Correspondence to: Stephen J. Korn, Department of Physiology and Neurobiology, Box U-156, University of Connecticut, 3107 Horsebarn Hill Rd., Storrs, CT 06269. Fax:860-486-3303 E-mail:korn{at}oracle.pnb.uconn.edu.
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Abstract |
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In the Kv2.1 potassium channel, binding of K+ to a high-affinity site associated with the selectivity filter modulates channel sensitivity to external TEA. In channels carrying Na+ current, K+ interacts with the TEA modulation site at concentrations 30 µM. In this paper, we further characterized the TEA modulation site and examined how varying K+ occupancy of the pore influenced the interaction of K+ with this site. In the presence of high internal and external [K+], TEA blocked 100% of current with an IC50 of 1.9 ± 0.2 mM. In the absence of a substitute permeating ion, such as Na+, reducing access of K+ to the pore resulted in a reduction of TEA efficacy, but produced little or no change in TEA potency (under conditions in which maximal block by TEA was just 32%, the IC50 for block was 2.0 ± 0.6 mM). The all-or-none nature of TEA block (channels were either completely sensitive or completely insensitive), indicated that one selectivity filter binding site must be occupied for TEA sensitivity, and that one selectivity filter binding site is not involved in modulating TEA sensitivity. At three different levels of K+ occupancy, achieved by manipulating access of internal K+ to the pore, elevation of external [K+] shifted channels from a TEA-insensitive to -sensitive state with an EC50 of ~10 mM. Combined with previous results, these data demonstrate that the TEA modulation site has a high affinity for K+ when only one K+ is in the pore and a low affinity for K+ when the pore is already occupied by K+. These results indicate that ionion interactions occur at the selectivity filter. These results also suggest that the selectivity filter is the site of at least one low affinity modulatory effect of external K+, and that the selectivity filter K+ binding sites are not functionally interchangeable.
Key Words: permeation, outer vestibule, conformation, repulsion
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INTRODUCTION |
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Voltage-gated K+ channels are multi-ion pores, and, while conducting, can be occupied by three to four K+ (
In the cloned potassium channel, Kv2.1, block by tetraethylammonium is cation dependent (
In Kv2.1, reduction of TEA sensitivity upon reduction of [K+] results primarily from a conformational change in the outer vestibule of the conduction pathway (
In this paper, we demonstrate that changes in K+ occupancy of the pore modulate TEA efficacy in Kv2.1 channels carrying K+ current. This observation allowed us to address the following questions: (a) what is the nature of the K+ binding site responsible for conferring TEA sensitivity? and (b) how does occupancy of the selectivity filter by different cations influence the affinity of the TEA modulation site for K+? Our data indicate that TEA sensitivity is modulated by occupancy of just one of the two K+ binding sites associated with the selectivity filter. Whereas the TEA modulation site binds K+ with relatively high affinity when only one K+ occupies the selectivity filter, it binds K+ with much lower affinity when a K+ ion also occupies the other selectivity filter site. Consequently, our data indicate that ionion interactions occur at the selectivity filter, and that the selectivity filter is the site of at least one low affinity interaction of external K+ with the pore.
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METHODS |
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Molecular Biology and Channel Expression
Experiments were done on two channels: wild-type Kv2.1 and a mutant channel derived from Kv2.1, Kv2.1 K356G, K382V. Mutagenesis and expression details are described in
Electrophysiology
Currents were recorded at room temperature in the whole cell patch clamp configuration. Patch pipets were fabricated from N51A glass (Garner Glass Co.), coated with Sylgard and firepolished. Currents were collected with an Axopatch 1D amplifier, pClamp 6 software, and a Digidata 1200 A/D board (Axon Instruments). Currents were filtered at 2 kHz and sampled at 2001,000 µs/pt. Series resistance ranged from 0.5 to 2.5 M and was compensated 8090%. The holding potential was -80 mV, and depolarizing stimuli were presented once every 515 s, depending on the experiment. In all experiments except Fig 4 B, currents were recorded at 0 mV. Fig 4 B experiments recorded currents at +20 mV. TEA inhibited currents identically at 0 and +20 mV (data not shown). Concentrationresponse curves were fit to Equation 1,
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(1) |
where X is the drug concentration, IC50 is the drug concentration that produced half maximal inhibition, bmax is the maximal level of block at saturation, and N is the slope of the curve. Data were analyzed with Clampfit (Axon Instruments); curve fitting and significance testing (unpaired Student's t test) were done with SigmaPlot 2.0. All plotted data are represented as mean ± SEM, with the number of data points denoted by n. For IC50 values, a range of values is given for n. This range represents the number of cells used for each data point in the complete concentrationresponse curve from which the IC50 was calculated.
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Electrophysiological Solutions
Currents were recorded in a constantly flowing, gravity fed bath. Solutions were placed in one of six reservoirs, each of which fed via plastic tubing into a single quartz tip (~100 µm diameter; ALA Scientific Instruments). The tip was placed within 20 µm of the cell being recorded before the start of the experiment. One solution was always flowing, and solutions were changed by manual switching (solution exchange was complete within 510 s). Control internal solutions contained (mM): 140 XCl (X = a combination of K+ and NMG+), 20 HEPES, 10 EGTA, 1 CaCl2, 4 MgCl2, pH 7.3, osmolality 285. Block by external TEA was identical when Mg2+ was omitted from the internal solution (data not shown). Control external solutions contained (mM): 165 XCl, 20 HEPES, 10 glucose, 2 CaCl2, and 1 MgCl2, pH 7.3, osmolality 325. In all experiments, internal and external [K+] are reported; osmotic balance was maintained with NMG+. Other additions and substitutions are listed in the figure legends.
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RESULTS |
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Inhibition by External TEA at Different [K+]
Fig 1A and Fig B, illustrates outward currents recorded through Kv2.1 with different internal and external [K+] (in this and all experiments, recordings were made in the absence of Na+). We previously demonstrated that in Kv2.1, inactivation rate was dependent on K+ occupancy of the pore (
Fig 1 A illustrates block by 3 mM TEA under these three conditions. Fig 1 B illustrates block of K+ currents by 100 mM TEA under the same conditions. As the [K+] that the channels were exposed to was decreased, block by TEA decreased. Fig 1 C illustrates complete [TEA]response curves under these three conditions. With 100 mM internal and 50 mM external K+, TEA blocked K+ currents with an IC50 of 1.9 ± 0.2 mM (n = 45; Fig 1, ). Importantly, 100% of the current through Kv2.1 could be blocked by TEA, which indicates that TEA interacted with 100% of the conducting channels. These data further indicate that if TEA block requires the binding of K+ to a site in the pore (see below), then the TEA modulation site is saturated with these internal and external [K+]. Under conditions of lower K+ occupancy, TEA potency was essentially unchanged, but not all channels could be blocked by external TEA. In recordings with 100 mM internal but 0 external K+, TEA blocked K+ currents with an IC50 of 3.1 ± 0.4 mM (n = 316; Fig 1, ). However, 100 mM TEA blocked just 83.9 ± 0.8% (n = 12) of the current and, according to the best fit of the experimental data, maximal possible block of total current by TEA would be just 91.6 ± 1.1%. When currents were carried by just 10 mM internal K+ (in the presence of 0 mM external K+), the change in TEA efficacy was clear. Under these conditions, there was little or no change in TEA potency (IC50 = 4.1 ± 0.9 mM, n = 314), but the maximum block was markedly reduced (Fig 1,
). At a concentration of 100 mM, TEA blocked just 51.1 ± 1.1% (n = 14) of the current, and the best fit of the data indicated that just 56.2 ± 3.0% of the total current could be blocked. In all cases, full recovery was achieved upon removal of TEA (Fig 1A and Fig B).
The reduction in TEA efficacy suggests that current-carrying channels could be in one of two states. Some channels could be blocked by TEA with an IC50 of 24 mM; other channels were completely insensitive to TEA at concentrations of up to 100 mM. Furthermore, these data suggest that interconversion between these two states depended on the occupancy of the pore by K+, such that at higher K+ occupancy, channels were in the TEA-sensitive state and at lower K+ occupancy, channels were insensitive to TEA.
Change in External TEA Efficacy with an Internal Channel Blocker
In the experiments in Fig 1, we altered K+ occupancy of the pore by reducing the [K+] to which the channels were exposed. An alternative approach to influencing K+ occupancy of the channel is to block the pore from the intracellular side of the channel (
Fig 2 A illustrates the effect of two external [TEA] on currents recorded with 100 mM intracellular K+ plus 20 mM intracellular TEA (0 external K+). Intracellular TEA at this concentration blocks inward current through Kv2.1 by 8590% in the presence of 30 mM external K+ () and absence () of internal TEA. Internal TEA had no effect on the IC50 for external TEA (IC50 = 2.0 ± 0.6; n = 36). However, in the presence of internal TEA, 100 mM external TEA blocked just 31.8 ± 0.7% (n = 6) of the current. According to the best fit of the data, the maximum possible block by external TEA was 32.4 ± 0.5%.
[K+] Dependence of External TEA Sensitivity in the Presence of Internal TEA
In studies with Kv1.x channels, it had been suggested that internal TEA reduced external TEA potency by a direct interaction between the internal and external TEA (
In Fig 3, we examined the [K+] dependence of external TEA sensitivity on outward K+ currents, in the presence of 100 mM internal K+ plus 20 mM internal TEA. In all experiments, external TEA was applied to cells exposed to at least three different external [K+]. In different cells, the order of [K+] exposure was varied to control for the possibility of long term consequences of exposure (or lack thereof) to external K+. Fig 3 A illustrates the results of one experiment. For this cell, external TEA was applied under three conditions in the following order: in the presence of 3, 5, and 1 mM external [K+]. Once current magnitude stabilized at a given [K+], TEA was applied briefly (), and then removed. After recovery to control current amplitude, external [K+] was changed and external TEA reapplied. TEA block increased as a function of [K+]. In the presence of 1, 3, and 5 mM external K+, TEA blocked currents by 26.9 ± 1.0%, 35.0 ± 1.6%, and 38.2 ± 1.1% (n = 3), respectively.
In the absence of external TEA (), current magnitude changed with changes in external [K+]. In Kv2.1, as in other voltage-gated K+ channels, the magnitude of outward K+ currents increases as external [K+] is elevated from 0 to 10 mM (
In the presence of 100 mM internal K+ plus 20 mM internal TEA, changes in external [K+] were expected to modulate block by 10 mM TEA between 25 and 83% for the following reasons. In the absence of external K+, 10 mM TEA blocked channels by 24.7 ± 0.9% (n = 3; Fig 2). Under conditions that saturated the TEA modulation site with [K+] (100 mM internal K+, 50 mM external K+, no internal TEA), 10 mM external TEA blocked 82.7 ± 1.5% (n = 4) of the total current (Fig 1). Consequently, in this experiment, the maximum block possible upon elevation of external [K+] was expected to be 83%, which would occur if the pore became fully saturated by K+. Fig 3 B illustrates that elevation of external [K+] between 0 and 50 mM produced a concentration-dependent increase in block, with an EC50 of 10.8 ± 1.1 mM (n = 37).
[K+] Dependence of External TEA Sensitivity in the Absence of Internal TEA
Our working hypothesis was that the [K+] dependence observed in Fig 3 B simply reflected the association of external K+ with a binding site in a K+-conducting pore. This interpretation depends on the assumption that internal TEA did not influence the binding of external K+ to this site, and that the [K+] dependence was not influenced by a direct interaction between external K+ and internal TEA. It was critical, therefore, to demonstrate that the concentration dependence observed was not related to the presence of internal TEA. To do this, we examined the [K+] dependence of external TEA block under conditions that did not include internal TEA.
With 100 mM K+ in the pipet and 0 external K+, TEA efficacy was submaximal (Fig 1). In the absence of external K+, 10 mM TEA blocked current carried by 100 mM K+ by 65.6 ± 1.2% (n = 16). As previously described, 10 mM TEA blocked current in K+-saturated channels by 83% (Fig 1). Fig 4 A illustrates the [K+]-dependent enhancement of block by 10 mM TEA from 66 to 83%. Even over this limited range of enhancement, the [K+] dependence is reasonably well fit by a sigmoidal function, with an EC50 that fell between 8 and 10 mM.
Fig 4 B illustrates the [K+]-dependent increase of TEA block when currents were carried by 10 mM internal K+. As described previously, the maximum possible block by 10 mM TEA in a K+-saturated channel was 83%. In the absence of external K+, 10 mM TEA blocked 37.0 ± 1.8% (n = 10) of the total current carried by 10 mM internal K+ (Fig 1). Current magnitude was significantly enhanced in a concentration-dependent manner by addition of both 5 and 10 mM external K+. Indeed, in the presence of 10 mM external K+, TEA blocked total current by 59.9 ± 1.0% (n = 3), which is the halfway point between 37 and 83%. Thus, under three different experimental conditions (10 Kin, 100 Kin, and 100 Kin plus 20 TEAin), which produced different K+ occupancies of the pore and different efficacies of external TEA, enhancement of TEA block by external K+ from the minimum (measured in the absence of external K+) to the maximum possible (measured in a K+-saturated pore) occurred with an EC50 of ~10 mM. In other words, in all conducting channels with an unoccupied TEA modulation site in the pore, this site was bound by K+ with an apparent Kd of ~10 mM.
Lysines in the External Vestibule Associated with Loss of TEA Sensitivity
Previous work demonstrated that in Kv2.1, the loss of TEA sensitivity in the presence of Na+ (and absence of K+) was associated with a conformational rearrangement in the outer vestibule of the channel (
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Fig 5 A illustrates the block by 0.3 mM TEA on currents carried through the mutant channel by 100 (top) and 10 (bottom) mM K+. With 100 mM internal K+ (and 0 external K+), TEA blocked currents through the channel with an IC50 of 0.21 ± 0.02 mM (n = 312; Fig 5 B, ). With 10 mM internal K+, TEA blocked currents with an IC50 of 0.67 ± 0.07 mM (n = 38; Fig 5 B, ). This shift in IC50 is consistent with the previously described K+-dependent change in TEA potency in this mutant channel (
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DISCUSSION |
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The interaction of K+ with the selectivity filter in Kv2.1 modulates a conformational change in the outer vestibule of the pore (
One Cation Binding Site Is Responsible for Modulation of TEA Efficacy
The crystal structure of KcsA suggests that the K+ channel pore contains three cation binding sites, two associated with the selectivity filter and one in a water-filled compartment internal to the selectivity filter (
Fig 6 illustrates a cartoon of the K+ channel pore that contains two cation binding sites associated with the selectivity filter, located between the internal and external TEA binding sites. In Fig 6 (1), the two selectivity filter binding sites are unoccupied. These channels are not carrying current and are invisible to the experiment. (3) represents a channel with a fully saturated selectivity filter. These latter channels can all be blocked by TEA, as demonstrated by the observation that TEA blocks 100% of channels at very high internal and external [K+] (Fig 1). (2a) and (2b)illustrate possible channel configurations, each with just one selectivity filter binding site occupied. Channels would exist in one or both of these configurations at [K+] that do not saturate the channel. There are two possible explanations for the observation that, at lower [K+], some channels cannot be blocked by TEA. First, it is possible that only fully saturated channels (3) can be blocked by TEA, and that the channel configurations represented in (2a) and (2b) are TEA-insensitive. Alternatively, it is possible that only one selectivity filter binding site must be occupied by K+ for block by TEA. Previous data indicate that, indeed, channels can be blocked by TEA when only a single selectivity filter site is occupied by K+. In Na+-conducting K+ channels, only 30 µM K+ is required for measurable inhibition of current by TEA ( 100 µM, K+ occupies the selectivity filter, but an insignificant number of channels will have selectivity filters that are doubly occupied by K+. Thus, TEA block requires K+ occupancy of just a single selectivity filter binding site.
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Because occupancy of the TEA modulation site by K+ influences TEA efficacy (as opposed to potency), there must also be a current-carrying channel that cannot be blocked by TEA. Since all channels in Fig 6 (3) can be blocked, and one of the channels in (2) can be blocked, the only other current carrying channel configuration available as a candidate for a TEA-insensitive channel is the other channel depicted in (2). Thus, only one of the two selectivity filter binding sites serves as the TEA modulation site.
The Affinity of the Second Selectivity Filter Cation Binding Site, When One Is Occupied, Is ~10 mM: Implications for Permeation Theory
Both functional and structural studies have provided convincing evidence that there are several discrete cation binding sites in the K+ channel conduction pathway (30 µM (
Is There a Low Affinity Cation Binding Site External to the Selectivity Filter Binding Sites in Voltage-gated K+ Channels?
Our results indicate that in K+ conducting channels, the low affinity modulation of TEA sensitivity by K+ occurs at a selectivity-filter binding site. The apparent affinity of ~10 mM for the interaction of K+ with the Kv2.1 pore already occupied by one K+ ion is similar to the apparent affinity of other low affinity interactions in K+-conducting channels. For example, external K+ slows inactivation rate in Shaker with an apparent Kd of 2 mM (
Additional Implications of Changes in TEA Efficacy Versus Potency
In Kv2.1, reduction of K+ occupancy of the pore in the absence of a substitute permeating ion produced little or no change in TEA potency, but had a dramatic impact on TEA efficacy (Fig 1 and Fig 2). The reduced efficacy indicates that occupancy of a site in the pore by a cation (K+ in this case) is an absolute requirement for TEA block of the wild-type Kv2.1 channel. When Na+ was substituted for K+, TEA was completely without effect (
Finally, our interpretation that the change in TEA efficacy reflects all-or-none channel block depends on the assumption that TEA-bound channels do not conduct under low [K+] conditions. There is no direct evidence that addresses this assumption. However, this assumption is strongly supported by the observations that TEA binds to all four channel subunits (
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Acknowledgements |
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We thank one of the anonymous reviewers and Dr. Olaf Andersen, whose insight helped clarify our interpretation and the manuscript.
Funding was provided by the National Science Foundation and the American Heart Association, Connecticut Affiliate.
Submitted: 22 November 1999
Revised: 29 February 2000
Accepted: 2 March 2000
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