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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 IRK1 channel in the presence of various concentrations of intracellular HEPES. (A) Steady state I-V curves with various concentrations of intracellular HEPES, obtained from the data shown in Fig. 3. (B) Ratios of the I-V curves with and without HEPES shown in A. The curves superimposed on the data are fits of the equation I/Io = Kd/(Kd + [HEPES]), where Kd = Kd(0 mV) e−ZFVm/RT. The fits yield Kd(0 mV) = 0.96 ± 0.04 M and Z = 1.0 ± 0.1 (mean ± SEM; n = 4).
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Figure 4: Current–voltage relationship of the IRK1 channel in the presence of various concentrations of intracellular HEPES. (A) Steady state I-V curves with various concentrations of intracellular HEPES, obtained from the data shown in Fig. 3. (B) Ratios of the I-V curves with and without HEPES shown in A. The curves superimposed on the data are fits of the equation I/Io = Kd/(Kd + [HEPES]), where Kd = Kd(0 mV) e−ZFVm/RT. The fits yield Kd(0 mV) = 0.96 ± 0.04 M and Z = 1.0 ± 0.1 (mean ± SEM; n = 4).

Mentions: Fig. 3 shows several series of IRK1 current traces in the presence of various concentrations of intracellular HEPES. The current relaxed more strongly with increasing HEPES concentration, which suggests that the relaxation results primarily from channel block by HEPES and/or some accompanying impurity. The I-V curves without and with various concentrations of HEPES are plotted in Fig. 4 A. Adding increasing amounts of HEPES to the phosphate-containing intracellular solution caused an increasingly pronounced downward deflection in the I-V curve at positive voltages. In Fig. 4 B, the fraction of unblocked current in the presence of various concentrations of HEPES is plotted against membrane voltage. The curves superimposed on the data are fits of the Woodhull equation (Woodhull 1973). The fit yields an apparent Kd (at 0 mV) equivalent to ∼1 M HEPES with an apparent valence (Z) of ∼1.


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 IRK1 channel in the presence of various concentrations of intracellular HEPES. (A) Steady state I-V curves with various concentrations of intracellular HEPES, obtained from the data shown in Fig. 3. (B) Ratios of the I-V curves with and without HEPES shown in A. The curves superimposed on the data are fits of the equation I/Io = Kd/(Kd + [HEPES]), where Kd = Kd(0 mV) e−ZFVm/RT. The fits yield Kd(0 mV) = 0.96 ± 0.04 M and Z = 1.0 ± 0.1 (mean ± SEM; n = 4).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2230623&req=5

Figure 4: Current–voltage relationship of the IRK1 channel in the presence of various concentrations of intracellular HEPES. (A) Steady state I-V curves with various concentrations of intracellular HEPES, obtained from the data shown in Fig. 3. (B) Ratios of the I-V curves with and without HEPES shown in A. The curves superimposed on the data are fits of the equation I/Io = Kd/(Kd + [HEPES]), where Kd = Kd(0 mV) e−ZFVm/RT. The fits yield Kd(0 mV) = 0.96 ± 0.04 M and Z = 1.0 ± 0.1 (mean ± SEM; n = 4).
Mentions: Fig. 3 shows several series of IRK1 current traces in the presence of various concentrations of intracellular HEPES. The current relaxed more strongly with increasing HEPES concentration, which suggests that the relaxation results primarily from channel block by HEPES and/or some accompanying impurity. The I-V curves without and with various concentrations of HEPES are plotted in Fig. 4 A. Adding increasing amounts of HEPES to the phosphate-containing intracellular solution caused an increasingly pronounced downward deflection in the I-V curve at positive voltages. In Fig. 4 B, the fraction of unblocked current in the presence of various concentrations of HEPES is plotted against membrane voltage. The curves superimposed on the data are fits of the Woodhull equation (Woodhull 1973). The fit yields an apparent Kd (at 0 mV) equivalent to ∼1 M HEPES with an apparent valence (Z) of ∼1.

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.

Show MeSH
Related in: MedlinePlus