Correspondence to: Jian Yang, Department of Biological Sciences, 915 Fairchild Center, MC2462, Columbia University, New York, NY 10027. Fax:(212) 531-0425 E-mail:jy160{at}columbia.edu.
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Abstract |
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We studied the effect of monovalent thallium ion (Tl+) on the gating of single Kir2.1 channels, which open and close spontaneously at a constant membrane potential. In cell-attached recordings of single-channel inward current, changing the external permeant ion from K+ to Tl+ decreases the mean open-time by 20-fold. Furthermore, the channel resides predominantly at a subconductance level, which results from a slow decay (
= 2.7 ms at -100 mV) from the fully open level immediately following channel opening. Mutation of a pore-lining cysteine (C169) to valine abolishes the slow decay and subconductance level, and single-channel recordings from channels formed by tandem tetramers containing one to three C169V mutant subunits indicate that Tl+ must interact with at least three C169 residues to induce these effects. However, the C169V mutation does not alter the single-channel closing kinetics of Tl+ current. These results suggest that Tl+ ions change the conformation of the ion conduction pathway during permeation and alter gating by two distinct mechanisms. First, they interact with the thiolate groups of C169 lining the cavity to induce conformational changes of the ion passageway, and thereby produce a slow decay of single-channel current and a dominant subconductance state. Second, they interact more strongly than K+ with the main chain carbonyl oxygens lining the selectivity filter to destabilize the open state of the channel and, thus, alter the open/close kinetics of gating. In addition to altering gating, Tl+ greatly diminishes Ba2+ block. The unblocking rate of Ba2+ is increased by >22-fold when the external permeant ion is switched from K+ to Tl+ regardless of the direction of Ba2+ exit. This effect cannot be explained solely by ionion interactions, but is consistent with the notion that Tl+ induces conformational changes in the selectivity filter.
Key Words: permeation, Tl+, conformational change, selectivity filter, backbone mutation
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INTRODUCTION |
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In many ion channels, gating is tightly coupled to ion permeation and is strongly influenced by the nature of the permeant ion. For example, gating of the Cl- channel of Torpedo electroplax is coupled to the transmembrane electrochemical gradient of Cl- ions (
The crystal structure of the prokaryotic KcsA channel shows that three permeant ions are bound in the pore (
Inward rectifier K+ (Kir)* channels are gated by a variety of mechanisms, including intracellular Mg2+ ions and polyamines, G proteins, and ATP (
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MATERIALS AND METHODS |
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Molecular Biology and Oocyte Expression
C169V mutation was made by oligonucleotide insertion method and was confirmed by sequencing. Tandem tetramers containing varying numbers of C169V mutant subunits were constructed using a method described previously (
Electrophysiological Recordings
Recordings were performed 17 d after RNA injection. Whole-cell current was measured by two-electrode voltage clamp (TEVC) with the oocyte clamp amplifier (model OC-725C; Warner Instruments). Recording glass electrodes were filled with 3 M KCl and had a resistance of 0.4-0.8 M. The external solution contained either 100 mM KNO3 or 100 mM TlNO3, with 2 mM MgCl2 and 1 mM HEPES (pH adjusted to 7.4 with NMDG). In some experiments, 2.3 mM EDTA was added to the solution. In anomalous mole fraction experiments, external solutions contained mixtures of different concentrations of KNO3 or TlNO3, with the total concentration of both salts being kept at 100 mM. Current was evoked from a holding potential of 0 or +10 mV either by a -100-mV voltage pulse or a voltage ramp from -100 to +80 mV with an increment of 0.5 mV/ms. In Ba2+ blocking experiments, the duration of the pulse or the interpulse interval was adjusted to achieve steady-state Ba2+ blockage and complete recovery from the blockage.
Single-channel recordings were obtained in cell-attached membrane patches. Electrodes were fabricated from Corning Pyrex glass tubes, coated with sylgard, and heat polished to a resistance of 58 M when filled with a solution containing 110 mM KCl, 10 mM HEPES, 9 mM EGTA, and 1 mM EDTA (pH adjusted to 7.4 with KOH). In some experiments, we also filled the pipette with a solution containing 120 mM KNO3, 1 mM KCl, 10 mM HEPES, 9 mM EGTA, and 1 mM EDTA (pH adjusted to 7.4 with NMDG). No significant difference in single-channel properties was observed with either K+ solutions, and, therefore, the data were pooled together. When Tl+ was used as the permeant ion, the solution contained 120 mM TlNO3, 1 mM TlCl, 10 mM HEPES, 9 mM EGTA, and 1 mM EDTA (pH adjusted to 7.4 with NMDG). In Ba2+ blockage experiments, the pipette solution contained either 130 mM KCl or 130 mM TlNO3 and 1 mM TlCl, with 10 mM HEPES and a known concentration of BaCl2 (pH adjusted to 7.4 with NMDG). All reagents were purchased from Sigma-Aldrich.
For TEVC recordings, data acquisition and analysis were performed with a customized program written in Axobasic (Axon Instruments Inc.). Single-channel current was recorded with the pClamp 6 program (Axon Instruments, Inc.) at a constant negative potential ranging from -40 to -200 mV. Unless indicated otherwise, currents were filtered at 1 KHz with an 8-pole Bessel filter, digitized at 210 KHz with a 12-bit A/D interface (Axon Instruments Inc.) and stored on a computer. All recordings were conducted at temperatures 2123°C.
Data Analysis
The permeability ratios, PTl/PK, was calculated from changes of the reversal potential upon switching from K+ to Tl+ solution according to the equation: Erev = Erev, Tl - Erev, K = 2.30RT/F log (PTl/PK), where R, T, and F have their usual thermodynamic meanings. The voltage dependence of Ba2+ blockage was determined using the
FV/RT), where Kd (0) is the apparent dissociation constant at 0-mV membrane potential, z is the valence of the blocking ion, and
is the fractional electrical distance of the blocking ion binding site from the outside.
Single-channel current was analyzed with the pClamp6 program. Only patches that contained one channel were included in the analysis. Dwell-time distributions were constructed from records of varying length (from 2 to 30 min depending on the frequency of open/closed or blocked transitions) using logarithmic binning and square-root transformation and were fit with one or more exponential functions. No corrections were made for filter dead-time and missed events. The Ba2+ unblocking rate was computed as the inverse of the blocked time. Data are presented as mean ± SD (number of observations). Statistical analysis was done with t test.
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RESULTS |
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Tl+ Is Highly Permeant in Kir2.1 Channels
We first examined Tl+ permeation properties in Kir2.1 channels expressed in Xenopus oocytes. Fig 1 A shows whole-cell currents evoked by voltage ramps from -100 to +60 mV, with either K+ or Tl+ as the external permeant ion. The currents displayed strong inward rectification with either species of ions. Upon switching the external solution from K+ to Tl+, the reversal potential was shifted to the positive direction (see close-up in Fig 1 B) and the inward current increased significantly. The average shift of reversal potential is 12.6 ± 3.6 mV, corresponding to a permeability ratio PTl/PK of 1.65, which is similar to the value of 1.5 reported for a native inward rectifier K+ channel (
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Kir2.1 channels also displayed a strong anomalous mole fraction effect: when Tl+ and K+ ions were mixed together (while total concentration of both ions was kept at a constant level of 100 mM), the whole-cell current became smaller than with either ion alone outside (Fig 1 C). This behavior is very similar to that observed in a native inward rectifier channel (
Tl+ Alters Single-channel Gating Kinetics
As we reported previously (
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Single-channel behavior was greatly altered when Tl+ was used as the external permeant ion. Representative single-channel Tl+ currents recorded at -100 mV from a cell-attached patch are shown in Fig 2 B. Compared with K+ currents, two major changes in the gating kinetics are readily seen. First, the channel closed more frequently and the open-time was decreased by 20-fold across the voltage range studied (Fig 2B and Fig E), indicating a dramatic destabilization of the open state by Tl+. Second, although there were still three closed states (Fig 2 D), the dwell-time of the two longer closed states were altered, and they remained relatively constant in the voltage range of -140 to -200 mV and increased rather than decreasing with hyperpolarization in the voltage range of -80 to -140 mV (Fig 2 G). Both dwell times were shorter than those obtained with K+ as the permeant ion at -100 mV. On the other hand, as the membrane potential was hyperpolarized to -200 mV, both dwell times became longer than those obtained with K+ as the permeant ion. Due to these two effects, the overall open probability at -100 mV decreased from
0.9 for K+ to
0.6 for Tl+. These results indicate that the single-channel gating kinetics is strongly influenced by the species of the permeant ion.
Tl+ Induces Decay of Single-channel Current and a Subconductance Level
In addition to its dramatic effect on open/close gating kinetics, Tl+ also induced marked changes in sublevel behavior (Fig 2 B). Notably, the channel resided primarily at a subconductance level (Fig 2 B, arrows) rather than the closed or fully open level. When the channel opened from the closed level, it often jumped to a large conductance level, from which it relaxed to the predominant sublevel within several milliseconds. Fig 3 A shows 12 superimposed single-channel current events with a slow decay, aligned by their opening to the full level. The ensemble average trace shows a decay phase that can be fitted by a single exponential function with a time constant of 2.7 ms (Fig 3 B). The unitary current at -100 mV is 4.2 ± 0.2 pA for the fully open level and 3.1 ± 0.2 pA (n = 10) for the subconductance level. Transitions to other subconductance levels were also observed (Fig 2 B, arrowheads) but were not investigated further due to their rare occurrence. We also recorded Tl+ single-channel current with filtering at 3 kHz and found no difference in single-channel properties.
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The Single-channel Current Decay and Subconductance Level Are Produced by Interactions between Tl+ and Residue C169
Interestingly, gating of Kir2.1 channels in the presence of external Tl+ exhibits a remarkable time asymmetry (Fig 2 B). The decay and subconductance state occur faithfully after nearly every opening to the fully open level. However, from the subconductance state, the channel predominantly enters a closed state and only occasionally transition back to the fully open level. This type of nonequilibrium gating is phenomenologically reminiscent of that seen in a Cl- channel, which is coupled to Cl- permeation (
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Kir2.1 channels expressed in Xenopus oocytes are homotetramers and, thus, have four identical C169 thiol groups. Does Tl+ need to interact with multiple thiolate groups or with only one thiolate group to induce the decay and subconductance level? If the latter were the case, one would expect that a channel containing one C169 still exhibits the decay but with one-fourth of the frequency observed in the wild-type channel. To address this question, we constructed tandem tetramers containing one to three C169V mutant subunits by using a strategy described previously (
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Kinetics of Single-channel Tl+ Current Was not Altered by C169V Mutation
Although Tl+ did not induce decay of single-channel current and subconductance level in C169V mutant channels, it still greatly destabilized the open state (Fig 4). The mean open time at -100 mV was decreased from 87 ± 3 ms (n = 4) with K+ as the permeant ion to 10.8 ± 0.4 ms (n = 4) with Tl+ as the permeant ion, which is similar to that for the wild-type channel. With Tl+, the dwell time of the three closed states at -100 mV was 0.63 ± 0.25, 5.1 ± 0.7, and 35 ± 6 ms (n = 4), respectively, which are also similar to those for the wild-type channel (0.7 ± 0.25, 4.5 ± 1.7, and 21 ± 11 ms, n = 10). These results suggest that the effect of Tl+ on single-channel gating kinetics is caused by interactions between Tl+ and other parts of the pore. The most likely region is the selectivity filter, where the more electron "hungry" Tl+ is expected to interact more strongly than K+ with the main chain carbonyl oxygens.
Tl+ Diminishes Ba2+ Blockage
To test the hypothesis that the effect of Tl+ on single-channel kinetics is exerted through its interactions with the selectivity filter, we investigated whether Tl+ induces conformational changes in the filter region. Such conformational changes, if there were any, would most likely be very subtle. To detect such conformational changes, we used Ba2+ as a probe and compared inhibition of K+ or Tl+ current by external Ba2+. Since Ba2+ binds to a high affinity site in the selectivity filter (
In whole-cell two-electrode voltage clamp recordings, 30 µM Ba2+ had little effect on the peak K+ current evoked by a test pulse to -100 mV from a holding potential of 0 mV, but it produced a time-dependent blockage of the current during the voltage pulse (Fig 6 A). As Ba2+ blocks K+ channels by one-to-one binding () derived from data points from -40 to -100 mV was identical (0.8) in the presence of K+ or Tl+, suggesting that in both cases Ba2+ blocks the channel by binding to a site similarly located in the membrane electrical field. This z
value is similar to the value of 1.08 reported previously for Kir2.1 channels (
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A much reduced Ba2+ blockage of Tl+ current was also evident at the single-channel level. With K+ as the external permeant ion, 10 µM external Ba2+ induced frequent long-lived blocking events lasting 5.27 ± 1.03 s (n = 5) at -100 mV (Fig 7 A). When Tl+ was used as the external permeant ion, however, a much higher concentration of Ba2+ (300 µM) was required to induce significant numbers of blocking events (Fig 7 B). The blocked state had a much shorter mean lifetime of 112 ± 19 ms (n = 8) at -100 mV, corresponding to a 47-fold increase in the Ba2+ unblocking rate (koff). The voltage dependence of Ba2+ koff was mild with either K+ or Tl+ as the permeant ion (Fig 8). Nevertheless, in both cases, the koff decreased significantly with increasing negative membrane potential in the range between -60 and -120 mV (or -140 mV for Tl+). With Tl+ as the permeant ion, the Ba2+ koff at -60 and -140 mV was 11.4 ± 2.3 s-1 and 7.8 ± 1.8 s-1 (n = 7), respectively, and was significantly different from each other (P < 0.01). This is indicative that the bound Ba2+ exits mainly to the external side in this voltage range. When the membrane potential was increased beyond -120 mV (or -140 mV for Tl+), the Ba2+ koff started to increase again, indicating that the bound Ba2+ now leaves mainly to the internal side. However, regardless of the direction of Ba2+ exit, its dissociation rate was increased by >22-fold when the permeant ion was switched from K+ to Tl+.
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Due to the long duration of the blocking events, we were unable to determine the Ba2+ blocking rate at the single-channel level. However, the apparent Ba2+ blocking rate can be obtained by using the equation kon = koff/Kd, where koff is obtained from single-channel recordings and Kd from whole-cell recordings. According to this method, the Ba2+ blocking rate at -100 mV is reduced by nearly sixfold when the external permeant ion is switched from K+ to Tl+.
Since C169 interacts with Tl+ and since the C169V mutation abolished Tl+-induced decay of single-channel current and subconductance level, we asked whether it also eliminated Tl+-induced changes in Ba2+ blockage. Ba2+ blocked whole-cell K+ or Tl+ current at -100 mV in C169V mutant channels with an apparent Kd of 2.2 ± 0.2 µM (n = 5) and 863 ± 405 µM (n = 8), respectively, similar to that obtained in the wild-type channel. Furthermore, in single-channel recordings, the Ba2+ dwell time at -100 mV was decreased from 5.4 ± 0.6 s (n = 4) in the presence of external K+ to 145 ± 16 ms (n = 4) in the presence of external Tl+, again similar to that observed in the wild-type channel. These results indicate that the Tl+-induced changes in Ba2+ blockage do not result from interactions between Tl+ and C169.
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DISCUSSION |
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Permeation and gating are two fundamental biophysical properties of ion channels. Although these two processes were originally thought to be independent, there is increasing evidence that they are tightly coupled and that permeant ions strongly affect gating (see INTRODUCTION). In this study, we demonstrate that substitution of K+ with another permeant ion (Tl+) dramatically alters gating of Kir2.1 channels in two ways. First, Tl+ induces a slow decay of the single-channel current, resulting in a dominant subconductance level. Second, Tl+ greatly destabilizes the open state of the channel and changes the open/close kinetics of gating.
Slow Decay of Single-channel Current
The slow decay of single-channel current immediately after channel opening (Fig 2 and Fig 3) is unusual and, to our knowledge, is unprecedented. Such decay was observed in every channel recorded with Tl+ (>30 patches with various recording conditions), but was never observed with K+ as the permeant ion (>100 patches including results from our previous studies), indicating that it is conferred by the unique chemical properties of Tl+. The observation that the C169V mutation eliminates the slow decay and the associated subconductance level indicates that they result from interactions between Tl+ and the thiolate group of C169. In KcsA, the residue (I100) occupying the same position as C169 points directly at the cavity ion (
How does such ionic interactions result in the slow decay of single-channel current and the subconductance level? One possibility is that each of the four thiolate groups binds one Tl+ independently, partially occluding the ion conduction pathway, and thereby reducing the single-channel current. Previous work has shown that covalent modification of one of the four subunits at a nearby position, 172, by a thiol reactive reagent does indeed produce partial inhibition of the single-channel current (
An alternative possibility is that when Tl+ permeates the cavity, it initiates an interaction with at least three thiolate groups of C169. This process involves reorientation of and optimal coordination by the thiolate groups. Since side chain rotation is generally extremely rapid (on the time scale of 10-1110-8 s;
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Fig 9 B depicts a model to qualitatively describe the slow decay of single-channel current and the subconductance level induced by Tl+. In this model, we postulate that, with Tl+ as the permeant ion, the channel can adopt a fully open state (Of), a subconductance state (Os), and two closed states, Cf and Cs. We also assume that in Cf the thiol group of C169 does not interact with Tl+, either because the side chain orientation or ionization is not optimal or because the cavity is devoid of Tl+. With this assumption, CfCs and Cf
Os transitions are not permitted. The closed channel can open to Of only from Cf (Fig 9 C). This transition generates full amplitude Tl+ inward current. Permeating Tl+ then binds to at least three thiolate groups of C169 and this binding triggers a slow conformational change of the ion conduction pathway, resulting in a slow decay of Tl+ current and a subconductance state Os. From Os, the channel can occasionally transition to Of, but most of the time it enters Cs (Fig 9 C), which still retains the conformational change induced by Tl+. Cs can subsequently open to Os or enter Cf (Fig 9 C). The latter transition (Cs
Cf) involves a conformation change opposite that of the Of
Os transition and enables the channel to conduct full amplitude Tl+ current when it undergoes the Cf
Of transition again.
According to this model, channel openings to the full level (CfOf transitions) should be, in most cases, preceded by two closed states (i.e., Cs and Cf) and those to the subconductance level (Cs
Os transitions) by only one closed state (i.e., Cs; Fig 9 C). We segregated these two types of openings and analyzed the closed-time distribution (at -100 mV) of their preceding closed states, and found that indeed full openings are preceded by two closed states with a dwell time of 6.7 ± 1.1 ms and 26 ± 6 ms (n = 3) and subconductance openings by a single closed state with a dwell time of 1.6 ± 0.2 ms (n = 3). These closed times are similar to those obtained with both types of openings mingled together (see RESULTS). The kinetics of the open
closed transitions are likely governed by interactions between permeant ions and the selectivity filter, as will be discussed below.
Permeant Ion-induced Conformational Changes in the Selectivity Filter
Our previous results with amide-to-ester mutations suggest that permeant ion-dependent conformational changes of the selectivity filter contribute directly to the spontaneous gating of single Kir2.1 channels (
The mechanism by which permeant ions produce channel closure has been studied by
In addition to the dramatic effects on gating, Tl+ also greatly diminishes Ba2+ blockage of the channel. In particular, the Ba2+ unblocking rate is increased by >22-fold within a wide range of membrane potential when the permeant ion is switched from K+ to Tl+ (Fig 8). What is the biophysical mechanism underlying this dramatic destabilization of Ba2+ binding? X-ray crystallography shows that in KcsA Ba2+ binds to a single site at the end of the selectivity filter close to the central cavity (
Assuming that the same set of ion binding sites is present in Kir2.1 channels, can the large difference in Ba2+ unblocking rate with either K+ or Tl+ as the permeant ion be explained by the difference in the occupancy of these sites? In a Ba2+-blocked channel, the internal lock-in site is in true equilibrium with the oocyte cytoplasm in the cell-attached recording configuration and, thus, is visited only by K+ regardless of the species of the external permeant ion. On the other hand, since the external lock-in site has a micromolar affinity for Tl+ and K+ (
Permeant ion-dependent conformational changes have been reported for other types of K+ channels. The C-type inactivation of voltage-gated K+ (Kv) channels involves a conformational change of the outer mouth of the pore, including the selectivity filter (
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Footnotes |
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* Abbreviations used in this paper: Kir channels, inward rectifier K+ channels; Kv channels, voltage-gated K+ channels; TEVC, two-electrode voltage clamp.
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Acknowledgements |
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This work was supported by grant HL58552 (to J. Yang) and fellowship NS11097 (to T. Lu) from the National Institutes of Health. J. Yang was a recipient of the McKnight Scholar Award.
Submitted: 12 July 2001
Revised: 24 August 2001
Accepted: 25 September 2001
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