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Pore block versus intrinsic gating in the mechanism of inward rectification in strongly rectifying IRK1 channels.

Guo D, Lu Z - J. Gen. Physiol. (2000)

Bottom Line: However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg(2+) and polyamines, the macroscopic I-V curve of the channels displays modest rectification.We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity.Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The IRK1 channel is inhibited by intracellular cations such as Mg(2+) and polyamines in a voltage-dependent manner, which renders its I-V curve strongly inwardly rectifying. However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg(2+) and polyamines, the macroscopic I-V curve of the channels displays modest rectification. This observation forms the basis of a hypothesis, alternative to the pore-blocking hypothesis, that inward rectification reflects the enhancement of intrinsic channel gating by intracellular cations. We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity. Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.

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Current–voltage relationship of the D172N mutant IRK1 channel in the presence of various pH buffers. (A and B) Current traces of the D172N channels recorded from inside-out patches with voltage protocol as in Fig. 1. Both intracellular and extracellular solutions contained either HEPES (A) or phosphate (B). (C) Normalized steady state I-V curves with intracellular solutions buffered by: phosphate (□), borate (○), MOPS (▵), and HEPES (▿). In each case, the extracellular pH buffer was the same as in the intracellular solution.
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Figure 2: Current–voltage relationship of the D172N mutant IRK1 channel in the presence of various pH buffers. (A and B) Current traces of the D172N channels recorded from inside-out patches with voltage protocol as in Fig. 1. Both intracellular and extracellular solutions contained either HEPES (A) or phosphate (B). (C) Normalized steady state I-V curves with intracellular solutions buffered by: phosphate (□), borate (○), MOPS (▵), and HEPES (▿). In each case, the extracellular pH buffer was the same as in the intracellular solution.

Mentions: We also found that the channel exhibited some slight, but clearly noticeable, inward rectification when we lowered the concentration of intracellular EDTA from 5 to 1 mM (Fig. 1 E, ⋄). Based on this finding and the fact that the channel has an extremely high affinity for intracellular cations, we surmise that the barely discernible residual current relaxation at +100 mV results from block of the channel by trace amounts of endogenous and/or exogenous cationic blockers, such as metal ions or amines, that we could not completely eliminate. If this is the case, the minimal remaining current relaxation should be further reduced or eliminated altogether by a mutation in the channel pore, D172N, that reduces the affinity of the channel for intracellular cations (Ficker et al. 1994; Lopatin et al. 1994; Lu and MacKinnon 1994; Stanfield et al. 1994; Wible et al. 1994; Fakler et al. 1995; Yang et al. 1995). Fig. 2 shows the behavior of the D172N channel in the presence of the various pH buffers. As expected, the I-V curve of this mutant channel with reduced affinity for intracellular cations exhibited somewhat reduced inward rectification when HEPES, or MOPS, was used as the pH buffer (Compare Fig. 2 C with 1 E), but became completely linear when phosphate or borate, was used (Fig. 2 C). Note the absence of current relaxation even at +100 mV when phosphate replaced HEPES as the pH buffer (compare Fig. 2 B with 1 B).


Pore block versus intrinsic gating in the mechanism of inward rectification in strongly rectifying IRK1 channels.

Guo D, Lu Z - J. Gen. Physiol. (2000)

Current–voltage relationship of the D172N mutant IRK1 channel in the presence of various pH buffers. (A and B) Current traces of the D172N channels recorded from inside-out patches with voltage protocol as in Fig. 1. Both intracellular and extracellular solutions contained either HEPES (A) or phosphate (B). (C) Normalized steady state I-V curves with intracellular solutions buffered by: phosphate (□), borate (○), MOPS (▵), and HEPES (▿). In each case, the extracellular pH buffer was the same as in the intracellular solution.
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Related In: Results  -  Collection

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Figure 2: Current–voltage relationship of the D172N mutant IRK1 channel in the presence of various pH buffers. (A and B) Current traces of the D172N channels recorded from inside-out patches with voltage protocol as in Fig. 1. Both intracellular and extracellular solutions contained either HEPES (A) or phosphate (B). (C) Normalized steady state I-V curves with intracellular solutions buffered by: phosphate (□), borate (○), MOPS (▵), and HEPES (▿). In each case, the extracellular pH buffer was the same as in the intracellular solution.
Mentions: We also found that the channel exhibited some slight, but clearly noticeable, inward rectification when we lowered the concentration of intracellular EDTA from 5 to 1 mM (Fig. 1 E, ⋄). Based on this finding and the fact that the channel has an extremely high affinity for intracellular cations, we surmise that the barely discernible residual current relaxation at +100 mV results from block of the channel by trace amounts of endogenous and/or exogenous cationic blockers, such as metal ions or amines, that we could not completely eliminate. If this is the case, the minimal remaining current relaxation should be further reduced or eliminated altogether by a mutation in the channel pore, D172N, that reduces the affinity of the channel for intracellular cations (Ficker et al. 1994; Lopatin et al. 1994; Lu and MacKinnon 1994; Stanfield et al. 1994; Wible et al. 1994; Fakler et al. 1995; Yang et al. 1995). Fig. 2 shows the behavior of the D172N channel in the presence of the various pH buffers. As expected, the I-V curve of this mutant channel with reduced affinity for intracellular cations exhibited somewhat reduced inward rectification when HEPES, or MOPS, was used as the pH buffer (Compare Fig. 2 C with 1 E), but became completely linear when phosphate or borate, was used (Fig. 2 C). Note the absence of current relaxation even at +100 mV when phosphate replaced HEPES as the pH buffer (compare Fig. 2 B with 1 B).

Bottom Line: However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg(2+) and polyamines, the macroscopic I-V curve of the channels displays modest rectification.We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity.Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.

ABSTRACT
The IRK1 channel is inhibited by intracellular cations such as Mg(2+) and polyamines in a voltage-dependent manner, which renders its I-V curve strongly inwardly rectifying. However, even in excised patches exhaustively perfused with a commonly used artificial intracellular solution nominally free of Mg(2+) and polyamines, the macroscopic I-V curve of the channels displays modest rectification. This observation forms the basis of a hypothesis, alternative to the pore-blocking hypothesis, that inward rectification reflects the enhancement of intrinsic channel gating by intracellular cations. We find, however, that residual rectification is caused primarily by the commonly used pH buffer HEPES and/or some accompanying impurity. Therefore, inward rectification in the strong rectifier IRK1, as in the weak rectifier ROMK1, can be accounted for by voltage-dependent block of its ion conduction pore by intracellular cations.

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