Correspondence to: Zhe Lu, University of Pennsylvania, Department of Physiology, D302A Richard Building, 3700 Hamilton Walk, Philadelphia, PA 19104. Fax:215-573-1940 E-mail:zhelu{at}mail.med.upenn.edu.
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Abstract |
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The IRK1 channel is inhibited by intracellular cations such as Mg2+ 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 Mg2+ 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.
Key Words: inward-rectifier K+ channel, channel block, ion permeation, polyamines, HEPES
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INTRODUCTION |
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Inward rectifiers, a subset of K+ channels, play many important biological roles by controlling and regulating the resting membrane potential (
Inward-rectifier K+ channels are inhibited by intracellular Mg2+ and polyamines (
We notice that the extent of the residual inward rectification in the absence of Mg2+ and polyamines varies among laboratories. For example, it is considerably smaller in our previously published report than in those of others (e.g., compare
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METHODS |
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Molecular Biology and Oocyte Preparation
IRK1 cDNA was cloned into the pcDNA1/AMP plasmid (Invitrogen) (
Patch Recording
Macroscopic IRK1 currents were recorded in the inside-out configuration from Xenopus oocytes (injected with IRK1 cRNA) with an Axopatch 200B amplifier (Axon Instruments, Inc.), filtered at 5 kHz, and sampled at 25 kHz using an analogue-to-digital converter (DigiData 1200; Axon Instruments, Inc.) interfaced with a personal computer. pClamp6 software (Axon Instruments, Inc.) was used to control the amplifier and acquire the data. During current recording, the voltage across the membrane patch was first hyperpolarized from the 0 mV holding potential to -100 mV for 25 ms, and then stepped to a test voltage between -100 and +100 mV for a period of 100 ms; the increment between consecutive test voltages was 10 mV. Background leak current correction was carried out as previously described (
Recording Solutions
All recording solutions contained 100 mM K+ contributed by: KCl, K2EDTA, K2HPO4, KH2PO4, K2B4O7, and KOH that was used to adjust pH. The HEPES-containing pipette (extracellular) solution contained (mM): 100 K+ (Cl- + OH-), 0.3 CaCl2, 1.0 MgCl2, and 10 HEPES, pH 7.6 (adjusted with KOH). In the MOPS-, phosphate-, and borate-buffered pipette solutions, pH 7.6, HEPES was replaced by an equal concentration of MOPS (pH adjusted with KOH), "K2HPO4 + KH2PO4" and "K2B4O7 + H3B03," respectively. The HEPES-containing bath (intracellular) solution contained (mM): 90 K+ (Cl- + OH-), 5 K2EDTA [or 98 K+ (Cl- + OH-) and 1 K2EDTA, when specified], and 10 HEPES, pH 7.6. In the MOPS-, phosphate-, and borate-buffered bath solutions, pH 7.6, HEPES was replaced by an equal concentration of MOPS (adjusted with KOH), "K2HPO4 + KH2PO4" and "K2B4O7 + H3B03," respectively. When its concentration dependence was examined, HEPES (free acid) at the specified concentration was included in the phosphate-containing bath solution (The final pH of the solution was adjusted to 7.6 with KOH). The bath solutions containing putrescine, spermidine, or spermine were prepared daily. All chemicals were purchased from Fluka Chemical Corp., except HEPES, which was purchased from either Fluka Chemical Corp. (A), Sigma-Aldrich (B), Calbiochem (C), ICN Biomedicals (D), or Fisher Scientific (E1 and E2).
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RESULTS |
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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.
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Interestingly, when phosphate replaced HEPES as the pH buffer in the intracellular (and extracellular) solutions, relaxation of the outward current at positive voltages vanished, except at +100 mV, where it is barely discernible (Fig 1 B). Consequently, the steady state I-V curve in phosphate is practically linear (Fig 1 E, ). A similarly linear I-V curve was also observed when borate replaced HEPES (Fig 1 E,
). In contrast, the I-V curve remained inwardly rectifying as HEPES was replaced by another organic zwitterionic buffer, MOPS (Fig 1 E,
).
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 (
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All remaining data were obtained with the wild-type IRK1 channel. Unless specified otherwise, they were collected with phosphate in both the intracellular and extracellular solutions, regardless of whether HEPES was also present in the intracellular solution.
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 (1 M HEPES with an apparent valence (Z) of
1.
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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.
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Previous studies have shown that intracellular polyamines block the IRK1 channel in a complex manner (e.g.,
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DISCUSSION |
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Under commonly employed experimental conditions, the outward IRK1 current at positive voltages exhibits significant relaxation, even in excised membrane patches exhaustively perfused with an artificial intracellular solution to remove endogenous blocking ions. As a result, the steady state I-V curve of the channel displays significant inward rectification. These phenomena led to the hypothesis that inward rectification results mainly from intrinsic (voltage-dependent) channel gating, which is enhanced when intracellular cations bind to the gating machinery, located at the intracellular side of the channel (e.g.,
In the present study, we found that the current relaxation and the resulting nonlinearity of the steady state I-V curve are primarily related to the use of HEPES (or a similar organic zwitterionic pH buffer, MOPS), because the current relaxation essentially vanished and the IV curve became practically linear when phosphate (or borate), instead of HEPES (or MOPS), was used as a pH buffer (Fig 1). The barely discernible current relaxation with phosphate at +100 mV most likely results from channel block by residual endogenous and/or contaminating exogenous blocking ions (Fig 1 B). This interpretation is supported by the fact that no current relaxation even at +100 mV was observed in the D172N mutant channel (Fig 2 B), which has a reduced affinity for intracellular cations (
The HEPES-related effects are concentration dependent (Fig 3 and Fig 4). The extent of channel block varied significantly when HEPES from different sources or even different lots of the same source was used (Fig 5 and Fig 6). Therefore, some impurity in HEPES, such as amines used or produced in its synthesis, must block the channel, although channel block by zwitterionic HEPES itself would not surprise. It is noteworthy that the negative portion of the I-V curve also exhibits a slight curvature when HEPES is used (Fig 1). However, when we replaced HEPES with phosphate in the extracellular solution, the I-V curve became practically linear. Therefore, the nonlinearity in the negative portion of the I-V curve results also from channel block by HEPES and/or some impurity.
We showed previously, in experiments using HEPES buffer, that the IRK1 current in the presence of intracellular putrescine decreased with increasing membrane voltage, but tended to a nonzero level at very positive voltages (
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Acknowledgements |
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We thank L.Y. Jan for the IRK1 channel cDNA clone, P. De Weer for critical review and discussion of our manuscript, and C.M. Armstrong for helpful discussion.
This study was supported by National Institutes of Health (NIH) grant GM55560. Z. Lu was a recipient of an Independent Scientist Award from NIH (HL03814).
Submitted: 29 June 2000
Revised: 25 August 2000
Accepted: 28 August 2000
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References |
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