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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 intracellular di- and polyamines. (A–C) Steady state I-V curves in the absence or presence of various concentrations of intracellular putrescine, spermidine, or spermine. (D–F) Ratios of the I-V curves with and without putrescine, spermidine, or spermine shown in A–C. The concentrations of the blockers were as indicated. The curves superimposed on the data in D or in E and F are fits of Eqs. 2 and 6, respectively, of Guo and Lu 2000a. Parameter values obtained from the fits are as follows (mean ± SEM; n = 3). For putrescine, K1 = 8.2 (± 0.9) × 10−4 M, Z1 = 1.9 ± 0.2; k−2/k−1 = 4.0 (± 0.5) × 10−2, “z−1 + z−2” = 1.6 ± 0.1. For spermidine, Ka1 = 3.9 (± 0.5) × 10−6 M, Za1 = 5.4 ± 0.4; ka−2/ka−1 = 2.7 (± 0.4) × 10−2, “za−1 + za−2” = 5.6 ± 0.4; Kb1 = 4.5 (± 0.6) × 10−5 M, Zb1 = 3.3 ± 0.4; kb−2/kb−1 = 2.0 (± 0.2) × 10−3, “zb−1 + zb−2” = 3.4 ± 0.5. For spermine, Ka1 = 2.4 (± 0.3) × 10−7 M, Za1 = 5.5 ± 0.4; ka−2/ka−1 = 3.5 (± 0.4) × 10−2, “za−1 + za−2” = 5.7 ± 0.5; Kb1 = 6.8 (± 0.7) × 10−6 M, Zb1 = 3.6 ± 0.3; kb−2/kb−1 = 6.9 (± 0.8) × 10−4, “zb−1 + zb−2” = 3.5 ± 0.4.
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Figure 7: Current–voltage relation of the IRK1 channel in the presence of intracellular di- and polyamines. (A–C) Steady state I-V curves in the absence or presence of various concentrations of intracellular putrescine, spermidine, or spermine. (D–F) Ratios of the I-V curves with and without putrescine, spermidine, or spermine shown in A–C. The concentrations of the blockers were as indicated. The curves superimposed on the data in D or in E and F are fits of Eqs. 2 and 6, respectively, of Guo and Lu 2000a. Parameter values obtained from the fits are as follows (mean ± SEM; n = 3). For putrescine, K1 = 8.2 (± 0.9) × 10−4 M, Z1 = 1.9 ± 0.2; k−2/k−1 = 4.0 (± 0.5) × 10−2, “z−1 + z−2” = 1.6 ± 0.1. For spermidine, Ka1 = 3.9 (± 0.5) × 10−6 M, Za1 = 5.4 ± 0.4; ka−2/ka−1 = 2.7 (± 0.4) × 10−2, “za−1 + za−2” = 5.6 ± 0.4; Kb1 = 4.5 (± 0.6) × 10−5 M, Zb1 = 3.3 ± 0.4; kb−2/kb−1 = 2.0 (± 0.2) × 10−3, “zb−1 + zb−2” = 3.4 ± 0.5. For spermine, Ka1 = 2.4 (± 0.3) × 10−7 M, Za1 = 5.5 ± 0.4; ka−2/ka−1 = 3.5 (± 0.4) × 10−2, “za−1 + za−2” = 5.7 ± 0.5; Kb1 = 6.8 (± 0.7) × 10−6 M, Zb1 = 3.6 ± 0.3; kb−2/kb−1 = 6.9 (± 0.8) × 10−4, “zb−1 + zb−2” = 3.5 ± 0.4.

Mentions: Previous studies have shown that intracellular polyamines block the IRK1 channel in a complex manner (e.g., Guo and Lu 2000a). Since we performed the experiments in the quoted study with HEPES as the pH buffer, we wondered whether HEPES and/or its impurity had significantly affected the general blocking behavior of polyamines. Fig. 7A–C, shows I-V curves of the channels in the absence and presence of various concentrations of intracellular putrescine, spermidine, or spermine obtained with phosphate, rather than HEPES, as the pH buffer. The fractions of unblocked current for each of the three polyamines are plotted in Fig. 7D–F. The blocking curve for putrescine tends to a nonzero level at positive membrane voltages, which is lower for higher putrescine concentrations; qualitatively similar features were seen with spermidine and spermine. Nevertheless, only the blocking curves for spermidine and spermine, but not for putrescine, exhibit a significant hump. These peculiar characteristics of polyamine block essentially mirror those previously observed when HEPES was used as the pH buffer (Guo and Lu 2000a). However, the blocking curves shown in Fig. 7 differ from the earlier ones by the absence of apparent voltage-independent channel inhibition by polyamines (compare Fig. 7, D–F, at negative voltages with Guo and Lu 2000a). Since, in the present study, we limited patch exposure to polyamine-containing solutions to <2 min, the apparent voltage-independent inhibition we previously observed may reflect a decrease in the level of membrane-associated PIP2, which is required for channel activity (Huang et al. 1998).


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 intracellular di- and polyamines. (A–C) Steady state I-V curves in the absence or presence of various concentrations of intracellular putrescine, spermidine, or spermine. (D–F) Ratios of the I-V curves with and without putrescine, spermidine, or spermine shown in A–C. The concentrations of the blockers were as indicated. The curves superimposed on the data in D or in E and F are fits of Eqs. 2 and 6, respectively, of Guo and Lu 2000a. Parameter values obtained from the fits are as follows (mean ± SEM; n = 3). For putrescine, K1 = 8.2 (± 0.9) × 10−4 M, Z1 = 1.9 ± 0.2; k−2/k−1 = 4.0 (± 0.5) × 10−2, “z−1 + z−2” = 1.6 ± 0.1. For spermidine, Ka1 = 3.9 (± 0.5) × 10−6 M, Za1 = 5.4 ± 0.4; ka−2/ka−1 = 2.7 (± 0.4) × 10−2, “za−1 + za−2” = 5.6 ± 0.4; Kb1 = 4.5 (± 0.6) × 10−5 M, Zb1 = 3.3 ± 0.4; kb−2/kb−1 = 2.0 (± 0.2) × 10−3, “zb−1 + zb−2” = 3.4 ± 0.5. For spermine, Ka1 = 2.4 (± 0.3) × 10−7 M, Za1 = 5.5 ± 0.4; ka−2/ka−1 = 3.5 (± 0.4) × 10−2, “za−1 + za−2” = 5.7 ± 0.5; Kb1 = 6.8 (± 0.7) × 10−6 M, Zb1 = 3.6 ± 0.3; kb−2/kb−1 = 6.9 (± 0.8) × 10−4, “zb−1 + zb−2” = 3.5 ± 0.4.
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Figure 7: Current–voltage relation of the IRK1 channel in the presence of intracellular di- and polyamines. (A–C) Steady state I-V curves in the absence or presence of various concentrations of intracellular putrescine, spermidine, or spermine. (D–F) Ratios of the I-V curves with and without putrescine, spermidine, or spermine shown in A–C. The concentrations of the blockers were as indicated. The curves superimposed on the data in D or in E and F are fits of Eqs. 2 and 6, respectively, of Guo and Lu 2000a. Parameter values obtained from the fits are as follows (mean ± SEM; n = 3). For putrescine, K1 = 8.2 (± 0.9) × 10−4 M, Z1 = 1.9 ± 0.2; k−2/k−1 = 4.0 (± 0.5) × 10−2, “z−1 + z−2” = 1.6 ± 0.1. For spermidine, Ka1 = 3.9 (± 0.5) × 10−6 M, Za1 = 5.4 ± 0.4; ka−2/ka−1 = 2.7 (± 0.4) × 10−2, “za−1 + za−2” = 5.6 ± 0.4; Kb1 = 4.5 (± 0.6) × 10−5 M, Zb1 = 3.3 ± 0.4; kb−2/kb−1 = 2.0 (± 0.2) × 10−3, “zb−1 + zb−2” = 3.4 ± 0.5. For spermine, Ka1 = 2.4 (± 0.3) × 10−7 M, Za1 = 5.5 ± 0.4; ka−2/ka−1 = 3.5 (± 0.4) × 10−2, “za−1 + za−2” = 5.7 ± 0.5; Kb1 = 6.8 (± 0.7) × 10−6 M, Zb1 = 3.6 ± 0.3; kb−2/kb−1 = 6.9 (± 0.8) × 10−4, “zb−1 + zb−2” = 3.5 ± 0.4.
Mentions: Previous studies have shown that intracellular polyamines block the IRK1 channel in a complex manner (e.g., Guo and Lu 2000a). Since we performed the experiments in the quoted study with HEPES as the pH buffer, we wondered whether HEPES and/or its impurity had significantly affected the general blocking behavior of polyamines. Fig. 7A–C, shows I-V curves of the channels in the absence and presence of various concentrations of intracellular putrescine, spermidine, or spermine obtained with phosphate, rather than HEPES, as the pH buffer. The fractions of unblocked current for each of the three polyamines are plotted in Fig. 7D–F. The blocking curve for putrescine tends to a nonzero level at positive membrane voltages, which is lower for higher putrescine concentrations; qualitatively similar features were seen with spermidine and spermine. Nevertheless, only the blocking curves for spermidine and spermine, but not for putrescine, exhibit a significant hump. These peculiar characteristics of polyamine block essentially mirror those previously observed when HEPES was used as the pH buffer (Guo and Lu 2000a). However, the blocking curves shown in Fig. 7 differ from the earlier ones by the absence of apparent voltage-independent channel inhibition by polyamines (compare Fig. 7, D–F, at negative voltages with Guo and Lu 2000a). Since, in the present study, we limited patch exposure to polyamine-containing solutions to <2 min, the apparent voltage-independent inhibition we previously observed may reflect a decrease in the level of membrane-associated PIP2, which is required for channel activity (Huang et al. 1998).

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