K+ Binding Sites and Interactions between Permeating K+ Ions at the External Pore Mouth of an Inward Rectifier K+ Channel (Kir2.1)*

Ru-Chi ShiehDagger §, Jui-Chu ChangDagger , and Chung-Chin Kuoparallel

From the Dagger  Institute of Biomedical Sciences, Academia Sinica, Taipei 11529 and the  Department of Physiology, National Taiwan University College of Medicine and Department of Neurology, National Taiwan University Hospital, Taipei 100, Taiwan, Republic of China

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The arginine at position 148 is highly conserved in the inward rectifier K+ channel family. Increases of external pH decrease the single-channel conductance in mutant R148H of the Kir2.1 channel (arginine is mutated into histidine) but not in the wild type channel. Moreover, in 100 mM external K+, varying external pH induced biphasic changes of open channel noise, which peaks at around pH 7.4 in the R148H mutant but not in the wild type channel. The maximum single-channel conductances are higher in the wild type channel and R148H mutant at pH 6.0 than those in the R148H mutant at pH 7.4. However, the maximal conductance is achieved with much lower external [K+] for the latter. Interestingly, the single-channel conductances and open channel noise of the wild type channel at pH 6.0 and the R148H mutant at pH 6.0 and 7.4 become the same in [K+] = 10 mM. These results indicate that the residue at position 148 is accessible to the external H+ and probably is involved in the formation of two K+ binding sites in the external pore mouth. Effective repulsion between permeating K+ ions in this area requires a positive charge at position 148, and such K+-K+ interaction is the essential mechanism underlying high K+ conduction rate through the Kir2.1 channel pore.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inward rectifier K+ channels are important in maintaining stable resting membrane potentials and controlling excitability of many cells. The physiological function of these channels is closely related to their unique inward rectification mechanisms, which allow much larger inward current than outward current through the channels. Another important "asymmetrical" feature of the inward rectifier K+ channels is that external but not internal K+ seems to interact with and thereby activate the channel (1). The mechanisms underlying inward rectification have been ascribed to voltage (Vm)-dependent blockade by internal cations such as Mg2+ (2, 3) and polyamines (4, 5) and to a pHi-dependent intrinsic gate (6). On the other hand, the ion permeating process itself, namely interactions between K+ ions and between the permeating K+ ion and the inward rectifier K+ channel, is less characterized.

The arginine at position 148 is highly conserved in the inward rectifier K+ channel family and seems to play an important role in the K+ ion permeation process. Kubo (7) showed that mutation of the arginine at position 148 into tyrosine (R148Y) in the cloned inward rectifier K+ channel (Kir2.1) (8) resulted in a reduction of the [K+]o-dependent activation of inward currents and a negative shift of the conductance-voltage relationship. Sabirov et al. (9) reported that although mutation of the arginine at position 148 into histidine (R148H) resulted in no channel activity, coexpression of the mutant with the wild type (WT)1 cRNA demonstrated populations of channels with reduced single-channel conductances (gamma ). Moreover, the single-channel conductance is dramatically increased in a recently cloned inward rectifier K+ channel (Kir7.1) when the neutral amino acid methionine at position 125 (the equivalent position of the arginine at site 148 in Kir2.1) was mutated into a positively charged amino acid (10). All these studies suggest that the positive charge at position 148 (148oplus ) in the inward rectifier K+ channel family is important in determining K+ ion conduction and channel activation, yet detailed mechanisms underlying the effects of arginine at position 148 with a positive charge on K+ ion permeation through the Kir2.1 channel pore remain unexplored.

With successful expression of the Kir2.1 mutant R148H in Xenopus oocytes, we studied the effect of pHo and [K+]o on the single-channel conductance and the open channel "noise" in WT channel and R148H mutant. We found that the side chain of the amino acid at position 148 is accessible to the external H+. Also, the amino acid at position 148 probably is involved in the formation of a set of two K+ binding sites in the external pore mouth. Effective repulsion between permeating K+ ions in this area requires a positive charge at position 148, and such K+-K+ interaction may be an important mechanism underlying high K+ conduction rate through the Kir2.1 channel pore.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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Molecular Biology and Preparation of Xenopus Oocytes-- Mouse macrophage Kir2.1 in pCDNAI/Amp (generously provided by Dr. Lily Jan (University of California at San Diego)) digested with HindIII and StuI was sublconed into the HindIII-SmaI sites in pALTER/Tet (Promega, Madison, WI). Site-directed mutation was then generated using the Altered Sites II in vitro mutagenesis systems (Promega, Madison, WI). The mutant DNA was sequenced with the ABI PrismTM dRhodamine Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Foster City, CA) to confirm the presence of histidine at position 148. The WT Kir2.1 subcloned in pBSIISK(+) (a generous gift from Drs. Scott A. John and James N. Weiss) and R148H DNAs were linearized with NotI and ScaI, respectively, and approximately 1 µg of purified linear DNA was used for in vitro T7 (for WT DNA) and Sp6 (for R148H DNA) transcription reactions (mMessage mMachine, Ambion, Dallas, TX).

Xenopus oocytes were prepared as described previously (11). In brief, Xenopus oocytes were isolated by partial ovariectomy from frogs anesthetized with 0.1% aminobenzoic acid ethyl ester. The day after isolation, Xenopus oocytes were pressure-injected with either 10-100 pg of WT cRNA or 100-1000 pg of R148H cRNA. Oocytes were maintained at 18 °C in Barth's solution containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM Ca(N2O6), 0.41 mM CaCl2, 0.82 mM MgSO4, 15 mM HEPES, 20 µg/ml gentamicin, pH 7.6, and used 1-3 days after RNA injection.

Single-channel Recordings-- Single-channel currents were recorded at room temperature (22-24 °C) on cell-attached or inside-out patches (12) using an Axopatch 200A amplifier (Axon Instruments, Burlingame, CA). Glass electrodes were pulled using a horizontal electrode puller (Sutter, Novato, CA) from borosilicate glass tubing (WPI Inc., Saratoga, FL). The diameters of the pipettes ranged from 1 to 5 µm. 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 sampled at 5-10 kHz and filtered at 1-5 kHz.

For experiments performed in cell-attached patches, the pipette solution contained 100 mM KCl + KOH, 2 mM EDTA. For experiments carried out in inside-out patches (except for Fig. 7; see Fig. 7 legend), the pipette and internal solutions were always kept symmetrical and both contained x mM KCl + KOH, 2 mM EDTA, where x = 10-1000. For x = 10-100, 90 mM of N-methyl-D-glucamine (NMG) was also added. Because the single-channel conductances obtained with 100 mM [K+] and 100 mM [K+] + 90 mM [NMG] are similar, NMG was not added for solutions containing [K+] > 100 mM. Because it has been shown that channels formed from coexpression of the WT and R148H subunits are sensitive to external divalent cations (9), in this study we used pipette solutions containing no divalent cations. The patch electrode solution was pH-buffered by the following buffers: 5 mM MES for pHo < 7.0; 5 mM HEPES for 7.0 <=  pHo <=  8.0; 5 mM CHES for pHo > 8.0. The rundown of channel activity was avoided by treating the inside-out patches with 25 µM of L-alpha -phosphatidylinositol-4,5-bisphosphate (Sigma) (11, 13).

Data Analysis-- Single-channel current amplitude was measured by manually placing a cursor at the center of the closed or the open state levels and from amplitude histograms. To compare the degree of open channel noise in the WT channel and R148H mutant under various recording conditions, current variance (sigma ) of single-channel recordings was calculated for the open and closed states using the Clampfit basic statistics:
&sfgr;=<FR><NU>1</NU><DE>N−1</DE></FR> <LIM><OP>∑</OP><LL>i<UP>=</UP>1</LL><UL>N</UL></LIM>(x<SUB><UP>i</UP></SUB>−<A><AC>x</AC><AC>&cjs1171;</AC></A>)<SUP>2</SUP> (Eq. 1)
where x is the mean of the samples, and N is the sample number. x was calculated from a segment in the open or closed level current of the sample sweeps (187-506 sample points). The open channel current variance from a sweep was normalized to the closed level current variance of the same sweep, and the normalized sigma (O)/sigma (C) values were then used to compare the magnitude of open channel noise among different experimental conditions. Averaged data are presented as the means ± S.E. Student's independent t test was used to assess statistical significance.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of the R148H Mutant in Xenopus Oocytes-- Fig. 1A shows a series of sample current traces recorded at testing pulses in a cell-attached patch containing one single R148H mutant channel at pHo 7.0. Fig. 1B shows the single-channel current amplitude histogram demonstrating a single predominant conducting state. The averaged single-channel current-voltage (i-Vm) relationship is plotted in Fig. 1C. The i-Vm relationship of the R148H mutant remains inwardly rectifying, and no outward single-channel current is recorded.


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Fig. 1.   The i-Vm relationship of the R148H mutant at pHo 7.0. A, single-channel currents were recorded at different Vm in a cell-attached patch exposed to a 100 mM [K+]o solution at pHo 7.0. Throughout this report, the solid lines indicate the closed state; the downward arrows identify the beginning of the voltage step, the upward arrows indicate the end of the voltage step (500 ms) from a holding potential of 0 mV, and capacitance transients were subtracted off-line using null traces. B, an amplitude histogram from single-channel currents recorded at Vm = -140 mV. C, the i-Vm curves were obtained by measuring single-channel current at various voltages from 4-11 patches. Throughout this study, single-channel conductances were obtained by linear fits to the i-Vm data between Vm = -140 and -60 mV. The solid line is the best fit to data with a slope of 20 pS.

Monotonously Decreased Single-channel Conductance of the R148H Mutant with Increasing pHo-- After successful expression of the R148H mutant in Xenopus oocytes, we explored the role of 148oplus in K+ permeation. First we examined whether the histidine residue is accessible to external H+ and thus the charge at position 148 can be controlled by varying pHo. Fig. 2A shows the single-channel currents recorded at Vm = -140 mV from a holding potential of 0 mV at various pHo in the WT channel and R148H mutant. Changes of pHo have little effect on the single-channel current in the WT channel, yet the single-channel current amplitude becomes smaller at higher (more alkaline) pHo in the R148H mutant. At low pHo, the single-channel current in the mutant is almost comparable with (although still slightly smaller than) the current in the WT channel. As pHo is increased, the single-channel current of the R148H mutant decreases in a pHo-dependent manner. The averaged single-channel currents recorded from several patches similar to that shown in Fig. 2A are plotted against membrane potentials and are shown in Fig. 2B. The i-Vm relationships are very similar at pHo 5.0 to 6.5. Further increase of pHo decreases single-channel conductance in a pHo-dependent manner. Fig. 2C shows that the conductance of the WT channel is little affected by changes in pHo, which is consistent with previous studies (9, 14). In contrast, the single-channel conductance of the R148H mutant is sensitive to pHo. The gamma -pHo relationship can be described by a simple one-to-one binding curve with a pKa of 7.3, and gamma  varies between 26 and 7 pS. These findings strongly support that the side chain of histidine at position 148 can be protonated by external H+, and 148oplus seems to enhance K+ ion permeation through the Kir2.1 channel pore.


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Fig. 2.   The effect of pHo on the i-Vm relationship of the WT channel and R148H mutant. A, representative single-channel current traces of the WT channel and R148H mutant were recorded at Vm = -140 mV, and the indicated pHo from cell-attached patches exposed to the 100 mM [K+]o solution. B, the i-Vm curves were obtained at the indicated pHo (n = 2-15). C, the single-channel conductances of the WT channel and R148H mutant are plotted against the corresponding pHo values. The curve superimposed on the R148H mutant data is the best fit to the data of the form: gamma max × [ [H+]/([H+]+Ka)]+ gamma min with gamma max = 26 pS, gamma min = 7 pS, and pKa = -logKa = 7.3. The dotted lines indicate gamma max and gamma min.

pHi Has No Effect on the Single-channel Conductance of R148H Mutant-- The effect of pHi on the R148H mutant was also examined. Fig. 3 shows that changes of pHi from 6.0 to 9.0 do not produce any change of the single-channel conductance of the R148H mutant. Thus the histidine at position 148 seems to be accessible only to external H+ but not to internal H+.


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Fig. 3.   Effects of pHi on the i-Vm relationships of the R148H mutant. Single-channel currents were measured in inside-out patches (n = 2-5) exposed to symmetrical 100 mM [K+] at pHo = 7.4 and the indicated pHi.

Lack of Vm Dependence of the pKa of R148H Mutant-- To determine whether the amino acid at position 148 is located within the electrical field, Vm dependence of the effects of pHo on the single-channel current of the R148H mutant was examined. At each Vm, the averaged single-channel currents recorded at different pHo were normalized to those at pHo 6.0 (Fig. 4A). The normalized i-pHo relationships are similar at different Vm. Fig. 4B shows that the pKa values determined from the i-pHo relationships in Fig. 4A remain very similar at different Vm. The lack of Vm dependence of pKa suggests that the amino acid at position 148 is probably located outside the electrical field.


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Fig. 4.   The Vm dependence of the effect of pHo on single-channel currents. A, the fractional currents (averaged single-channel currents recorded at various pHo normalized to that at pHo 6.0), i/ipH 6.0, are plotted against pHo. The curves are the best fits to data of the form: [H+]/([H+]+Ka) + C where C = 0.15 (-140 mV), 0.08 (-120 mV), 0.11 (-100 mV), 0.09 (-80 mV), or 0.08 (-60 mV). B, pKa values obtained from panel A are very similar at Vm = -60 to -140 mV.

Biphasic Changes of Open Channel Noise with pHo-- It is interesting to note that other than single-channel conductances, the open channel noises also vary with pHo in the R148H mutant. For example, it is evident in Fig. 2A that the open channel noise increases when pHo is increased from 5.0 to 7.4. However, further increases of pHo result in a decrease in open channel noise. To compare open channel noise we calculated single-channel current variance at the open channel level and then normalized it to that at the closed level in the same sweeps (for details see under "Experimental Procedures"). Fig. 5 demonstrates the normalized variance of single-channel currents in the WT channel (Fig. 5A) and in the R148H mutant (Fig. 5B) at different pHo. The open channel noise varies with pHo in a biphasic manner and is peaked at pHo 7.4 in the R148H mutant, whereas the open channel noise remains small and does not change with pHo in the WT channel. The biphasic change of the open channel noise in the R148H mutant seems to indicate that the unitary conductance of the mutant channel quickly fluctuates between two levels. The two levels probably are correlated with protonation and deprotonation of residue 148 of the channel, because the fluctuation is smallest either at pHo 6.0 (where the channel is mostly in the protonated and high conductance state) or at pHo 9.0 (where the channel is mostly in the deprotonated and low conductance state) yet is most prominent at pHo 7.4, the pKa of the histidine residue at position 148 in the R148H mutant (Fig. 4).


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Fig. 5.   Effects of pHo on open channel noise. Single-channel currents were recorded (with a sampling rate of 10 kHz and filtering rate of 1 kHz) in cell-attached patches exposed to 100 mM [K+]o at different pHo. Open channel noises were then quantified and expressed as sigma (O)/sigma (C) (see "Experimental Procedures") for the WT channel (A) and R148H mutant (B). Data groups shown in panel A are not statistically different from each other (p > 0.05), whereas the particular data group indicated by an asterisk in panel B is statistically different from that of the R148H mutant at pHo 7.4 (p < 0.001; n = 18 in each data group).

Higher Affinity between Permeating K+ Ions and the R148H Mutant Channel Pore at pHo 7.4 than at pHo 6.0-- We have demonstrated that histidine at position 148 of the R148H mutant can be accessed by H+ from the external space and that protonation of this site may have a significant effect on ion permeation through the Kir2.1 channel. The biphasic effect of pHo on the open channel noise furthermore argues for a direct rather than an indirect or allosteric effect of 148oplus on K+ permeation through the Kir2.1 channel (see "Discussion" for details). A direct effect of a positive charge (148oplus ) on the permeating K+, a positively charged ion, most likely would be an elevation in the free energy experienced by K+. Because the single-channel conductance is higher in channel with 148oplus , what is likely to be elevated is the minimum rather than the maximum of the energy profile. Because an increase in energy minimum may be manifested as a decrease in binding affinity for K+ in the pore, we investigated the effects of varying [K+] on the single-channel conductance of the WT channel and R148H mutant. The i-Vm relationships were measured in symmetrical [K+] ranging from 10 to 1000 mM in inside-out patches exposed to internal Mg2+- and polyamine-free solutions. Fig. 6 shows the i-Vm curves for the WT channel at pHo 6.0, R148H mutant at pHo 6.0, and R148H mutant at pHo 7.4 (panels A-C, respectively). Fig. 6D plots the single-channel conductance against [K+] (gamma -[K+] plot) for the WT channel at pHo 6.0, R148H mutant at pHo 6.0, and R148H mutant at pHo 7.4. For the WT channel at pHo 6.0, the single-channel conductance steeply increases as [K+] is increased and reaches a plateau of ~53 pS with a Kd = 49 mM. The [K+] dependence of the single-channel conductance of the R148H mutant at pHo 6.0 is similar to the WT channel except that the single-channel conductance saturates at 35 pS with a Kd = 30 mM. In contrast, the single-channel conductance of the R148H mutant at pHo 7.4 saturates at ~14 pS. Because the single-channel conductance is already ~10 pS in 10 mM [K+], it is plausible that the Kd for K+ binding in the R148H mutant is smaller than 10 mM (an accurate estimate of Kd is difficult in this condition because it is hard to accurately measure the single-channel conductance with [K+] < 10 mM). Thus the affinity between K+ and the R148H mutant channel pore seems to be higher at pHo 7.4 than at pHo 6.0. This is consistent with the foregoing prediction that 148oplus elevates the energy minimum experienced by the permeating K+ in the pore.


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Fig. 6.   Effects of [K+] on the single-channel conductances of the WT channel and R148H channel. A-C, i-Vm relationships for the WT channel at pHo 6.0, R148H mutant at pHo 6.0 and R148H mutant at pHo 7.4, respectively. Single-channel currents were recorded from inside-out patches exposed to the indicated [K+] (n = 2-6). D, single-channel conductances are shown as a function of [K+]. Solid lines are the best fits to data in the form: gamma  = gamma max/(1+Kd/[K+]) with Kd = 49 mM and gamma max = 53 pS for the WT channel at pHo 6.0, Kd = 30 mM, and gamma max = 35 pS for the R148H mutant at pHo 6.0 and Kd = 4 mM and gamma max = 14 pS. E, effects of [K+]o on open channel noise. Normalized open channel noise values (sigma (O)/sigma (C)) in 10 mM [K+] were obtained from single-channel currents recorded in inside-out patches perfused with symmetrical [K+] (n = 18). The open channel noises in 100 mM [K+]o are taken from Fig. 5 for comparison.

It is also interesting to note in Fig. 6D that the single-channel conductances for the three experimental conditions are the same in [K+] = 10 mM. If the increased open channel noise in 100 mM [K+] (Fig. 5) is indeed ascribed to the fluctuations of the single-channel conductance between two conducting levels and thus results in the decreased conductance of the R148H mutant in 100 mM [K+] at pHo 7.4, it would be desirable to check whether the open channel noises would also be the same in 10 mM [K+]o in different experimental conditions, just like the single-channel conductance. Fig. 6E shows this is indeed the case. The open channel noises are about the same for the WT channel at pHo 6.0, R148H mutant at pHo 6.0 and R148H mutant at pHo 7.4 in 10 mM [K+]. Moreover, the open channel noises in these conditions are all small and similar to the noises in the WT channel and R148H mutant at pHo 6.0 in 100 mM [K+]o. Thus the effect of protonation at position 148 in the R148H mutant, or 148oplus , on K+ conductance seems to be different in different bulk K+ concentration, suggesting that the free energy profile of the permeating K+ ion in the pore actually varies with bulk K+ concentration.

Internal [K+] Has No Effect on the pHo-dependent Changes of the Single-channel Conductance in the R148H Mutant-- We have demonstrated that 148oplus has a significant effect on ion permeation through Kir2.1 channel pore, and the effect is closely correlated with bulk K+ concentration. Because the results in Fig. 6 are obtained in symmetrical [K+] and the results in Figs. 1-4 suggest that the residue at position 148 is located in the external pore mouth, it is desirable to further examine whether it is external K+ or internal K+ that is crucial for the effect of 148oplus on K+ permeation. Fig. 7 shows the effects of asymmetrically varying [K+] on the single-channel conductance of the R148H mutant at pHo 7.4. In these experiments, the pipette solution contained either 10 or 100 mM [K+], and the inward currents were recorded at various Vm in inside-out patches perfused with either 10 mM [K+] or 100 mM [K+]. The results indicate that the single-channel conductance of the R148H mutant is dependent on [K+]o but not internal [K+] ([K+]i). The Vm independence of the effect of pHo (Fig. 4) and the lack of effect of pHi (Fig. 3) as well as [K+]i (Fig. 7) on the single-channel conductance of the R148H mutant all suggest that position 148 is located very close to the external pore mouth.


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Fig. 7.   The pHo-dependent changes of the single-channel conductance of the R148H mutant is sensitive to [K+]o but not [K+]i. Inward single-channel currents from inside-out patches exposed to four different combinations of external/internal solutions were recorded at pHo = 7.4. The junction potential change produced by changing solutions between 10 mM [K+]i + 90 mM [NMG+]i and 100 mM [K+]i was less than 2 mV and was not corrected.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we report successful expression of the Kir2.1 mutant R148H in Xenopus oocytes. We find that to obtain the same level of functional channel expression, the amounts of the R148H cRNA injected into the Xenopus oocytes have to be about 10-100 times more than those of the WT cRNA. The fact that the R148H mutant retains inward rectification (because of Vm-dependent block of the channels by intracellular Mg2+ and polyamines), sensitivity to extracellular Ba2+ block (data not shown), and K+ selectivity (9) suggests the absence of major conformational changes caused by the mutation. Our results show that the side chain of the amino acid residue at position 148 is accessible to external H+ and that this amino acid is probably located outside the electrical field. Furthermore, protonation of this position is essential for a faster transportation of permeating K+ ions. Because changes of pHo from 5.0 to 9.0 do not have any significant effect on the single-channel conductance of the WT channel (Fig. 2), external H+ (or OH-) binding to a part of the channel other than at position 148 probably can be neglected as a first approximation in the discussion of the findings here. Basically there are two possible mechanisms underlying the effect of pHo at position 148: protonation-deprotonation at this position may have an indirect or allosteric effect on channel protein and thus on K+ permeation, or it may have a direct effect on K+ permeation through the Kir2.1 channel pore.

If pHo modulated K+ permeation through the pore by an allosteric effect, the single-channel conductance in the R148H mutant at pHo 6.0 presumably should have a more constant relationship with that at pHo 7.4 in any given [K+]. It is therefore difficult to envisage why the single-channel conductances would be the same for the R148H mutant at pHo 6.0 and 7.4 in 10 mM [K+] yet are quite different in 100 mM or higher [K+]o (Fig. 6D). Moreover, in Figs. 2A and 5 we have noted that pHo has an effect on both the open channel noise and the single-channel conductance. Most interestingly, the open channel noise has a biphasic change. When pHo is increased from 6.0 to 7.4 the open channel noise increases. However, further increasing pHo results in a decrease in the open channel noise. With an allosteric model, it is hard to envisage the biphasic effect on the open channel noise by pHo. Also, with a sampling interval of 100 µs, we still could not resolve the open channel noise into discrete conducting states, indicating that the effect of protonating the channel has very fast kinetics. Because protein conformational change usually is a much slower process, it seems unlikely that an allosteric effect involving large scale protein conformational changes is responsible for the pHo effect in the R148H mutant.

In contrast to the indirect or allosteric model, a direct effect of external H+ on K+ ion conduction seems much more plausible. A direct effect of protonation-deprotonation in the pore will certainly fit the very fast kinetics of the modulatory effect of external H+ (revealed by the increased open channel noise). In view of higher single-channel conductance in channels with 148oplus (Fig. 2), we have argued that the direct effect of 148oplus may involve elevation of some energy minimum rather than energy maximum of the permeating K+ ion. The findings in Fig. 6 are also consistent with the notion that direct protonation of the pore at residue 148 elevates some free energy minimum of the K+ ion in the pore. It is very interesting, then, that the effect of protonation of position 148 in the R148H mutant (the positive charge at position 148, 148oplus ) on K+ ion conductance or the free energy profile of permeating K+ may be so different in different [K+]o (Figs. 6 and 7). Because changes of free energy minimum of permeating K+ ion by protonation-deprotonation of the R148H mutant channel pore (manifested as increased open channel noise at pHo 7.4) are evident only in high (100 mM) but not in low (10 mM) [K+]o, it seems that 148oplus would have a significant effect on K+ ion permeation only when the pore is "crowded" with permeating K+ ions. In other words, ion-ion interaction is very likely to be involved in the effect of 148oplus on K+ ion permeation, which would in turn imply simultaneous existence of at least two permeating K+ ions around position 148. We therefore propose that there is a set of ligand groups around position 148 in the external pore mouth to accommodate two permeating K+ ions (Fig. 8). When the ligand at position 148 has an uncharged histidine (Fig. 8B), the ligand may participate in coordinating the partly dehydrated permeating K+, and the set of ligands represents two "complete" binding sites for K+ ions. Because the energy minimum is always low, the Kd and maximal single-channel conductance are both small. However, when the ligand at position 148 is positively charged (Fig. 8C), it can no longer participate in coordinating K+ ions. The sites are now somewhat insufficient to accommodate two K+ ions, and the second K+ would have to adopt a position (site b in Fig. 8A) competing for some coordinating ligands with the first K+ (at site d in Fig. 8A). Both the Kd for loading the second K+ to site b in Fig. 8A and the maximal single-channel conductance would therefore be larger. When [K+]o is low (e.g. <=  10 mM) there would be no significant occupancy by the second K+ of site b (whose apparent Kd with site d already occupied by a K+ presumably is ~49 mM, Fig. 6D). The permeation of K+ thus would always happen on an "unenhanced" basis whether position 148 is charged or not (if there is little occupancy of site b by the other K+ ion there would be no significant ion-ion interaction to enhance the exit of the K+ at site d). The unitary conductance and open channel noise level would therefore remain very similar in 10 mM [K+]o, no matter whether it is WT channel or R148H mutant, and no matter whether pHo is 6.0 or 9.0. 


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Fig. 8.   Schematic illustration of the effect of 148oplus on K+ permeation through the Kir2.1 channel pore. A, several ligand groups around position 148 form a set of K+ binding sites in the external pore mouth. If the set contains only one K+ ion (the single occupancy state), the free energy level would be similar for the K+ ion at site a, b, c, or d, and thus the K+ ion may move among these sites freely. B, if the residue at position 148 is an uncharged histidine, the set would have sufficient ligand to "comfortably" accommodate two K+ ions at sites a and d (the double occupancy state). There is little interaction between the two ions at these sites, and the free energy level of each of the K+ ions would not be significantly higher than the K+ ion in the single occupancy state. Although it does not take high [K+]o to achieve the double occupancy state (the dissociation constant of the second K+ ion to the set is small), the exit rates of the K+ ion at site d (exiting inwardly to make inward K+ current) are not significantly different whether the set is doubly or singly occupied. C, when the residue at position 148 is charged (an arginine or a protonated histidine), site a no longer represents a low energy configuration to accommodate a K+ ion. The set of binding ligands can still accommodate two K+ ions, but now the two ions would be forced to take sites b and d. Because there would be competition for a common ligand (black oval) between the two ions, the free energy level of the K+ ions in such a double occupancy state is significantly higher than the K+ ion in the single occupancy state. It would therefore take higher [K+]o to achieve the double occupancy state (the dissociation constant of the second K+ ion to the set is larger), but the exit rate of the K+ ion at site d would be significantly enhanced when the set is doubly occupied. Note that the relative position of residue 148 to the other binding ligands is arbitrarily assigned. Also, we are not sure whether the uncharged histidine at position 148 is directly involved in K+ ion binding. However, all major points would remain the same as long as the positive charge at position 148 serves to decrease the ability of other nearby ligands to coordinate permeating K+ ions in the set of K+ binding sites.

The multi-ion nature of inward rectifier K+ channels has been supported by several lines of evidence including anomalous greater fraction effect (15), a flux-ratio exponent of 2.2 (16), steep Vm-dependent block by monovalent ions (17), and Vm- and [K+]o-dependent inward rectification (1). However, it is unclear whether and how these multiple K+ ions in the pore interact with each other. In this study, we add a new line of evidence for the multi-ion nature of the Kir2.1 channel pore. Furthermore, we suggest that the interaction between the permeating K+ ions near position 148 is essential for rapid transportation of the K+ ion through the pore and that position 148 probably is directly involved in or close to a set of two K+ binding sites in the external pore mouth. The arginine residue at position 148 is highly conserved in the Kir family, and it is likely that this highly conserved arginine at position 148 will also influence K+ permeation through other inward rectifier K+ channels in a way similar to what is described in this study. The existence of a set of two closely associated K+ binding sites in the external mouth of the Kir2.1 channel is intriguing, because it is very similar to what is described for the L-type Ca2+ channels (18-20). It would be interesting to see whether this is a more general attribute in the molecular design of these cationic channels in the future. In addition to the set of K+ binding sites located near the external mouth of the pore, it has recently been suggested that K+ and Ba2+ may compete for a binding site located in the electrical field in the Kir2.1 channels (11). Further characterization of how K+ ions in these different binding sites interact with each other or with the channel may provide us a more complete picture of the ion permeation process through the pore of the Kir2.1 channel.

    ACKNOWLEDGEMENTS

We thank Drs. Jorge Arreola, Chyuan-Yih Lee, and Kenneth K.-Y. Wu for helpful discussions.

    FOOTNOTES

* This work was supported by Academia Sinica and National Science Council Grants 87-2314-B-001-039, 88-2314-B-001-042, and 88-2314-B-002-212 in Taiwan, R.O.C.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence may be addressed: Inst. of Biomedical Sciences, Academia Sinica, 128 Yen-Chiu Yuan Rd., Section 2, Taipei 11529, Taiwan, R.O.C. Tel.: 886-2-2789-9024; Fax: 886-2-2785-3569; E-mail: ruchi{at}novell.ibms.sinica.edu.tw.

parallel To whom correspondence may be addressed: Dept. of Physiology, National Taiwan University, College of Medicine, 1 Jen-Ai Rd., Section 1, Taipei 100, Taiwan, R. O. C. Tel.: 886-2-2312-3456, Ext. 8236; Fax: 886-2-2396-4350; E-mail: cckuo{at}ha.mc.ntu.edu.tw.

    ABBREVIATIONS

The abbreviations used are: WT, wild type; 148oplus , the positive charge at position 148; NMG, N-methyl-D-glucamine; MES, 2-morpholinoethanesulfonic acid; CHES, 2-(cyclohexylamino)ethanesulfonic acid; pS, picosiemens.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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