Correspondence to: Diane M. Papazian, Department of Physiology, Box 951751, UCLA School of Medicine, Los Angeles, CA 90095-1751. Fax:310-206-5661 E-mail:papazian{at}mednet.ucla.edu.
Released online: 28 February 2000
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Abstract |
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We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.
Key Words: gating currents, prepulse hyperpolarization, activation model, voltage clamp
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Introduction |
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Voltage-gated ion channels play a crucial role in determining the resting membrane potential, shaping the action potential, and controlling secretion in excitable cells. In response to depolarization of the membrane, voltage-gated channels undergo conformational changes that lead to the opening of the ion conduction pore. This sensitivity of protein conformation to changes in the transmembrane electric field is conferred by an intrinsic, charged voltage sensor (
The voltage sensor and its conformational changes have been extensively studied in Shaker K+ channels. K+ channels comprise four similar or identical subunits that surround the water-filled pore for K+ permeation (
The Drosophila ether-à-go-go K+ channel (eag)1 and its vertebrate homologues constitute a unique subfamily of voltage-gated K+ channels (
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MATERIALS AND METHODS |
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Molecular Biology
Wild-type eag channels and the mutants L342H and 333-337 were expressed in Xenopus oocytes as previously described (
Electrophysiology
Ionic and gating currents from wild-type and L342H channels were measured using the cut-open oocyte vaseline gap technique (333337 channels were recorded using a conventional two-electrode voltage clamp (
In experiments using a two-electrode voltage clamp, pipettes were filled with 3 M KCl. The bath contained normal Ringer's solution, composed of 118 mM NaCl, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.2. As noted, NaCl was replaced by KCl to record eag tail currents, and MgCl2 was added to investigate the effect of Mg2+ on activation of eag channels. Voltage-clamp protocols were applied and data were acquired using pCLAMP v.5.5.1 software and a TL-1 Labmaster Interface (Axon Instruments). Linear leak and capacitative currents were subtracted using the P/-4 protocol (
In experiments using the cut-open oocyte voltage clamp, pipettes were filled with 3 M KCl or 3 M NaCl. Electrical access to the interior of the oocyte was achieved by permeabilizing the membrane with 0.1% saponin applied in the lower chamber. To record ionic currents, the extracellular solution contained 120 mM Na-methanesulfonate (MES) or 120 mM N-methylglucamine (NMG)-MES, 1.8 mM CaCl2, and 10 mM HEPES, pH 7.2. The internal solution contained 120 mM K-MES, 1 mM EGTA, and 10 mM HEPES, pH 7.2. Extracellular Na-MES or NMG-MES was replaced by K-MES to record tail currents, and 2 mM MgCl2 was added to the extracellular solution as noted. To measure gating currents, ionic currents were blocked by replacing the external Na-MES or NMG-MES and internal K-MES with TEA-MES. Linear leak and capacitative currents were subtracted using the P/-4 protocol with a subtraction holding potential of -110 mV (
To study the effect of hyperpolarizing prepulses on gating current kinetics, leak and capacitative currents were compensated electronically at 0 mV. In control experiments, we attempted to detect gating charge movement between -200 and -100 mV using a variety of alternative subtraction protocols, including (a) P/-4 with subtracting holding potentials as negative as -180 mV, (b) P/+4 with subtracting holding potentials up to +10 mV, and (c) no subtraction (electronic compensation at 0 mV). Substantially similar results were obtained regardless of the subtraction protocol (data not shown).
The kinetics of eag ionic and gating currents were described by fitting specified regions of the current traces with a single exponential function using our own analysis software or Origin software (Microcal Software, Inc.). For gating currents, the decaying phases of both ON and OFF gating currents were fitted. For ionic currents, the late rising phase (close to the peak) of activation currents and the decaying phase of tail currents were fitted.
ON and OFF gating charge was measured by time integration of the ON and OFF gating currents using our own analysis software.
Computer Simulations
The SCOP v. 3.5 simulation program (Simulation Resources) was used to simulate eag ionic and gating currents using a modified kinetic scheme based on the class D model for the Shaker channel proposed by i) and backward (ßi) rate constants were assumed to take the form:
i =
i0exp(zfieV/kT), and ßi = ßi0exp(-zbieV/kT), where
i0 and ßi0 are the rates at 0 mV, zfi and zbi are the valences of the moving charge in the forward and backward directions, e is the elementary charge (1.6 x 10-19 C), V is the membrane potential, and k and T are the Boltzmann constant and absolute temperature, respectively.
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RESULTS |
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Effect of Prepulse Hyperpolarization on eag Ionic Current Kinetics
As originally shown by
The Drosophila eag K+ channel was expressed in Xenopus oocytes to investigate the effects of prepulse hyperpolarization on the ionic current. Hyperpolarizing prepulses delayed the onset of channel opening and, in addition, dramatically slowed the kinetics of the ionic currents evoked by a subsequent depolarization (Fig 1 A). As a result, eag ionic currents elicited after various prepulses could not be superimposed by shifting the traces along the time axis (Fig 1 B). This phenomenon has been attributed to the existence of rate-limiting transitions between remote closed states reached only during hyperpolarizations (
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The initial phase of activation in the eag channel and its mammalian homologues displays sigmoid kinetics (
Effect of Extracellular Mg2+ on eag Ionic Currents
Without applying hyperpolarizing prepulses, eag ionic currents were dramatically slowed in the presence of 2 mM extracellular Mg2+ (Fig 2 A). Steady state current amplitudes were unchanged. The effect of Mg2+ on channel kinetics was quantified by fitting a single exponential component to the late phase of ionic current activation (Fig 2 B). Mg2+ slowed eag activation kinetics in a voltage-dependent manner, with a larger effect after smaller depolarizing steps (Fig 2 C). At -10 mV, 2 mM Mg2+ increased the time constant of activation ~11-fold, from ~13 to 148 ms. Higher concentrations of extracellular Mg2+ resulted in even slower activation kinetics (data not shown).
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In addition to decelerating activation initiated from a holding potential of -90 mV, Mg2+ dramatically slowed activation kinetics after hyperpolarizing prepulses (Fig 3 A). The interaction of Mg2+ and prepulse hyperpolarization was complex and particularly prominent during the initial phase of activation (compare Fig 3 B with 1 C). Because the initial phase of activation was poorly fitted by a single exponential component, the effect was quantified by measuring the time required to reach half peak current amplitude at +50 mV as a function of prepulse potential in the presence and absence of Mg2+ (Fig 3 C). The time to half peak is sensitive to changes in both the delay and time course of the ionic currents. The range of prepulse potentials that elicited the steepest change in the time to half peak appeared to be shifted to more depolarized values in the presence of Mg2+ (Fig 3 C). The interaction of Mg2+ and prepulse hyperpolarization suggests that Mg2+ further slows rate-limiting gating transitions between closed states that are populated at hyperpolarized potentials, and may shift the voltage dependence of these transitions in the depolarized direction.
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The late phase of ionic current activation was fitted with a single exponential component (Fig 3 B), and the resulting time constants were plotted versus prepulse potential (Fig 3 D). During the late phase of activation, the dramatic slowing of activation kinetics by Mg2+ was virtually independent of prepulse potential.
A reactivation protocol was used to investigate the effect of Mg2+ on other transitions in the activation pathway (
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To determine whether the opening transition was rate limiting, the time courses of activation during the first and second depolarizing pulses were compared. At very short interpulse intervals, eag opened more quickly in response to the second pulse compared with the first in both the presence and absence of Mg2+ (Fig 4 A). Therefore, the opening transition is not the rate limiting step for eag activation.
The cut-open oocyte voltage clamp provides excellent temporal resolution, making it feasible to measure reactivation time constants after interpulse intervals as short as 0.1 ms. During extremely short interpulse intervals, many channels do not close. The experiment was therefore performed using a bath solution nominally free of K+ to eliminate inward currents at -90 mV, which would interfere with determining reactivation kinetics after short interpulse intervals.
In the absence of external Mg2+, the fitted reactivation time constant was ~0.5 ms after an interval of 0.1 ms, and reached 3 ms after an interval of 1 ms. In contrast, in the presence of 2 mM Mg2+, the reactivation time constant was ~5 ms within the same range of interpulse intervals (Fig 4 B), and remained at this value for intervals as long as 30 ms (data not shown). These results suggest that Mg2+ modulates the kinetics of channel opening in eag. In contrast, Mg2+ did not change the kinetics of deactivation, indicating that Mg2+ does not modulate the transition from the open state to the most accessible closed state(s) (Fig 4 C).
In the absence of external Mg2+, activation kinetics during the second pulse matched those of the first pulse after interpulse intervals longer than 2 ms. In contrast, in the presence of 2 mM Mg2+, interpulse intervals of >600 ms were required for the activation kinetics to return to their original rate (Fig 4 D). These results indicate that one or more back transitions between closed states are slowed by extracellular Mg2+.
The results presented so far indicate that Mg2+ modulates activation gating in eag K+ channels, as previously suggested for the rat homologue of eag (
Measurement of eag Gating Currents
eag ionic currents were blocked by perfusing the oocyte both externally and internally with TEA, and gating currents were recorded with the cut-open oocyte voltage clamp (Fig 5 A). In response to depolarizing pulses, a small rising phase was observed in the ON gating currents, indicating that initial transitions in the activation pathway move less charge than subsequent transitions. Rising phases have also been observed in gating currents recorded from Shaker and Kv2.1 K+ channels (
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The steady state activation properties of eag channels were characterized by deriving open probabilityvoltage (Po-V) and gating chargevoltage (Q-V) curves from ionic and gating currents, respectively (Fig 5 C, and Table 1). In Fig 5, the Q-V curve plots the OFF gating charge (QOFF), obtained as the time integral of the OFF gating current, as a function of pulse potential. Each curve was fitted with a single Boltzmann distribution to derive a midpoint potential (Vmid) and apparent gating valence (z). Consistent with the existence of several closed states in the activation pathway, Vmid for the Q-V curve was shifted by -20 mV relative to Vmid for the Po-V curve. The Q-V curve was slightly steeper, as reflected in its higher z value. Lower estimates of the gating valence for the Q-V and Po-V curves corresponded to ~2.5 and 2.1 e0, respectively. Extracellular Mg2+ (2 mM) shifted the Po-V and Q-V curves by <5 or 10 mV in the depolarized direction (Fig 5 D).
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Q-V curves derived from ON and OFF gating currents are expected to be identical. In some types of channels, however, QON and QOFF cannot be measured with equal accuracy. In the eag channel, the kinetics of the ON gating current were slow, particularly for depolarizations to 0 mV or less, leading to underestimates of the ON gating charge. The slow movement of the ON charge could be inferred from a gradual increase in the OFF gating charge with longer pulse durations, reflecting the return of additional charge (see Fig 7 B). Therefore, to characterize the gating chargevoltage relationship of the eag channel, we measured the OFF gating charge evoked by repolarization after 70-ms pulses (Fig 5 C). This procedure should provide an accurate estimate of the total charge because at this time point, the OFF charge movement has saturated. In eag, unlike other channels such as Shaker, fast inactivation and TEA do not delay the return of the OFF gating charge, a phenomenon that has been called charge immobilization (
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In eag, ionic tail currents and OFF gating currents had similar time courses over a wide range of potentials (Fig 5 E). The closing valence (zc) as determined from the slope of a semilogarithmic plot of time constant versus tail potential (Fig 5 E, right) was ~0.37 in both cases. Importantly, these similarities suggest that ionic current tails and OFF gating currents are measuring the same molecular event.
Prepulse Hyperpolarization and Extracellular Mg2+ Modulate eag Gating Current Kinetics
Hyperpolarizing prepulses slowed the time course of the ON gating currents. Decay kinetics of ON gating currents obtained after prepulses to different potentials could not be superimposed (Fig 6 A). At potentials more negative than -130 mV, the effect of prepulse hyperpolarization on the ON gating currents was enhanced by extracellular Mg2+ (Fig 6 B). These results indicate that Mg2+ directly modulates the activation gating process in eag channels. In contrast, OFF gating current kinetics were unaffected by Mg2+ (Fig 6 A).
Hyperpolarizing prepulses in the presence and absence of Mg2+ have qualitatively similar effects on the kinetics of eag ionic currents and ON gating currents (Fig 1, Fig 3, and Fig 6). Quantitatively, however, the change in gating current kinetics was significantly less than that seen in the ionic currents (compare Fig 1 D, 3 B, and 6 B). This suggests that prepulse hyperpolarization accesses gating transitions that occur slowly and/or move relatively little charge, and are therefore difficult to detect with gating current measurements. Attempts to detect gating charge movement between -200 and -100 mV were unsuccessful.
In the Absence of Hyperpolarizing Prepulses, the Activation Kinetics of eag Gating and Ionic Currents Show Differential Sensitivity to Extracellular Mg2+
In the absence of hyperpolarizing prepulses, Mg2+ had significantly different effects on the ionic and gating currents of eag channels. Although extracellular Mg2+ dramatically slowed the activation of eag ionic currents (Fig 2), the kinetics of the decay phase of the ON gating currents evoked by depolarizing from a holding potential of -90 mV were insensitive to Mg2+ (Fig 7 A). This suggests that channel opening requires transitions that are not well represented in the gating current measurements.
ON gating currents are slow in eag channels, and therefore the effects of Mg2+ may be difficult to resolve. To investigate whether a slow, Mg2+-sensitive component of gating charge movement is present, the magnitude of the OFF gating charge (QOFF) was measured as a function of pulse duration. Longer depolarizing pulses provide an opportunity for slow components of the ON gating charge to move. Although this charge may be lost in the baseline during integration of ON gating currents, a slow component of charge movement should be detectable as a gradual increase in QOFF as a function of pulse duration. Indeed, this protocol revealed a slow component of charge movement that was more prominent in the presence of Mg2+ (Fig 7 B). The kinetics of this component were voltage dependent and Mg2+ sensitive. QOFF reached its maximum value more quickly in the absence than in the presence of Mg2+ at all test potentials (Fig 7 B). These results reveal that Mg2+ slows the kinetics of ON gating currents in eag, but the effect is much smaller than on ionic current kinetics (Fig 2 C).
A quantitative discrepancy between the effect of Mg2+ on ionic and gating current kinetics was also seen using the reactivation protocol. Although Mg2+ dramatically slowed the reactivation time course of ionic currents (Fig 4), reactivation kinetics of ON gating currents were not sensitive to extracellular Mg2+ (Fig 8).
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The results described in this section suggest that Mg2+ modulates gating transitions that occur slowly and/or move relatively little charge. These are likely to include the rate-limiting transitions accessed by prepulse hyperpolarization.
Role of the S3S4 Loop in Mg2+ Modulation of eag Gating
In the bovine homolog of eag, alternatively spliced variants differing in the length of the S3S4 loop are differentially sensitive to extracellular Mg2+ (
When bound to proteins, Mg2+ is often chelated by carboxylate side chains (333337) (
333337 slows the activation kinetics of eag (Fig 9 A). In contrast to their effect on wild-type eag, hyperpolarizing prepulses increased the delay but did not affect the time course of
333337 ionic currents elicited by a subsequent depolarization (Fig 9 B). Activation time constants, estimated by fitting a single exponential function to the late phase of ionic current records, did not differ significantly as a function of prepulse potential (Fig 9 B). The increase in the delay was reflected by an increase in the time to half maximal current amplitude after more negative prepulses (Fig 9 B). These results suggest that the transitions between closed states populated at hyperpolarized potentials are no longer rate limiting for activation in
333337 channels. However, activation kinetics were still modulated by extracellular Mg2+ in
333337 channels (Fig 9 C). Thus, the DRDED sequence does not contribute significantly to the Mg2+ binding site in eag channels. Measurement of gating currents from the
333337 mutant was not feasible because of the slow activation kinetics and low expression of this construct.
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Consistent with these results, we note that Mg2+ does not significantly modulate the gating of Shaker or other voltage-dependent K+ channels that are not members of the eag subfamily. It is worth noting that negatively charged sequences are also found in the analogous location in other voltage-dependent K+ channels, including Shaker (
In contrast to the 333337 deletion, another mutation in the S3S4 loop, L342H, eliminated the modulation of eag activation gating by prepulse hyperpolarization and extracellular Mg2+. As previously described, the steady state and kinetic properties of activation in L342H, measured from ionic current recordings, are similar to those of wild-type eag (
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To determine whether the opening transition was still sensitive to Mg2+ in the L342H mutant, a reactivation experiment was performed. The kinetics of opening, estimated at very short interpulse intervals, were unaffected by Mg2+ in L342H channels (Fig 10 C). Furthermore, activation kinetics during the second pulse returned to that seen in the first pulse at the same rate in the presence and absence of Mg2+ (Fig 10 D). These results indicate that the L342H mutation abolishes the effect of Mg2+ on activation of the ionic currents in eag.
We also investigated whether the L342H mutation eliminates modulation of gating current kinetics by prepulse hyperpolarization and Mg2+ (Fig 11). In the presence or absence of extracellular Mg2+, hyperpolarizing prepulses did not significantly change the kinetics of ON gating currents (Fig 11 A). The normalized decay kinetics of the ON gating currents in mutant and wild-type channels are compared as a function of prepulse potential in Fig 11 B. Furthermore, Mg2+ did not modulate the development of a slow component of QOFF after long depolarizations (Fig 12). These results confirm that the L342H mutation greatly attenuates or eliminates the modulation of activation gating by Mg2+ and prepulse hyperpolarization.
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DISCUSSION |
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Qualitative Model of Activation Gating in eag
Unlike other voltage-dependent K+ channels, activation gating in the Drosophila eag channel and its mammalian homologues is dramatically modulated by extracellular Mg+2 (
Models for the gating of voltage-dependent K+ channels generally postulate sequential, charge-moving transitions between closed states. These steps prime the channel for opening (
To address this question, we adapted a qualitative model for eag gating from the class D model previously proposed by C1 and C1
C2 in the condensed model). Once all four subunits are in the C2 state, the channel is in a conformation permissive for opening, designated C2*. A final, concerted conformational change opens the channel (C2*
O).
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In the model, the C0 conformation is populated at hyperpolarized potentials. The first gating transition, C0 C1, is the rate-limiting step in the pathway, in accordance with the effects of hyperpolarizing prepulses on activation kinetics. This transition moves less gating charge than the second transition, C1
C2, which is responsible for the bulk of the detectable gating charge movement in eag. Opening of the pore, C2*
O, is the fastest transition in the pathway, consistent with the results of reactivation experiments, which demonstrate that opening is not rate limiting in eag channels. We have assumed that pore opening occurs much faster than the reverse transition, O
C2*.
The idea that the opening transition is much faster than the reverse reaction has previously been incorporated into models describing the gating of Shaker channels (
Our data suggest that Mg2+ slows at least two steps in the activation pathway, including rate-limiting transitions between closed states populated at hyperpolarized potentials and the transition from a nearby closed state(s) to the open state. Although the kinetics of ionic tail currents were unaffected by Mg2+, the reactivation experiment demonstrated that Mg2+ decelerates one or more back transitions between closed states. Mg2+ modulation of activation kinetics was simulated by reducing the rates of the forward and backward transitions between C0 and C1, and to a lesser extent the forward transition from C1 to C2. According to the model, once the channel reaches the C2* state, the pore opens rapidly. Therefore, slowing the C1 C2 transition can account for the effect of Mg2+ on pore opening. In the simulation, the fast kinetics of the C2* to O transition were unchanged by Mg2+.
This model is not intended to provide a quantitative or complete description of eag gating. In particular, it does not reproduce the complicated, sigmoid kinetics of the initial phase of activation of eag ionic currents, which would require additional transitions. Importantly, however, simulations using this model can reproduce the quantitatively larger effects of Mg2+ and hyperpolarizing prepulses on ionic than on gating currents and key features of eag ionic and gating currents, including the deceleration of activation kinetics after hyperpolarizing prepulses and the effect of Mg2+ on activation and reactivation kinetics (Fig 14).
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Shown in Fig 14 A is the simulated effect of a prepulse to -200 mV before a test pulse to +50 mV. The time courses of ionic currents evoked with and without the prepulse cannot be superimposed by sliding the traces along the time axis (Fig 14 A, left). Thus, the model confirms that this feature can result from rate-limiting transitions between closed states populated at hyperpolarized potentials. Importantly, the model predicts that the same prepulse protocol results in a much smaller effect on the kinetics of ON gating currents (Fig 14 A, right).
The simulated effect of Mg2+ on activation kinetics is shown in Fig 14 B. In accord with our experimental results, the model predicts that Mg2+ slows activation kinetics of ionic currents without changing tail current kinetics (Fig 14 B, left). Significantly, Mg2+ has a minimal effect on gating current kinetics in the simulation (Fig 14 B, right). Also in agreement with the data, Mg2+ has little effect on the steady state voltage dependence of the channel (Fig 14 C).
The model predicts that the effect of Mg2+ on ionic and gating currents is less prominent at more positive test potentials and that Mg2+ enhances the effects of prepulse hyperpolarization (not shown). These features are consistent with our experimental findings (Fig 2, Fig 3, Fig 6, and Fig 7).
The model also predicts the effect of Mg2+ on the reactivation kinetics of eag ionic currents (not shown). In particular, slowing the back transition from C1 to C0 can account for our observation that a longer interpulse interval is required for activation kinetics to return to their original rate in the presence of Mg2+. Furthermore, decelerating the C1 C2 transition in the presence of Mg2+ results in slower reactivation kinetics even at very short interpulse intervals, accounting for the apparent effect of Mg2+ on pore opening.
The simulation results indicate that a sequential activation model can account for the differential effects of hyperpolarization and Mg2+ on eag ionic and gating currents.
It is worth noting that our data do not rule out the possibility that the quantitatively larger effect of Mg2+ on ionic than gating currents is due to some kind of antagonistic interaction between Mg2+ and TEA. Such an interaction would affect gating current but not ionic current measurements. To address this possibility, we attempted to record gating currents after complete replacement of internal and external K+ by NMG. Such attempts were unsuccessful. Using the cut-open oocyte approach, eag ran down before K+ was thoroughly replaced, making it infeasible to measure gating currents uncontaminated by ionic currents. Run down of eag in excised patches has been previously reported (
Role of the S3S4 Loop in the Voltage-dependent Gating of eag
Mutations in the S3S4 loop strongly influence the steady state voltage dependence of eag (
The L342H mutation virtually eliminates the modulation of eag ionic and gating currents by Mg2+. It is unlikely that the original leucine side chain is directly involved in coordination of the Mg2+ ion, although mutating the residue may indirectly alter the architecture of the binding site. Alternatively, the mutation may block access to the site or dramatically reduce the kinetic prominence of the Mg2+-sensitive steps in the gating mechanism. At present, we cannot distinguish between these possibilities.
Comparison of Activation Gating in eag and Other Voltage-dependent K+ Channels
The properties of gating currents in eag channels resemble those previously described in other voltage-dependent K+ channels such as Shaker. Gating currents recorded from Shaker and eag channels are characterized by a rising phase, indicating that early steps in the activation pathway move less charge than later transitions (
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Footnotes |
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Portions of this work were previously published in abstract form (Tang, C.-Y., D. Sigg, F. Bezanilla, and D.M. Papazian. 1996. Biophys. J. 70:A406; Tang, C.-Y., F. Bezanilla, and D.M. Papazian. 1998. Biophys. J. 74:A240).
1 Abbreviations used in this paper: eag, Drosophila ether-à-go-go K+ channel; MES, methanesulfonate.
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Acknowledgements |
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We are grateful to Dr. Sally Krasne and members of the Papazian laboratory for their comments on the manuscript.
This work was supported by grants from the National Institutes of Health to F. Bezanilla (GM30376) and D.M. Papazian (GM43459).
Submitted: 25 October 1999
Revised: 20 January 2000
Accepted: 21 January 2000
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