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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 relation of the IRK1 channel in the presence of 10 mM HEPES from different sources. (A) The linear steady state I-V curve was obtained in the presence of 10 mM intracellular phosphate without HEPES; the others were obtained in the presence of HEPES from the various sources used for the data shown in Fig. 5. All seven I-V curves were obtained from the same patch as in Fig. 5. (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 in Fig. 4. The fitted Kd values (M) are: 1.11 ± 0.06, 0.89 ± 0.10, 0.36 ± 0.04, 0.39 ± 0.02, 0.34 ± 0.02, and 0.65 ± 0.07 (mean ± SEM; n = 4) for sources A, B, C, D, E1, and E2, respectively. The fitted Z values are: 1.02 ± 0.01, 0.97 ± 0.01, 0.97 ± 0.01, 1.00 ± 0.01, 0.98 ± 0.01, and 0.97 ± 0.01 for 0.07 (mean ± SEM; n = 4) for sources A, B, C, D, E1, and E2, respectively.
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Figure 6: Current–voltage relation of the IRK1 channel in the presence of 10 mM HEPES from different sources. (A) The linear steady state I-V curve was obtained in the presence of 10 mM intracellular phosphate without HEPES; the others were obtained in the presence of HEPES from the various sources used for the data shown in Fig. 5. All seven I-V curves were obtained from the same patch as in Fig. 5. (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 in Fig. 4. The fitted Kd values (M) are: 1.11 ± 0.06, 0.89 ± 0.10, 0.36 ± 0.04, 0.39 ± 0.02, 0.34 ± 0.02, and 0.65 ± 0.07 (mean ± SEM; n = 4) for sources A, B, C, D, E1, and E2, respectively. The fitted Z values are: 1.02 ± 0.01, 0.97 ± 0.01, 0.97 ± 0.01, 1.00 ± 0.01, 0.98 ± 0.01, and 0.97 ± 0.01 for 0.07 (mean ± SEM; n = 4) for sources A, B, C, D, E1, and E2, respectively.

Mentions: HEPES from different commercial sources or even from different lots of the same source affects the channel differently. Fig. 5 shows several series of current traces recorded from the same patch in the presence of HEPES from various common commercial sources or, for the data labeled E1 and E2, from different lots of the same source. Since both the extent and the rate of current relaxation vary significantly among preparations, the observed channel block must at least in part be caused by a contaminant(s). The I-V curves with either phosphate or HEPES from the various sources tested are plotted in Fig. 6 A, while ratios of the I-V curves with HEPES relative to that with phosphate are plotted in Fig. 6 B. The curves superimposed on the data in Fig. 6 B are fits of the Woodhull equation. Regardless of the source of the HEPES used, the apparent valence of channel block varies little, whereas the blocking potency varies considerably, most likely reflecting different amounts of contaminant(s) present.


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 relation of the IRK1 channel in the presence of 10 mM HEPES from different sources. (A) The linear steady state I-V curve was obtained in the presence of 10 mM intracellular phosphate without HEPES; the others were obtained in the presence of HEPES from the various sources used for the data shown in Fig. 5. All seven I-V curves were obtained from the same patch as in Fig. 5. (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 in Fig. 4. The fitted Kd values (M) are: 1.11 ± 0.06, 0.89 ± 0.10, 0.36 ± 0.04, 0.39 ± 0.02, 0.34 ± 0.02, and 0.65 ± 0.07 (mean ± SEM; n = 4) for sources A, B, C, D, E1, and E2, respectively. The fitted Z values are: 1.02 ± 0.01, 0.97 ± 0.01, 0.97 ± 0.01, 1.00 ± 0.01, 0.98 ± 0.01, and 0.97 ± 0.01 for 0.07 (mean ± SEM; n = 4) for sources A, B, C, D, E1, and E2, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2230623&req=5

Figure 6: Current–voltage relation of the IRK1 channel in the presence of 10 mM HEPES from different sources. (A) The linear steady state I-V curve was obtained in the presence of 10 mM intracellular phosphate without HEPES; the others were obtained in the presence of HEPES from the various sources used for the data shown in Fig. 5. All seven I-V curves were obtained from the same patch as in Fig. 5. (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 in Fig. 4. The fitted Kd values (M) are: 1.11 ± 0.06, 0.89 ± 0.10, 0.36 ± 0.04, 0.39 ± 0.02, 0.34 ± 0.02, and 0.65 ± 0.07 (mean ± SEM; n = 4) for sources A, B, C, D, E1, and E2, respectively. The fitted Z values are: 1.02 ± 0.01, 0.97 ± 0.01, 0.97 ± 0.01, 1.00 ± 0.01, 0.98 ± 0.01, and 0.97 ± 0.01 for 0.07 (mean ± SEM; n = 4) for sources A, B, C, D, E1, and E2, respectively.
Mentions: HEPES from different commercial sources or even from different lots of the same source affects the channel differently. Fig. 5 shows several series of current traces recorded from the same patch in the presence of HEPES from various common commercial sources or, for the data labeled E1 and E2, from different lots of the same source. Since both the extent and the rate of current relaxation vary significantly among preparations, the observed channel block must at least in part be caused by a contaminant(s). The I-V curves with either phosphate or HEPES from the various sources tested are plotted in Fig. 6 A, while ratios of the I-V curves with HEPES relative to that with phosphate are plotted in Fig. 6 B. The curves superimposed on the data in Fig. 6 B are fits of the Woodhull equation. Regardless of the source of the HEPES used, the apparent valence of channel block varies little, whereas the blocking potency varies considerably, most likely reflecting different amounts of contaminant(s) present.

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