated on the wall of the transmembrane pore (D172) and the cytoplasmic pore (e.g. E224) of Kir2.1 reduces the channel's sensitivity to high-affinity block by internal cations, which suggests that these sites are involved in the high-affinity binding related to block (see Introduction for references). In Fig. 7, we show that the susceptibility of Kir2.1(D172N) to the SPM block was comparable to that of the wild-type Kir2.1 to low-affinity SPM block. Figure 7A shows Kir2.1(D172N) currents recorded in the absence and presence of SPM. In the control (SPM-free) solution, the instantaneous amplitudes of the outward currents showed a nearly ohmic I–V relation, and the outward currents showed a slow decay, which were similar to wild-type Kir2.1 currents (Fig. 7A, cf. Fig. 4A), the inward rectification of Kir2.1(D172N) currents caused by 0.1–10 µM SPM was weaker than the rectification of the wild-type currents (Fig. 7B left, cf. Fig. 4C). Moreover, in contrast to the wild-type currents, the I–V relations of the outward currents obtained with 0.1 µM, 1 µM and 10 µM SPM all showed a single peak. Interestingly, the voltage at which the outward component of Kir2.1(D172N) currents showed the largest amplitude at each SPM concentration was comparable to that at which the outward currents mediated by the low-affinity conductance of the wild-type channel appeared to have the largest amplitude (Fig. 7B, right panel, red lines). Accordingly, in the presence of 10 µM SPM, the shape of the I–V relation of the outward Kir2.1(D172N) currents was very similar to that of the corresponding wild-type currents, which are mediated mostly by the conductance susceptible to low-affinity block. The G–V relations of Kir2.1(D172N) did not show two phases like those of the wild-type, but seemed to be approximated by a single-Boltzmann relation (Fig. 7C). The steepness of the voltage dependence and the half-blocking voltages of the fitted Boltzmann relations were similar to those for the low-affinity block of the wild-type channel (Fig. 7C and D).
Low-affinity block is markedly weakened in Kir2.1(E224G)
By contrast, E224G mutation of Kir2.1 greatly reduced the channel's sensitivity to low-affinity block without markedly altering its susceptibility to high-affinity block or the fractional conductance susceptible only to the low-affinity block. Consistent with previously reported findings (Yang et al. 1995a; Xie et al. 2003), the E224G mutation induced a weak instantaneous inward rectification in control (SPM-free) solution (Fig. 8Aa). Moreover, it dramatically slowed the time courses of the block and unblock by SPM – i.e. the decay phase of the outward currents and the activation phase of the inward currents (Fig. 8Aa) (Yang et al. 1995a; Taglialatela et al. 1995; Kubo & Murata, 2001; Panama & Lopatin, 2006). We therefore used long pulses to obtain the steady-state amplitudes of the outward currents (Fig. 8Ab). The steady-state I–V relations showed unique shapes at different SPM concentrations (Fig. 8B). Even though 1 µM SPM largely blocked the outward currents in the voltage range close to Vrev, a nearly ohmic I–V relation was observed at positive voltages, suggesting that the currents around Vrev and at the positive voltages had different susceptibilities to SPM block. When the SPM concentration was increased (10–500 µM), the inward and outward currents in the positive voltage range were both blocked in a concentration-dependent manner.
The G–V relations of Kir2.1(E224G) plotted on a semilogarithmic scale (Fig. 8Ca) clearly showed two phases, like those of the wild-type channel (Fig. 5B). Because the instantaneous inward rectification of Kir2.1(E224G) currents in the control (SPM-free) solution (Fig. 8Ca, open circles) may be a property of the unblocked channel (Yang et al. 1995a), we further normalized these G–V relations with the instantaneous G–V relation acquired in SPM-free solution and then fitted them with the sum of two Boltzmann relations (eqn (1)) (Fig. 8Cb). The fractional conductance susceptible to low-affinity block was 0.15, which was slightly larger than that of the wild-type channel. The E224G mutation caused the voltage dependence of the high-affinity block to become slightly shallower, but the Kd(V) values for the high-affinity block were similar to those of the wild-type channel (Fig. 8D). By contrast, the sensitivity of low-affinity block was significantly reduced; the Kd(V) value at +60 mV was estimated to be increased by more than 104-fold, as compared to the wild-type.
When we tested the effects of a D172N and E224G double mutation, we found that the susceptibility of the channel to the SPM block was dramatically reduced (Fig. 9A and B), such that SPM concentrations of 1 µM and below did not noticeably affect the currents (Fig. 9Ca). The degree of the instantaneous rectification of Kir2.1(D172N and E224G) observed in the control (SPM-free) solution (Fig. 9Ca, open circles) was nearly identical to that of Kir2.1(E224G) (Fig. 8Ca). The G–V relations of Kir2.1(D172N and E224G) corrected for the instantaneous inward rectification were similar to those for the low-affinity component of the Kir2.1(E224G) (Fig. 9Cb). This suggests that addition of the D172N mutation made almost all of the Kir2.1(E224G) channels susceptible only to the low-affinity block, as it did in the wild-type channel.
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Comparison of Kir2.1 and Kir2.2
We previously showed that the characteristic shapes of the I–V relations of the outward component of Kir2.1 currents observed in the presence of different concentrations of internal SPM/SPD are explained by the presence of two (large and small) conductances, which are susceptible to the high-affinity and low-affinity modes of polyamine block, respectively (Ishihara & Ehara, 2004). In the present study, the same finding was more clearly obtained with Kir2.2 and SPM because the SPM in the concentration range between 0.1 and 10 µM did not diminish the maximum conductance (Fig. 1B), enabling us to determine the fractional size of the two conductances in a straightforward manner. In the case of Kir2.1, there was a ‘shallow block’, which reduced the maximum conductance in a concentration-dependent manner by negatively shifting the I–V relation of the inward currents (Figs 5A and 4C in this study; Lopatin et al. 1995; Xie et al. 2002; Panama & Lopatin, 2006). Xie et al. (2002) showed that this shallow component of block is likely to be caused by a charge screening effect of SPM due to binding to the negatively charged residues (E224 and E299) within the cytoplasmic pore. This shallow block was also observed under the low external [K+] condition, suggesting it depends on the difference between the membrane potential and EK (Fig. 4C). We ignored this shallow component in fitting the G–V relations of Kir2.1 with eqn (1) (Fig. 5 in this study; Ishihara & Ehara, 2004) by tentatively assuming that its effect on the inward current conductance is additive with the high- and low-affinity block we analysed. Since E224 and E299 are conserved in Kir2.2, the difference in the behaviour of Kir2.1 and Kir2.2 may reflect a slight structural difference in the cytoplasmic pore.
The SPM-induced inward rectification of Kir2.2 currents was stronger than that of Kir2.1 currents (Fig. 1C) because the fractional conductance susceptible to low-affinity block is smaller in Kir2.2 than in Kir2.1 (0.065 versus 0.1), and the susceptibility to block is greater in Kir2.2 (Fig. 3A). This is consistent with earlier observations that in mammalian cells exogenously expressing Kir2.1 or Kir2.2, whole-cell Kir2.2 currents showed stronger inward rectification than Kir2.1 currents (Dhamoon et al. 2004; Panama & Lopatin, 2006). The outward-to-inward ratios of the Kir2.2 current amplitudes were about 2-fold smaller than those for Kir2.1 at the largest outward current observed at each SPM concentration (Fig. 1C). This difference is substantial for cells such as cardiac ventricular myocytes, in which the density of the outward IKir is significantly larger than those of other current systems (see Matsuoka et al. 2003).
The findings discussed above may be of particular importance in terms of cardiac electrophysiology because the outward IKir, which mediates membrane repolarization in the working cardiac muscle, is thought to be carried mainly by channels composed of Kir2.1 and Kir2.2 subunits, though the precise contributions of the respective subunits are still not clear and may differ among different human races (Kaibara et al. 2002). Although Kir2.3 also has been shown to be expressed in the cardiac myocytes (Liu et al. 2001), it likely does not significantly contribute to the cardiac IKir, as the cardiac IKir does not exhibit the sensitivity to external pH characteristic of the Kir2.3 channel (Yan et al. 2005). The results shown in the present study indicate that the characteristic I–V relation of the cardiac IKir is not reconstituted with the Kir2.2 homotetramer; the outward currents flowed in narrower voltage range than the native cardiac IKir (Fig. 1Ca). This finding may be indicative of the crucial role played by Kir2.1 in mediating the outward component of the cardiac IKir at relatively positive voltages, where a change in IKir could easily affect cardiac repolarization, thereby increasing the risk of arrhythmia. Indeed, mutations of Kir2.1(KCNJ2) have been implicated in disorders of cardiac repolarization that predispose patients to arrhythmias (Plaster et al. 2001; Priori et al. 2005).
Low external K+ experiments and SPM block in asymmetrical K+
We attempted to verify whether the parameters of the SPM block of Kir2.1 obtained in symmetrical [K+] could be adopted under conditions in which the external [K+] was at a physiological level. For this purpose, we used low [K+] pipette solutions without compensating for the reduced osmolarity so as to eliminate previously observed relaxation of inward currents (Shieh, 2000), which we found to be mainly caused by NMDG included in the pipette solution to compensate for the osmolarity. Internal NMDG also blocks Kir2.1 in a voltage-dependent manner, and the same step was taken to examine the internal [K+] dependence of the Kir2.1 conductance (Lopatin & Nichols, 1996). Elimination of Hepes and Ca2+, which also have been shown to cause relaxation of inward Kir2.1 currents (Guo & Lu, 2002), did not clearly reduce that observed with NMDG (see Fig. 6A). Even with the low-K+ pipette solutions devoid of NMDG, Hepes and Ca2+, we sometimes still saw some relaxation and thought it may be caused by depletion of K+ at the external rim of a patch membrane, which occurred especially when a large patch of membrane was drawn deeply into the pipette. Those data were discarded from the analysis of the G–V relations (Fig. 5A). The Vrev values of currents thus obtained at 50 mM and 20 mM
[K+] were more positive than the predicted EK by 2.5 and 5 mV, respectively (see the legend to Fig. 5 for the mean Vrev values). This was not due to the contribution of leak currents (see Method), but is explained by the increased activity of K+ ion in the hypotonic low-K+ solutions: the activity coefficients for K+ in the 50 mM and 20 mM K+ solutions were calculated to be higher than in the 150 mM K+ solution by a factor of 1.2, which seems reasonable for a mixture of KCl and K-phosphate solutions (Robinson & Stokes, 1959; Akiyama & Fozzard, 1975). Therefore, it is likely that both the high- and low-affinity block depended very much on the difference between the membrane potential and EK, when the external [K+] was altered at a constant internal [K+]. We find it noteworthy that the fractional conductance susceptible to low-affinity SPM block measured in 150 mM external [K+] using Hepes- and Ca2+-free pipette solution as a control for the low external [K+] data was smaller than that obtained in the presence of external Hepes and Ca2+ (0.075 versus 0.1). The difference may be caused in part by an apparently time-independent block of the inward currents by Hepes and Ca2+ in the external solution.
The mechanisms of the two modes of block
Given that the crystal structures of Kir channels (Nishida & MacKinnon, 2002; Kuo et al. 2003; Pegan et al. 2005) show that there are not two pathways for K+ through a single Kir2 channel (with different affinities for SPM block, one conducting 9 times more K+ than the other), we speculate that Kir2 channels can exist in two states that are susceptible to the high-affinity and low-affinity modes of SPM block, respectively, and that there is a higher probability (90%) that the channel will be in the ‘high-affinity state’ (Ishihara & Ehara, 2004) (Fig. 10). We showed that the D172N mutation in the transmembrane pore of Kir2.1, which is known to reduce its affinity for block by internal cationic particles, made almost all of the Kir2.1 channels susceptible only to low-affinity block (Fig. 7), suggesting that the block of the Kir2.1(D172N) channel occurred in the same manner as that of the wild-type channel in the ‘low-affinity state’. This is consistent with the observations that the D172N mutation eliminates the time-dependent gating of the wild-type channel (Wible et al. 1994; Stanfield et al. 1994) and that relief of high-affinity SPM block is time dependent, while relief of low-affinity SPM block is virtually instantaneous, in the wild-type channel (Ishihara & Ehara, 2004). When we assume that a channel in the ‘high-affinity state’ possesses both high- and low-affinity binding sites related to block (to which SPM binds either mutually exclusively or non-exclusively) and their affinities for block are as shown in Fig. 3A, the blocked fractions of currents are negligibly different from those of a channel possessing only the high-affinity site for block (calculations not shown). Thus, our finding fits with the idea that a channel in the ‘high-affinity state’ is also susceptible to low-affinity block and that the D172N mutation eliminated the binding site for high-affinity block, rendering the channel blockable only at the low-affinity site. Indeed, recent evidence suggests that the interaction of the trailing amine group of SPM with a negative charge provided by D172 stabilizes the blockade of K+ permeation by its head amine at a deeper site, probably at the selective filter (John et al. 2004; Kurata et al. 2004; Kurata et al. 2006). A different and more imaginative possibility is that a polyamine binding to D172 in a non-blocking configuration is crucial for stabilizing the conformational state susceptible to high-affinity block. Recently, Xie et al. (2005) proposed that such interaction of a polyamine with D172 may allosterically strengthen the interaction of the channel with PIP2 (which stabilizes the open configuration of the channel) without blocking the channel. In that case, however, the channel bound at D172 with a polyamine in a non-blocking configuration may not be the ‘high-affinity state’ of the channel, because our analyses of G–V relations showed that the ratio of low/high affinity states is almost constant at different SPM concentrations (e.g. Fig. 2), and the voltage-dependent increase in the ‘high-affinity state’ is inconsistent with the G–V relations obtained from the experiments.
|
A more important observation was that E224G mutation dramatically reduced the affinity for low-affinity block without significantly affecting the fractional conductance susceptible to low-affinity block (Fig. 8C), which is in good agreement with the data reported by Panama & Lopatin (2006). This observation indicates that the marked attenuation of the low-affinity block was not accompanied by an increase in the fraction of the high-affinity block. This supports our hypothesis that the conductance susceptible to low-affinity block is not susceptible to high-affinity block. The great reduction in the affinity for the low-affinity bock caused by the E224G mutation indicates that E224 significantly contributes to forming the binding site for the low-affinity block. This finding also reveals that the site for the polyamine block of Kir2.1(D172N) is at the cytoplasmic pore.
The mechanisms of the high-affinity and low-affinity block that we propose based on our previous and present studies are summarized in Fig. 10. Our results suggest that in the ‘low-affinity state’ polyamines cannot access the high-affinity blocking site in the transmembrane pore, probably due to some kind of steric hindrance. The steric hindrance may be induced by a conformational change in the narrow part of the cytoplasmic pore (Pegan et al. 2005). It is unlikely to be caused by polyamines binding within the cytoplasmic pore, because the proportion of the ‘low-affinity channel’ seems to be almost constant at different SPM concentrations. Another important possibility we considered for the mechanism of the two modes of block, which originates from the proposal of Yang et al. (1995b), is that the high-affinity block does not completely block the current flow but reduces the single channel conductance by the amount corresponding to the fractional conductance susceptible to the high-affinity block, and that the channel being blocked at the high-affinity site becomes susceptible to the low-affinity block. The model is,
|
| (3) |
|
| (4) |
|
Footnotes |
|---|
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Dhamoon AS, Pandit SV, Sarmast F, Parisian KR, Guha P, Li Y, Bagwe S, Taffet SM & Anumonwo JM (2004). Unique Kir2.x properties determine regional and species differences in the cardiac inward rectifier K+ current. Circ Res 94, 1332–1339.
Fabiato A & Fabiato F (1979). Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 75, 463–505.[Medline]
Fakler B, Brandle U, Glowatzki E, Weidemann S, Zenner HP & Ruppersberg JP (1995). Strong voltage-dependent inward rectification of inward rectifier K+ channels is caused by intracellular spermine. Cell 80, 149–154.[CrossRef][Medline]
Guo D & Lu Z (2000). Mechanism of IRK1 channel block by intracellular polyamines. J Gen Physiol 115, 799–814.
Guo D & Lu Z (2002). IRK1 inward rectifier K+ channels exhibit no intrinsic rectification. J Gen Physiol 120, 539–551.
Guo D & Lu Z (2003). Interaction mechanisms between polyamines and IRK1 inward rectifier K+ channels. J Gen Physiol 122, 485–500.
Guo D, Ramu Y, Klem AM & Lu Z (2003). Mechanism of rectification in inward-rectifier K+ channels. J Gen Physiol 121, 261–275.
Hagiwara S & Takahashi K (1974). The anomalous rectification and cation selectivity of the membrane of a starfish egg cell. J Membr Biol 18, 61–80.[CrossRef][Medline]
Hayashi M, Komazaki S & Ishikawa T (2003). An inwardly rectifying K+ channel in bovine parotid acinar cells: possible involvement of Kir2.1. J Physiol 547, 255–269.
Ishihara K & Ehara T (2004). Two modes of polyamine block regulating the cardiac inward rectifier K+ current IK1 as revealed by a study of the Kir2.1 channel expressed in a human cell line. J Physiol 556, 61–78.
Ishihara K, Hiraoka M & Ochi R (1996). The tetravalent organic cation spermine causes the gating of the IRK1 channel expressed in murine fibroblast cells. J Physiol 491, 367–381.
Ishihara K, Mitsuiye T, Noma A & Takano M (1989). The Mg2+ block and intrinsic gating underlying inward rectification of the K+ current in guinea-pig cardiac myocytes. J Physiol 419, 297–320.
Ishihara K, Yan D-H, Yamamoto S & Ehara T (2002). Inward rectifier K+ current under physiological cytoplasmic conditions in guinea-pig cardiac ventricular cells. J Physiol 540, 831–841.
John SA, Xie LH & Weiss JN (2004). Mechanism of inward rectification in Kir channels. J Gen Physiol 123, 623–625.
Kaibara M, Ishihara K, Doi Y, Hayashi H, Ehara T & Taniyama K (2002). Identification of human Kir2.2 (KCNJ12) gene encoding functional inward rectifier potassium channel in both mammalian cells and Xenopus oocytes. FEBS Lett 531, 250–254.[CrossRef][Medline]
Kubo Y, Baldwin TJ, Jan YN & Jan LY (1993). Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362, 127–133.
Kubo Y & Murata Y (2001). Control of rectification and permeation by two distinct sites after the second transmembrane region in Kir2.1 K+ channel. J Physiol 531, 645–660.
Kuo A, Domene C, Johnson LN, Doyle DA & Venien-Bryan C (2005). Two different conformational states of the KirBac3.1 potassium channel revealed by electron crystallography. Structure 13, 1463–1472.[Medline]
Kuo A, Gulbis JM, Antcliff JF, Rahman T, Lowe ED, Zimmer J, Cuthbertson J, Ashcroft FM, Ezaki T & Doyle DA (2003). Crystal structure of the potassium channel KirBac1.1 in the closed state. Science 300, 1922–1926.
Kurachi Y (1985). Voltage-dependent activation of the inward-rectifier potassium channel in the ventricular cell membrane of guinea-pig heart. J Physiol 366, 365–
