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Dynamic control of deactivation gating by a soluble amino-terminal domain in HERG K(+) channels.

Wang J, Myers CD, Robertson GA - J. Gen. Physiol. (2000)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison Medical School, 53706, USA.

ABSTRACT
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.

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Multiple amino termini are required to slow deactivation in the HERG channel. (A) Tail currents from S620T Δ2-354 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) Dose–response curve plotted as a fractional slowing of deactivation rate [Δ(1/τ)/Δ(1/τ)max] against peptide concentration (n = 3 for each peptide concentration up to 1 mM, n = 2 for 5 mM). τ 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 − y)/y] = −N(lnK − 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).
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Figure 3: Multiple amino termini are required to slow deactivation in the HERG channel. (A) Tail currents from S620T Δ2-354 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) Dose–response curve plotted as a fractional slowing of deactivation rate [Δ(1/τ)/Δ(1/τ)max] against peptide concentration (n = 3 for each peptide concentration up to 1 mM, n = 2 for 5 mM). τ 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 − y)/y] = −N(lnK − 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).

Mentions: 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).


Dynamic control of deactivation gating by a soluble amino-terminal domain in HERG K(+) channels.

Wang J, Myers CD, Robertson GA - J. Gen. Physiol. (2000)

Multiple amino termini are required to slow deactivation in the HERG channel. (A) Tail currents from S620T Δ2-354 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) Dose–response curve plotted as a fractional slowing of deactivation rate [Δ(1/τ)/Δ(1/τ)max] against peptide concentration (n = 3 for each peptide concentration up to 1 mM, n = 2 for 5 mM). τ 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 − y)/y] = −N(lnK − 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).
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Related In: Results  -  Collection

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Figure 3: Multiple amino termini are required to slow deactivation in the HERG channel. (A) Tail currents from S620T Δ2-354 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) Dose–response curve plotted as a fractional slowing of deactivation rate [Δ(1/τ)/Δ(1/τ)max] against peptide concentration (n = 3 for each peptide concentration up to 1 mM, n = 2 for 5 mM). τ 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 − y)/y] = −N(lnK − 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).
Mentions: 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).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison Medical School, 53706, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus