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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 pH buffers. (A and B) Current traces recorded from inside-out patches at membrane voltages between −100 and +100 mV in 10-mV increments, corrected for the background currents shown in C and D, respectively. Both intracellular and extracellular solutions contain either HEPES (A) or phosphate (B). The dashed lines identify the zero-current levels. (E) Normalized steady state I-V curves with intracellular solutions containing (mM): 10 phosphate and 5 EDTA (□), 10 borate and 5 EDTA (○), 10 phosphate and 1 EDTA (⋄), 10 MOPS and 5 EDTA (▵), and 10 HEPES and 5 EDTA (▿). In each case, the extracellular pH buffer was the same as in the intracellular solution.
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Figure 1: Current–voltage relationship of the IRK1 channel in the presence of various pH buffers. (A and B) Current traces recorded from inside-out patches at membrane voltages between −100 and +100 mV in 10-mV increments, corrected for the background currents shown in C and D, respectively. Both intracellular and extracellular solutions contain either HEPES (A) or phosphate (B). The dashed lines identify the zero-current levels. (E) Normalized steady state I-V curves with intracellular solutions containing (mM): 10 phosphate and 5 EDTA (□), 10 borate and 5 EDTA (○), 10 phosphate and 1 EDTA (⋄), 10 MOPS and 5 EDTA (▵), and 10 HEPES and 5 EDTA (▿). In each case, the extracellular pH buffer was the same as in the intracellular solution.

Mentions: Fig. 1 A shows a series of IRK1 current traces at membrane voltages between −100 and +100 mV in 10-mV increments, with 100 mM K+ on both sides of the membrane (pH 7.6, buffered with HEPES). All current traces are corrected for the background currents shown in Fig. 1 C. As previously shown, the outward IRK1 current after a step to positive voltages exhibited significant relaxation and, consequently, the corresponding steady state I-V curve (determined at the end of the voltage steps) exhibited inward rectification even in a patch exhaustively perfused with the artificial intracellular solution (Fig. 1 E, ▿). Furthermore, a slight curvature was also present in the negative portion of the steady state I-V curve.


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 pH buffers. (A and B) Current traces recorded from inside-out patches at membrane voltages between −100 and +100 mV in 10-mV increments, corrected for the background currents shown in C and D, respectively. Both intracellular and extracellular solutions contain either HEPES (A) or phosphate (B). The dashed lines identify the zero-current levels. (E) Normalized steady state I-V curves with intracellular solutions containing (mM): 10 phosphate and 5 EDTA (□), 10 borate and 5 EDTA (○), 10 phosphate and 1 EDTA (⋄), 10 MOPS and 5 EDTA (▵), and 10 HEPES and 5 EDTA (▿). In each case, the extracellular pH buffer was the same as in the intracellular solution.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Current–voltage relationship of the IRK1 channel in the presence of various pH buffers. (A and B) Current traces recorded from inside-out patches at membrane voltages between −100 and +100 mV in 10-mV increments, corrected for the background currents shown in C and D, respectively. Both intracellular and extracellular solutions contain either HEPES (A) or phosphate (B). The dashed lines identify the zero-current levels. (E) Normalized steady state I-V curves with intracellular solutions containing (mM): 10 phosphate and 5 EDTA (□), 10 borate and 5 EDTA (○), 10 phosphate and 1 EDTA (⋄), 10 MOPS and 5 EDTA (▵), and 10 HEPES and 5 EDTA (▿). In each case, the extracellular pH buffer was the same as in the intracellular solution.
Mentions: Fig. 1 A shows a series of IRK1 current traces at membrane voltages between −100 and +100 mV in 10-mV increments, with 100 mM K+ on both sides of the membrane (pH 7.6, buffered with HEPES). All current traces are corrected for the background currents shown in Fig. 1 C. As previously shown, the outward IRK1 current after a step to positive voltages exhibited significant relaxation and, consequently, the corresponding steady state I-V curve (determined at the end of the voltage steps) exhibited inward rectification even in a patch exhaustively perfused with the artificial intracellular solution (Fig. 1 E, ▿). Furthermore, a slight curvature was also present in the negative portion of the steady state I-V curve.

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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