Correspondence to: Chih-Yung Tang, UCLA School of Medicine, Los Angeles, CA 90095. Address correspondenceto Diane M. Papazian, Ph.D., Department of Physiology, UCLA School of Medicine, Los Angeles, CA 90095-1751. Fax:(310) 206-5661 E-mail:papazian{at}mednet.ucla.edu.
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Abstract |
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Extracellular Mg2+ directly modulates voltage-dependent activation in ether-à-go-go (eag) potassium channels, slowing the kinetics of ionic and gating currents (Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 2000. J. Gen. Physiol. 115:319-337). To exert its effect, Mg2+ presumably binds to a site in or near the eag voltage sensor. We have tested the hypothesis that acidic residues unique to eag family members, located in transmembrane segments S2 and S3, contribute to the Mg2+-binding site. Two eag-specific acidic residues and three acidic residues found in the S2 and S3 segments of all voltage-dependent K+ channels were individually mutated in Drosophila eag, mutant channels were expressed in Xenopus oocytes, and the effect of Mg2+ on ionic current kinetics was measured using a two electrode voltage clamp. Neutralization of eag-specific residues D278 in S2 and D327 in S3 eliminated Mg2+-sensitivity and mimicked the slowing of activation kinetics caused by Mg2+ binding to the wild-type channel. These results suggest that Mg2+ modulates activation kinetics in wild-type eag by screening the negatively charged side chains of D278 and D327. Therefore, these residues are likely to coordinate the bound ion. In contrast, neutralization of the widely conserved residues D284 in S2 and D319 in S3 preserved the fast kinetics seen in wild-type eag in the absence of Mg2+, indicating that D284 and D319 do not mediate the slowing of activation caused by Mg2+ binding. Mutations at D284 affected the eag gating pathway, shifting the voltage dependence of Mg2+-sensitive, rate limiting transitions in the hyperpolarized direction. Another widely conserved residue, D274 in S2, is not required for Mg2+ sensitivity but is in the vicinity of the binding site. We conclude that Mg2+ binds in a water-filled pocket between S2 and S3 and thereby modulates voltage-dependent gating. The identification of this site constrains the packing of transmembrane segments in the voltage sensor of K+ channels, and suggests a molecular mechanism by which extracellular cations modulate eag activation kinetics.
Key Words: voltage clamp, structural model, kinetics, voltage sensor, metal binding site
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INTRODUCTION |
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The Drosophila ether-à-go-go (eag) gene and its homologues encode a distinct subfamily of voltage-gated K+ channels (
In proteins, bound Mg2+ ions are often coordinated by the acidic side chains of aspartate and glutamate residues (
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The mechanism of voltage-dependent activation has been extensively studied in K+ channels, notably Shaker channels (
The voltage sensor is contained in a domain of the channel that includes S2 through S4 at a minimum, with a possible structural contribution from segment S1 (
Voltage-dependent activation in eag can be described reasonably well by a sequential model in which each subunit undergoes two voltage-dependent transitions that prime the channel for opening, followed by a cooperative and less voltage-dependent step that opens the pore (
To test the hypothesis that eag-specific acidic residues in S2 and S3 contribute to the Mg2+-binding site, these residues and others were individually mutated to determine the effect on sensitivity to Mg2+. The results indicate that Mg2+ binds between segments S2 and S3, and thereby modulates activation gating in eag channels.
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MATERIALS AND METHODS |
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Molecular Biology
Mutations in eag were generated by PCR using a three or four primer strategy (
Constructs were linearized with NotI before in vitro transcription using the mMessage mMachine kit (Ambion). RNA encoding wild-type and mutated eag channels was injected into Xenopus oocytes for electrophysiological analysis (
Electrophysiology
Ionic currents from wild-type and mutant eag channels were recorded at room temperature (2022°C) using a two electrode voltage clamp as described previously (.
Voltage pulse protocols were applied and data were acquired using pClamp v5.5.1 software and a TL-1 Labmaster interface (Axon Instruments, Inc.). The sampling rate was varied as needed to resolve activation kinetics. Data were filtered at 0.2 to 2 kHz using an 8-pole Bessel filter (Frequency Devices), and digitized at a frequency five times higher than the filter frequency. Linear capacitive and leak currents were subtracted using a P/-4 protocol (
Activation kinetics were compared in the absence and presence of MgCl2, using concentrations up to 20 mM. Ionic currents were evoked by pulsing from a holding potential of -80 or -90 mV to voltages ranging from -60 mV to +60 or +100 mV in 20 mV increments. Activation kinetics were quantified in two ways as previously described (. Alternatively, the time to half maximal current amplitude was measured to obtain a value for t1/2. These methods gave compatible results. Values of
and t1/2 are provided as mean ± SEM.
The probability of opening as a function of voltage was determined by measuring the amplitude of isochronal tail currents in 89 mM KCl bath solution. Ionic currents were evoked by pulsing from a holding potential of -90 mV to voltages ranging from -70 mV to +80 mV in 10 mV increments. Tail currents were recorded upon return to -90 mV. Tail current amplitudes were determined at isochronal points ranging from 4 to 10 ms following repolarization, normalized to the maximum value, and plotted versus test potential. The data were fitted with a single Boltzmann equation using Origin 5.0 software (Microcal) to obtain a slope factor and midpoint potential.
The effect of prepulse hyperpolarization on activation kinetics was investigated in the presence and absence of Mg2+ (
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RESULTS |
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Modulation of Activation Kinetics by Mg2+
The Drosophila eag K+ channel was expressed in Xenopus oocytes. Ionic currents were recorded using a two electrode voltage clamp in the presence and absence of 10 mM extracellular Mg2+, a concentration that produced a near maximal slowing of activation kinetics (see Fig 4 B). Eag kinetics were quantified in two ways () for the process (Fig 2 A). However, this
value does not adequately describe the initial time course of the ionic current, which displays sigmoid kinetics (
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Mg2+ slowed eag kinetics throughout the activation voltage range, with a larger effect after smaller depolarizations (Fig 2) (10-fold at 0 mV, compared with 4-fold at +100 mV. To investigate whether eag activation kinetics become Mg2+-independent at very depolarized voltages, plots of
and t1/2 versus test potential were extrapolated beyond the experimentally accessible voltage range. Both plots were well fitted by single exponential functions (Fig 2), which were used to predict limit values for
and t1/2 at infinite positive voltage. This procedure resulted in estimates of 6 ms for
lim in the presence of Mg2+, compared with
2 ms in its absence. Similarly, t1/2lim was estimated to be 12 ms in the presence and
4 ms in the absence of Mg2+. Thus, Mg2+ is predicted to slow activation kinetics by about threefold at infinite positive voltage.
As previously described, hyperpolarizing prepulses also decelerate eag activation, increasing the delay and decreasing the rate of onset of ionic currents evoked by a subsequent test pulse ( and t1/2 as a function of prepulse potential in the presence and absence of Mg2+ (Fig 3 B). The data reveal the existence of rate-limiting, Mg2+-sensitive transitions that occur at hyperpolarized potentials during the activation of wild-type eag channels (
D278 Mutations Eliminate Mg2+ Sensitivity
Eag-specific acidic residues in S2 and S3 are good candidates to bind Mg2+ because of their location in the voltage sensing domain and their conservation in the eag subfamily. Neutralization of D278, the eag-specific acidic residue in S2, in the mutant D278V eliminated Mg2+ modulation of activation kinetics (Fig 4). Fig 4 A (left) shows currents evoked by depolarizing from -80 mV to +60 mV in the presence and absence of 10 mM Mg2+. Whereas wild-type eag showed measurable changes in activation kinetics upon exposure to 1 mM Mg2+, D278V was insensitive to Mg2+ concentrations as high as 20 mM (Fig 4 B). Ionic current kinetics in D278V were unaffected by extracellular Mg2+ throughout the voltage range for activation (data not shown).
Interestingly, the conservative mutation D278E also had Mg2+-insensitive activation kinetics (Fig 4 A, right, and B). This is significant because the glutamate substitution in D278E retains a carboxylic acid side chain, but is longer by one methylene group than the original aspartate residue. These data suggest that the mutation prevents Mg2+ from binding because the binding site is sterically constrained in the vicinity of D278.
Activation kinetics in D278V and D278E were quantified as a function of prepulse potential (Fig 4 C). In these mutants, rate-limiting transitions were accessed at hyperpolarized potentials, as indicated by an increase in after hyperpolarizing prepulses (Fig 4 C, left). Values for t1/2 showed a similar trend (Fig 4 C, right). Extracellular Mg2+ did not modulate the kinetics of these transitions, however (Fig 4 C). An extended range of prepulses was used for D278V because this mutation shifted the probability of opening versus voltage (PoV) curve in the depolarized direction compared with wild-type eag and the mutant D278E (Fig 4 D). The transition to slower activation kinetics occurred at more depolarized potentials in D278V than in wild-type or D278E channels (Fig 4 C).
Significantly, the D278V and D278E mutations had differential effects on eag activation kinetics. Although both mutants were insensitive to extracellular Mg2+, the activation kinetics of D278E, which retains a negatively charged side chain, were rapid, resembling those of the wild-type channel in the absence of Mg2+ (Fig 4A and Fig C). In contrast, the kinetics of D278V were slow, similar to those of the wild-type channel in the presence of Mg2+ (Fig 4A and Fig C). These results suggest that Mg2+ slows activation kinetics in wild-type eag in part by screening the negative charge at position D278. The neutralization mutation D278V mimics the Mg2+-bound state of wild-type eag, in which the charge at D278 is shielded by the ion, whereas D278E resembles the Mg2+-free state of wild-type eag, in which the charge is unshielded. These data support the conclusion that D278 in S2 contributes to the binding site by coordinating the Mg2+ ion.
D327 Mutations Dramatically Reduce Mg2+ Sensitivity
D327, the eag-specific acidic residue in S3, was replaced by alanine or phenylalanine to generate the mutants D327A and D327F. Phenylalanine is found in the analogous position in Shaker channels (Fig 1 B).
Extracellular Mg2+ concentrations up to 20 mM had no significant effect on activation kinetics in D327A channels (Fig 5A and Fig B). In this mutant, the late phase of activation was not well fitted by a single exponential component (data not shown), so activation kinetics were estimated by measuring t1/2 (Fig 5 C, right). Mg2+ had no significant effect on t1/2 over the tested prepulse range (Fig 5 C), which was extended to -40 mV because D327A shifted the PoV curve in the depolarized direction (Fig 5 D).
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In contrast, Mg2+ slightly slowed the kinetics of D327F channels (Fig 5 C). Values of and t1/2 were determined as a function of prepulse potential in the presence and absence of Mg2+ (Fig 5 C). In D327F, the value of t1/2 increased from
33 ms in the absence of Mg2+ to
53 ms in the presence of 20 mM Mg2+, a difference that is statistically significant (P < 0.05, ANOVA) (Fig 5 B). A small residual sensitivity to Mg2+ was also apparent when
was plotted as a function of prepulse potential (Fig 5 C). One interpretation of these results is that the negatively charged
electrons associated with the phenyl ring in phenylalanine may be able to interact weakly with Mg2+ in D327F (
D327F and D327A channels exhibited slow activation kinetics whether Mg2+ was present or absent (Fig 5 A). In D327F, values for and t1/2 resembled those of wild-type eag measured in the presence of Mg2+, whereas t1/2 values in D327A were even longer (Fig 5 C). Thus, neutralization of D327 resulted in activation kinetics that were qualitatively similar to those of the Mg2+-bound state of wild-type eag. These data suggest that Mg2+ slows activation kinetics in the wild-type channel in part by screening the charge at D327. The results support the conclusion that D327, like D278, contributes to the Mg2+-binding site by coordinating the ion.
Activation kinetics of the conservative mutant, D327E, were sensitive to extracellular Mg2+ (data not shown), in contrast to the results obtained with D278E (Fig 4). Values for and t1/2 measured either in the presence or absence of Mg2+ resembled those of the wild-type channel (data not shown). Mg2+ sensitivity of D327E suggests that the ion-binding site may be less sterically constrained near D327 than near D278.
D274 Is Not Required for Mg2+ Sensitivity, but Lies Near the Binding Site
D274 in S2 corresponds to an acidic residue that is widely conserved among voltage-dependent K+ channels (-helical conformation, as suggested by perturbation analysis of Shaker and Kv2.1 K+ channels (
The mutations D274A and D274E were generated and expressed in Xenopus oocytes. In the absence of Mg2+, the activation kinetics of D274A and D274E were similar to wild-type eag. And, as in wild-type, activation of D274A and D274E was significantly slowed by Mg2+, indicating that D274 is not required for Mg2+ binding (Fig 6 A). In fact, the neutralization mutation D274A was as sensitive to Mg2+ as wild-type eag. Values of measured as a function of prepulse potential in the presence and absence of Mg2+ were similar in wild type and D274A (data not shown). In addition, the change in t1/2 was virtually identical for Mg2+ concentrations up to 20 mM (Fig 6 B). These results indicate that D274 does not contribute to eag's Mg2+-binding affinity, and is therefore unlikely to interact with the bound ion.
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Interestingly, two lines of evidence suggest that substituting D274 with the longer acidic amino acid, glutamate, increased the binding affinity for Mg2+. First, Mg2+ had a larger effect on t1/2 in D274E than in wild-type eag, with a shift in the dose response curve to lower Mg2+ concentrations (Fig 6 B). Second, after removing Mg2+ from the bath solution, activation kinetics recovered more slowly in D274E than in wild-type eag (Fig 6 C) or other Mg2+-sensitive mutants such as D274A (data not shown). This effect was quantified by measuring the time interval between 10 and 90% of the peak current amplitude as a function of perfusion time during wash in and wash out of a bath solution containing 20 mM Mg2+ (Fig 6 C). These results indicate that the mutation D274E decreases the Mg2+ dissociation rate, thereby increasing the apparent binding affinity. The data suggest that the longer glutamate residue is able to interact with the bound ion, unlike the original aspartate. We conclude that D274 is close to the binding site, consistent with the idea that D274 is exposed in the same water-filled pocket that contains the ion-binding site.
Hyperpolarizing Prepulses Reveal Mg2+-sensitive Activation Kinetics in D284N
The position corresponding to D284 in eag is highly conserved among voltage gated K+ channels (Fig 1 B). In Shaker channels, the homologous residue E293 plays a key role in the mechanism of voltage-dependent activation. It contributes to the single channel gating charge, either by moving relative to the transmembrane electric field or by determining the profile of the field traversed by charge-moving residues in S4 (
In the eag mutants D284N and D284A, activation kinetics were fast, similar to wild-type eag, and unlike neutralization mutations at D278 and D327 (Fig 7; compare to Fig 4 and Fig 5). Mg2+ had little effect on activation kinetics in D284N and D284A when currents were evoked by depolarizing from a holding potential of -80 mV (data not shown). However, hyperpolarizing prepulses revealed altered kinetics in the presence of Mg2+ in D284N (Fig 7A and Fig B). A similar trend was evident in D284A channels (Fig 7A and Fig C). These results indicate that activation kinetics in D284 mutants remain sensitive to hyperpolarizing prepulses, and that transitions occurring at hyperpolarized voltages retain their sensitivity to Mg2+. Therefore, D284 is not required for Mg2+ binding.
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Interestingly, the results indicate that the sensitivity of activation kinetics to prepulse voltages has been shifted to more hyperpolarized potentials in the D284 mutants, despite the fact that their PoV curves were shifted in the depolarized direction (Fig 7 D). The data suggest that mutations at D284 separate the voltage dependence of transitions in the eag activation pathway, shifting rate-determining, Mg2+-sensitive transitions in the hyperpolarized direction, and channel opening in the depolarized direction. In the presence of Mg2+, these transitions appeared to be faster in the mutants than in wild type (compare Fig 2 and Fig 7). This may explain the observation that ionic current kinetics in D284N and D284A were unaffected by Mg2+ at voltages where these channels opened. In wild type, in contrast, activation kinetics measured in the presence and absence of Mg2+ did not converge even at +100 mV (see Fig 2). These results are consistent with the idea that D284 makes an important contribution to the gating mechanism, as does its homologue, E293, in Shaker channels (
D319N Is Insensitive to Extracellular Mg2+
D319, a residue near the intracellular end of S3, is highly conserved in different types of voltage-gated K+ channels (Fig 1 B). In Shaker, the analogous residue, D316, has been implicated in structural interactions with K374 in S4 that are essential for proper folding of the voltage sensor (
In the eag mutant, D319N, ionic current kinetics were fast, similar to those of D284 neutralization mutants and wild-type eag in the absence of Mg2+ (Fig 8 A; compare to Fig 2 and Fig 7). Mg2+ had no significant effect on the rate of activation of ionic currents evoked by depolarizing from -80 mV or after hyperpolarizing prepulses (Fig 8A and Fig B). An extended range of prepulses was used because the D319 mutation shifted the PoV curve in the depolarized direction (Fig 8 C). In contrast to the wild-type channel, a transition to slower activation kinetics was not detected after hyperpolarized prepulses (Fig 8 B).
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The properties of D319N resemble those of D284N and D284A, except that hyperpolarizing prepulses did not reveal Mg2+-sensitive, rate-limiting transitions. Significantly, mutations that neutralized either D284 or D319 resulted in fast kinetics that did not mimic the Mg2+-bound state of wild-type eag, in contrast to neutralization mutations of D278 and D327. These results strongly suggest that D284 and D319 do not mediate the slowing of activation caused by Mg2+ binding. One possibility is that D319N, like D284N and D284A, primarily affects the eag gating pathway, and shifts the Mg2+-sensitive, slow transitions in the hyperpolarized direction. If so, the shift would be greater in D319N than in D284N or D284A. Because D319N was Mg2+ insensitive in the experimentally accessible voltage range, however, we cannot rule out the possibility that D319 is required for Mg2+ binding.
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DISCUSSION |
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Mg2+-binding Site between S2 and S3
Extracellular Mg2+ dramatically slows voltage-dependent activation in eag K+ channels (
Several lines of evidence support the conclusion that D278 and D327 coordinate the Mg2+ ion. Neutralization mutations at these positions eliminated or significantly reduced Mg2+ modulation of activation kinetics. Importantly, neutralization of either D278 or D327 dramatically slowed activation, mimicking the kinetics of the Mg2+-bound state of wild-type eag. The data suggest that Mg2+ slows activation in the wild-type channel by screening these negatively charged residues. The conclusion that D278 and D327 bind the ion is further strengthened by evidence that D274 in S2, one -helical rung above D278, is unlikely to coordinate Mg2+, but is quite close to the binding site. The mutation D274E increased the apparent affinity for Mg2+, suggesting that glutamate, which has an acidic side chain
1.5 Å longer than that of the original aspartate residue, is able to interact with an ion in the site.
The role of eag-specific residues in forming the binding site explains why modulation of activation kinetics by Mg2+ is unique to the eag family. Neutralizing either eag-specific aspartate residue eliminated Mg2+ sensitivity. It is worth noting that we have previously shown that a short sequence of primarily acidic residues in the S3S4 loop is not required for Mg2+ binding, consistent with the presence of negatively charged residues at the end of S3 in a variety of K+ channels (
In the structures of various Mg2+-binding sites, four, six, or eight ligands coordinate the ion (
Interestingly, a variety of cations slow activation in eag (
Packing Arrangement of Transmembrane Segments in the Voltage Sensor
Residues that coordinate the same Mg2+ ion must be within atomic distance of one another. Therefore, our results suggest an important structural constraint for the packing arrangement of transmembrane segments in the eag voltage sensor. In a variety of high resolution protein structures containing bound Mg2+ ions, the center-to-center distance between Mg2+ and coordinating oxygen atoms is approximately 2.2 Å (
Structural proximity between D278 and D327 is compatible with previously inferred structural constraints between S2, S3, and S4 in the Shaker voltage sensor (-helical, consistent with the results of perturbation analysis in Shaker and Kv2.1 channels (
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In Fig 9, the S4 segment is shown tilted relative to S2 and S3, a feature introduced in our earlier model to satisfy the structural constraints inferred for Shaker (
Results obtained with both Shaker and eag strongly suggest that there is an externally facing, water-filled pocket next to S2. In eag, extracellular Mg2+ is able to access a site formed by D278 in S2 and D327 in S3, which would project into the pocket to coordinate the ion. In an -helix, D274 in S2, located one rung above D278, would also be exposed in the same cavity. Significantly, previous work in Shaker channels indicates that E283, the S2 residue analogous to D274 in eag, is located in a cavity near the extracellular surface of the protein (
Interestingly, this pocket may be nearly isopotential with the extracellular solution. In Shaker, a cysteine substituted for E283 in S2 shows very similar reactivity at depolarized and hyperpolarized potentials (
Possible Mechanism of Mg2+ Action
Results obtained in a variety of laboratories suggest that activation of K+ channels involves at least two voltage-dependent conformational changes per subunit (
Based on this picture of activation, we propose a speculative model of the mechanism by which Mg2+ modulates voltage-dependent gating in eag. We previously suggested that Mg2+ modulates two different voltage-dependent transitions during eag activation, corresponding to the two phases described above (
Our results are consistent with the expectation that the overall structure and function of the voltage sensor is conserved in K+ channels that have a wide range of kinetics and differential regulation. Presumably, the detailed kinetic properties of activation in individual types of K+ channels are conferred by sequence differences that result in subtle structural changes despite a conserved architecture.
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Acknowledgements |
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We are grateful to Dr. James Bowie for helpful discussions and to members of the Papazian lab for their comments on the manuscript.
This work was supported by grants to D.M. Papazian from the National Institutes of Health (NIH; GM43459); the American Heart Association, Western States Affiliate; and the Laubisch fund for cardiovascular research at UCLA. W.R. Silverman was partially supported by an NIH predoctoral training grant (GM08496).
Submitted: 24 August 2000
Revised: 2 October 2000
Accepted: 2 October 2000
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