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Spermine block of the strong inward rectifier potassium channel Kir2.1: dual roles of surface charge screening and pore block.

Xie LH, John SA, Weiss JN - J. Gen. Physiol. (2002)

Bottom Line: We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability.We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate.This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Laboratory, Department of Medicine, University of California at Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

ABSTRACT
Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg(2+). Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

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Effect of neutralizing D172 on spermine block. (A) Representative macroscopic current recordings from oocytes expressing wild-type and D172N channels in the absence and presence of 100 μM spermine. (B) Ratio of the steady-state current in the presence of spermine (I) to that in its absence (Ictl) vs. voltage for WT (the same values as Fig. 6 B) and D172N. The smooth lines are fitted value to model 2 in Fig. 8 B. (C) Bar graph comparing the reduction in the currents at −80 mV by 100 μM spermine, in WT (n = 6), D172N (n = 3).
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fig7: Effect of neutralizing D172 on spermine block. (A) Representative macroscopic current recordings from oocytes expressing wild-type and D172N channels in the absence and presence of 100 μM spermine. (B) Ratio of the steady-state current in the presence of spermine (I) to that in its absence (Ictl) vs. voltage for WT (the same values as Fig. 6 B) and D172N. The smooth lines are fitted value to model 2 in Fig. 8 B. (C) Bar graph comparing the reduction in the currents at −80 mV by 100 μM spermine, in WT (n = 6), D172N (n = 3).

Mentions: The negatively charged residue D172, which is also know to be crucial for polyamine or Mg2+ block (Lu and MacKinnon, 1994; Stanfield et al., 1994; Wible et al., 1994; Yang et al., 1995), is thought to be located deep in the channel pore and therefore would not necessarily contribute to surface charge in the inner vestibule. Consistent with these predictions, Fig. 7 shows that neutralization of D172 had no effect on the shallow charge screening component of spermine block at −80 mV, whereas it did significantly affected the pore block component at −20 mV.


Spermine block of the strong inward rectifier potassium channel Kir2.1: dual roles of surface charge screening and pore block.

Xie LH, John SA, Weiss JN - J. Gen. Physiol. (2002)

Effect of neutralizing D172 on spermine block. (A) Representative macroscopic current recordings from oocytes expressing wild-type and D172N channels in the absence and presence of 100 μM spermine. (B) Ratio of the steady-state current in the presence of spermine (I) to that in its absence (Ictl) vs. voltage for WT (the same values as Fig. 6 B) and D172N. The smooth lines are fitted value to model 2 in Fig. 8 B. (C) Bar graph comparing the reduction in the currents at −80 mV by 100 μM spermine, in WT (n = 6), D172N (n = 3).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2311403&req=5

fig7: Effect of neutralizing D172 on spermine block. (A) Representative macroscopic current recordings from oocytes expressing wild-type and D172N channels in the absence and presence of 100 μM spermine. (B) Ratio of the steady-state current in the presence of spermine (I) to that in its absence (Ictl) vs. voltage for WT (the same values as Fig. 6 B) and D172N. The smooth lines are fitted value to model 2 in Fig. 8 B. (C) Bar graph comparing the reduction in the currents at −80 mV by 100 μM spermine, in WT (n = 6), D172N (n = 3).
Mentions: The negatively charged residue D172, which is also know to be crucial for polyamine or Mg2+ block (Lu and MacKinnon, 1994; Stanfield et al., 1994; Wible et al., 1994; Yang et al., 1995), is thought to be located deep in the channel pore and therefore would not necessarily contribute to surface charge in the inner vestibule. Consistent with these predictions, Fig. 7 shows that neutralization of D172 had no effect on the shallow charge screening component of spermine block at −80 mV, whereas it did significantly affected the pore block component at −20 mV.

Bottom Line: We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability.We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate.This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized.

View Article: PubMed Central - PubMed

Affiliation: Cardiovascular Research Laboratory, Department of Medicine, University of California at Los Angeles, School of Medicine, Los Angeles, CA 90095, USA.

ABSTRACT
Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg(2+). Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

Show MeSH
Related in: MedlinePlus