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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 I(Kr), 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. 112:637-647). 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. 95:649-655). 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.
Figure 1. A soluble peptide corresponding to the first 16 amino acids (H16) reversibly restores slow deactivation to HERG channels lacking amino termini. (A) Tail currents from S620T Î2-354 channels at â140 mV after a 1-s voltage step to 60 mV. (See methods regarding use of the S620T background.) Three current traces are shown, immediately after the patch was excised into the internal bath solution, after a 3-min incubation with 1 mM H16 peptide solution, and after a 5-min washout with peptide-free internal bath solution, respectively. The initial tail current amplitude, indicated by the long arrow, is unaffected by application of the peptide. The bottom panel, with expanded time scale, shows the initial tail current amplitude and includes dotted lines showing the fit with two exponential components. (B) Fast and slow mean time constants of deactivation extracted from exponential fits to the deactivating tail currents as shown in A. Values are Ïfast = 24.5 ± 6.5 ms, Ïslow = 309.8 ± 95.4 ms, Af (weight of the amplitude of the fast component represented as a percentage of total current) = 82.0 ± 8.2% for control; Ïfast = 54.2 ± 5.9 ms, Ïslow = 488.2 ± 143.5 ms Af = 73.4 ± 9.3% with the added peptide; and Ïfast = 29.9 ± 4.5 ms, Ïslow = 280.1 ± 48.3 ms, Af = 75.6 ± 6.9% upon washout of the peptide (n = 4). Using ANOVA (P < 0.01, at confidence interval = 0.05), there is a significant difference between the control and the experiment with peptide applied. (C) Tail currents before and after a 10-min incubation in the scrambled H16s peptide solution. (D) The mean time constants of both fast and slow components of the deactivation was not significantly changed by the scrambled peptide (ANOVA, P > 0.3 at confidence interval = 0.05). Values are Ïfast = 20.4 ± 2.9 ms, Ïslow = 381.9 ± 62.0 ms, Af = 89.7 ± 5.5% for control; Ïfast = 24.0 ± 4.2 ms, Ïslow = 414.7 ± 68.5 ms, Af = 81.6 ± 7.6% for the scrambled peptide (n = 3).
Figure 2. The H16 peptide has no effect on deactivation rate in channels with intact amino termini, indicating that the peptide acts by the same mechanism as the native amino terminus. (A) Tail currents from S620T channels evoked at â140 mV after a voltage step to 60 mV, before and after a 5-min incubation in 1 mM H16 peptide. (Arrow) Initial tail current amplitude. (B) Time constants of dominant fast components of the deactivation extracted from exponential fits to the tail currents were 137.6 ± 16.5 ms, Af = 90.0 ± 2.3% for the controls, and 167.7 ± 21.5 ms, Af = 87.6 ± 4.7% after application of the peptide (n = 3). There is no significant difference when tested with ANOVA. (P > 0.3 at confidence level of 0.05.)
Figure 4. The amino terminus does not alter the ability of TEA to block current or to slow deactivation. (A, top) Tail currents from S620TÎ2-354 truncated channels evoked at â140 mV after depolarization to 60 mV, before and after application of 1 mM TEA to the internal bath solution. Arrows point to the instantaneous tail current levels. (Bottom) The same currents scaled to their peak amplitudes to illustrate the slowing of apparent deactivation rate by TEA. (B, top) Tail currents from channels with amino termini intact under same conditions as in A. (Bottom) The same currents scaled. (C) Box plot shows that mean values for percent slowing of deactivation (left) are predicted by corresponding percent current block (right) for both S620TÎ2-354 truncated channels and S620T channels with intact amino termini, indicating a lack of competition between the amino terminus and TEA in open-channel block (n = 3 for each construct, P > 0.2). The competition models describe a channel that cannot close until the blocking particle vacates its binding site (Scheme I), where C, O, and B are the closed, open, and blocked states, respectively, and β is the rate constant of the open-to-closed transition. SCHEME I Channels in the blocked state are unavailable for closing until they return to the open, unblocked state, and so the apparent deactivation rate βapp is reduced according to the expression βapp = f · β, where f is the fraction of channels in the open state. At the very negative voltages studied, we assume little return from the closed to the open state and βapp â 1/Ïfast (see methods). The forward and reverse rates of block are assumed to be fast relative to β.
Figure 3. Multiple amino termini are required to slow deactivation in the HERG channel. (A) Tail currents from S620T Î2-354 channels show progressive slowing in solutions of increasing H16 peptide concentration. From top to bottom, the concentrations are 0, 100, 250, 500, 750, and 1,000 μM. (B) Doseâresponse curve plotted as a fractional slowing of deactivation rate [Î(1/Ï)/Î(1/Ï)max] against peptide concentration (n = 3 for each peptide concentration up to 1 mM, n = 2 for 5 mM). Ï is the time constant of the dominant fast component, which contributes >90% of the deactivating current. Fitting the data to the Hill equation [ln(1 â y)/y] = âN(lnK â ln[peptide]) yielded a Hill coefficient of 2.2 ± 0.1, indicating possible cooperative interactions among at least three amino termini. (C) High concentrations (5 mM) of peptide blocked the current (top), but caused no further slowing of deactivation (scaled currents, bottom).
Figure 5. Single-channel activities at negative (repolarizing) potentials display longer open times when the amino terminus is present. During a continuous voltage command of â80 mV, lasting for 3â5 min, single-channel activities were recorded in cell-attached patch with 100 mM potassium in the pipette. Representative segments of the records from S620T and S620TÎ2-354 channels are shown in A and B, with longer openings apparent in S620T channels (n = 3 for each construct). (C and D) Ensemble tail currents and representative single trials of activities from which the ensemble averages were constructed for S620T and S620TÎ2-354 channels, respectively, during a 2-s repolarizing command to â80 mV after depolarization to 60 mV. Deactivation of the ensemble currents displays typical slow phenotype for S620T channel and fast phenotype for S620TÎ2-354 channels (n = 3 for each construct).
Figure 6. The amino terminus specifically increases the mean open time of the channel. Representative histograms of open- (A) and closed-time (B) distribution for both S620T and S620TÎ2-354 channels are shown with the fitting of the second-order exponential superimposed onto the plot (solid smooth lines). Time constants extracted from the fittings were taken as the mean open and closed dwell time and plotted in C. Both fast and slow mean open times are reduced when the amino terminus are deleted (values are Ïfast = 2.4 ± 0.2 ms, Ïslow = 14.4 ± 1.1 ms for S620T channels; Ïfast = 1.1 ± 0.3 ms, Ïslow = 6.3 ± 1.7 ms for S620TÎ2-354 channels; n = 3 for each construct, P < 0.01), while the two mean closed times remain unaffected (values are Ïfast = 0.8 ± 0.1 ms, Ïslow = 17.1 ± 2.9 ms for S620T channels; Ïfast = 0.9 ± 0.1 ms, Ïslow = 14.6 ± 2.7 ms for S620TÎ2-354 channels; n = 3 for each construct, P > 0.3).
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