Correspondence to: Gail A. Robertson, Dept. of Physiology, 1300 University Ave., Madison, WI 53706. Fax:608-265-5512 E-mail:robertson{at}physiology.wisc.edu.
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Abstract |
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K+ channels encoded by the human ether-à-go-go-related gene (HERG) are distinguished from most other voltage-gated K+ channels by an unusually slow deactivation process that enables cardiac IKr, the corresponding current in ventricular cells, to contribute to the repolarization of the action potential. When the first 16 amino acids are deleted from the amino terminus of HERG, the deactivation rate is much faster (Wang, J., M.C. Trudeau, A.M. Zappia, and G.A. Robertson. 1998. J. Gen. Physiol. 112:637647). In this study, we determined whether the first 16 amino acids comprise a functional domain capable of slowing deactivation. We also tested whether this "deactivation subdomain" slows deactivation directly by affecting channel open times or indirectly by a blocking mechanism. Using inside-out macropatches excised from Xenopus oocytes, we found that a peptide corresponding to the first 16 amino acids of HERG is sufficient to reconstitute slow deactivation to channels lacking the amino terminus. The peptide acts as a soluble domain in a rapid and readily reversible manner, reflecting a more dynamic regulation of deactivation than the slow modification observed in a previous study with a larger amino-terminal peptide fragment (Morais Cabral, J.H., A. Lee, S.L. Cohen, B.T. Chait, M. Li, and R. Mackinnon. 1998. Cell. 95:649655). The slowing of deactivation by the peptide occurs in a dose-dependent manner, with a Hill coefficient that implies the cooperative action of at least three peptides per channel. Unlike internal TEA, which slows deactivation indirectly by blocking the channels, the peptide does not reduce current amplitude. Nor does the amino terminus interfere with the blocking effect of TEA, indicating that the amino terminus binding site is spatially distinct from the TEA binding site. Analysis of the single channel activity in cell-attached patches shows that the amino terminus significantly increases channel mean open time with no alteration of the mean closed time or the addition of nonconducting states expected from a pore block mechanism.We propose that the four amino-terminal deactivation subdomains of the tetrameric channel interact with binding sites uncovered by channel opening to specifically stabilize the open state and thus slow channel closing.
Key Words: excised macropatch, single channel recordings, electrophysiology, Xenopus oocyte, ion channels
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INTRODUCTION |
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Repolarization of the ventricular cardiac action potential depends on a collection of potassium currents that control the duration of the action potential and the QT interval during each heartbeat. Temporally, IKr is the last of these currents to exert its repolarizing influence (
Work in several laboratories has contributed to our understanding of how the gating mechanisms of HERG channels enable IKr to fulfill its physiological role in the heart. Like other S4-containing channels, HERG channels activate and inactivate upon depolarization, and return to rest (deactivate) upon repolarization (
Critical to the production of the resurgent current are a rapid, C-type inactivation mechanism (2-354) (
In contrast, a peptide corresponding to the first 135 amino acids of HERG, and encompassing a Per-Arnt-Sim (PAS) domain identified within its crystal structure, gradually reconstituted slow deactivation over a 24-h period when injected into oocytes expressing HERG channels lacking the amino terminus (
In this study, we tested the hypothesis that the small segment of 16 amino acids required for slow deactivation can act as an independent, soluble domain to dynamically restore slow deactivation to a channel lacking an amino terminus. This approach is analogous to that used to restore fast inactivation gating in Shaker channels with an amino-terminal peptide (
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METHODS |
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Expression of Channels in Xenopus Oocytes
Preparation of oocytes and RNA synthesis and injection were performed as previously described (
Unless otherwise noted, all experiments were carried out in an S620T background. This mutation dramatically increased expression levels. Deactivation in S620T was shown in a previous study to be regulated by the amino terminus in a manner indistinguishable from that in wild-type channels (
Current Recording and Data Analysis
All currents were recorded using an Axopatch 200A integrating patch clamp and pClamp 6.0.3 data acquisition software (Axon Instruments, Inc.). Recordings were digitized at 2.5 kHz unfiltered for macropatch experiment and 10 kHz filtered at 1 kHz for single channel experiments. Single channel activities were recorded in the cell-attached configuration, while the macropatch data were obtained in the inside-out patch configuration. Patch electrodes were fabricated from borosilicate glass using a Flaming/Brown micropipette puller (Sutter Instrument Co.). The tip of the electrode was heat polished with a microforge (Narishige Scientific Instruments) immediately before the recordings. For single-channel recordings, the electrode was also coated with Sylgard as close to the tip as possible to reduce capacitance. The tip opening was 36 µm for macropatch and 1 µm for single channel recordings. The resistance of the electrodes was 12 M for macropatch and 510 M
for single channel recordings with the solutions indicated below. Seal resistance was at least 10 G
for macropatch and 50 G
for the single channel recordings. In macropatch experiments, once a gigaseal was formed and on-cell currents were recorded as controls, the patch was excised from the oocyte into internal bath solution.
PClamp and Origin 4.1 (Microcal Software, Inc.) were used for data analysis and generating statistic plots. Time constants ( s) for deactivation were measured in Clampfit with a Chebyshev fit to the deactivating tail current using the equation y = A0 + A1e-t/
1 + A2e-t/
2. Deactivation rate was estimated from the time constant
= 1/(
+ ß), where
is the activation rate constant and ß is the deactivation rate constant for a two-state system. At -140 mV, there is little return from the closed to the open state and so deactivation rate can be reasonably inferred from
1/ß. Time constants are represented in figures as box plots, in which the box top and bottom represent the range of the data between 1 and 99% and the middle line represents the mean value. Histograms of the open- and closed-time distribution were generated in pStat from the continuous recordings obtained with Fetchex program. Second-order exponential fitting to the histograms using the Marquardt-LSQ method generated the time constants as the mean dwell time of each state.
We observed faster deactivation kinetics in the macropatch recordings compared with those in our previous whole-cell recordings. The faster deactivation rate occurred in both on-cell and excised patch configurations, and in both S620T and S620T2-354 channels. Therefore, the interpretation of our data is unaffected. Likely reasons for this change to fast kinetics include alteration of membrane mechanics and negative pressure associated with patch formation (
Solutions
Unless otherwise noted, the pipette and bath solution contained (mM): 100 KCl, 0.3 CaCl2, 1 MgCl2, 40 N-methyl-glucamine, and 10 HEPES, pH 7.4. The internal bath solution contained (mM): 140 KCl, 2 MgCl2, 10 EGTA, 5 HEPES, and 5 MgATP, pH 7.4. TEA-Cl was dissolved in internal bath solution, and then applied to the bath solution directly.
Peptide Synthesis and Handling
The H16 and H16s peptides were synthesized using the facilities at the University of Wisconsin Biotechnology Center. Both peptides were purified with reverse-phase HPLC. The peptide was stored as lyophilized powder at -20°C. Before each experiment, the peptide was then dissolved in internal bath solution to desired concentration, and then applied to the bath solution directly.
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RESULTS |
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Restoration of Slow Deactivation by an Amino-Terminal Peptide
In a previous study, we found that the first 16 amino acids are necessary for slow deactivation in HERG channels (2-354 (
2-354. Fig 1 A is a composite of three recordings of tail currents evoked at -140 mV after an activating step to 60 mV (only partially shown). The currents were sequentially recorded from the same patch, first immediately after excision, then after application of peptide (H16), and finally after wash out of the peptide. Deactivation was rapid in the on-cell recording and unchanged by excision. Application of the peptide slowed deactivation without reducing the initial tail current, an effect that was rapidly reversible upon washout (Fig 1A). Consistent results were obtained with four patches, indicating that the first 16 amino acids comprise an independent, soluble domain that reversibly slows deactivation without pore blocking. Both exponential components characteristic of HERG deactivation were slowed by the peptide (Fig 1 B), consistent with the effect of the native amino terminus in whole cell recordings (
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To determine whether the H16 peptide mimics the intact amino terminus or instead slows deactivation by an independent mechanism not intrinsic to the native channel, we tested the effect of the peptide in HERG channels with intact amino termini. In contrast to its slowing of truncated channels shown in Fig 1 A, the peptide had no effect on the deactivation rate in channels with an intact amino terminus (Fig 2 A), indicating that the peptide and the native amino terminus slow deactivation by the same process. The values for the mean time constants for the dominant fast component extracted from exponential fits to the deactivating tail currents from three such experiments are summarized in Fig 2 B.
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Multiple Amino Terminal Peptides Cause Slow Deactivation
How many amino termini are required to slow the deactivation rate of a single channel? The peptide slowed deactivation in a dose-dependent manner (Fig 3A and Fig B), giving a Hill coefficient of 2.2 ± 0.1. Thus, three or more amino termini probably mediate the slowing effect. Restoration of slow deactivation was not complete, with the time constant saturating at a value approximately halfway between the corresponding values for the wild-type and truncated channels (see DISCUSSION).
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At significantly higher concentrations (5 mM), the peptide has an inhibitory effect (Fig 3 C). We wondered whether channel block by the peptide might reflect a physiological process that could account for the fractional slowing of deactivation not restored by the peptide. Based on a model of open-channel block, in which the degree of slowing of deactivation is directly proportional to the degree of current block (see detailed description in Fig 4, legend), the resulting inhibition of current predicts that the deactivation rate will be slowed by 82.3 ± 6.8%. We found that deactivation is slowed only by 47.6 ± 9.3%, similar to that observed with the lower, saturating concentration of peptide. The inhibition is therefore an artifact not unexpected from such high concentrations of synthetic peptide and cannot account for the additional component of slowing.
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The Amino Terminus Does Not Slow Deactivation by a Pore-blocking Mechanism
Note that the slowing of deactivation by the peptide shown in Fig 1 and Fig 3 is accompanied by an increase in the current integral, or total charge moving through the channels, implying that the peptide increases the channel mean open time. If, in contrast, the amino terminus had slowed the apparent deactivation rate by occluding the pore, as proposed for Shaker channels (
Like other voltage-gated K+ channels, HERG channels are blocked by internal TEA (2-354 truncated channels in excised macropatches. TEA slowed the apparent deactivation rate by 44.1 ± 10.9%, close to the 50.5 ± 5.4% predicted by the fractional current block obtained in this experiment (Fig 4A and Fig C). The tail currents have been scaled to their peaks in Fig 4A, bottom, for comparison of deactivation rates.
To determine whether the amino terminus competes with TEA to slow deactivation, reflecting overlapping binding sites in the permeation pathway, we applied TEA to the channels with intact amino termini (Fig 4 B). TEA slowed the apparent deactivation rate by 39.6 ± 4.0%, similar to the 42.4 ± 5.5% predicted by current block. Quantitatively similar effects were observed in the truncated channel (summarized in Fig 4 C). The additive and noncompetitive effects of TEA and the amino terminus indicate that they slow deactivation using different mechanisms and different sites.
The Amino Terminus Slows Deactivation by Stabilizing the Open State
The slowing of macroscopic currents by peptide in the excised macropatch, with no evidence of channel block, suggests that a preferential stabilization of the open state underlies slow deactivation. To test this hypothesis directly, we determined the life time of each conformational state inferred from recordings of single channels with intact (S620T) or truncated (S620T2-354) amino termini. Under steady state conditions at -80 mV, bursting behavior is prominent in both channels, but the truncated channels exhibit shorter openings (Fig 5A and Fig B). Ensemble tail currents constructed from multiple trials of single channel recordings elicited by a repolarizing step are consistent with the corresponding whole-cell currents for each construct, confirming the identity of the single channels (Fig 5C and Fig D).
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Fits to the histograms of the open- and closed-time distributions (Fig 6A and Fig B) yielded two exponential components for open and closed states for both channels, indicating two open and two closed states. Both mean open times, as represented by the time constants of the open time histogram, are significantly prolonged in the presence of the amino terminus, whereas neither mean closed time is affected. This selective increase of the mean open time is consistent with a mechanism by which the amino terminus stabilizes the open state to hold the channel open longer. The lack of additional nonconducting states attributable to the presence of the amino terminus also further confirms the absence of pore block.
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DISCUSSION |
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In this study, we found that a small amino terminal domain of HERG can reversibly reconstitute slow deactivation as a soluble peptide. The amino terminal domain slows deactivation directly by increasing channel open time, rather than indirectly slowing apparent deactivation rate by a pore blocking mechanism. We envision the amino terminus interacting with the open "teepee" conformation suggested by recent structural and functional studies of the KcsA bacterial K+ channel (
It remains to be determined why we were able to rapidly restore slow deactivation using the 16-mer peptide in the excised macropatch when a longer, 135-amino acid polypeptide, which included the initial 16 amino acids, had no effect under the same conditions (
Another puzzle is why the 16-mer peptide only partially restores slow deactivation to the truncated channel. Although deletion of this region is sufficient to fully disable the amino-terminal slowing of deactivation (2-16, but unfortunately we were unable to express this mutant at sufficiently high levels for excised macropatch recordings. The larger, 135 amino acid peptide containing the PAS domain gave quantitatively similar results, slowing deactivation to a rate about halfway between that of wild-type and truncated channels (
Analysis of the dose dependence of the peptide effect indicates that three or more amino termini bind to hold each channel open. This finding is consistent with our previous study of heteromeric mouse ether-à-go-gorelated gene 1 (Merg1) channels composed of two types of subunits arising from alternative splicing, one lacking what we now recognize as the deactivation subdomain (
Previous studies have shown that deactivation of voltage-dependent K+ currents can be slowed by an amino-terminal blocking mechanism, as in Shaker (
The slow deactivation process in HERG channels has provided an opportunity to investigate an unusual gating mechanism that was not previously apparent in studies of rapidly deactivating channels more commonly studied. We do not yet know exactly how the first 16 amino acids stabilize the open state in HERG, or whether the action of the deactivation subdomain will emerge as a common gating mechanism used by members of other channel families. However, it is clear that for many channels, including HERG, the amino terminus is a hub of activity for a diverse array of mechanisms modulating gating such as phosphorylation (e.g.,
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Footnotes |
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1 Abbreviations used in this paper: HERG, human ether-à-go-go-related gene; PAS, Per-Arnt-Sim.
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Acknowledgements |
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We thank Erin McCarthy for preparation of oocytes, Dr. Jeff Walker for advice on peptide synthesis, and Drs. Cynthia Czajkowski, Anne Lynn Gillian, and Robert Pearce for critically reading a previous version of this manuscript.
This work was supported by National Institutes of Health grant HL-55973, a National Science Foundation Career award, and an American Heart Association (AHA) Established Investigator Award (G.A. Robertson), as well as by a predoctoral fellowship from AHA-Wisconsin (J. Wang).
Submitted: 27 March 2000
Revised: 24 April 2000
Accepted: 26 April 2000
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