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Figure 3. . Other causes underlying apparent inward rectification. (AâD) Currents were recorded from the same inside-out patch with the voltage pulse protocol shown in Fig. 1. The intracellular solution composition (besides KCl) and pH for each panel are indicated. (E) Normalized I-V curves constructed from the currents determined at the end of each test voltage-pulse. The I-V curves aâd correspond to the currents from AâD, respectively. All data points are mean ± SEM (n = 5).
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Figure 1. . Comparisons of the effects of intracellular HEPES, HEP, and piperazine on IRK1 currents (structures shown at top). (AâC) Currents were recorded with 10 mM HEPES, 3 μM HEP and 0.3 μM piperazine, respectively, from the same inside-out patch with the voltage pulse protocol shown below. In all cases the intracellular solutions contained 100 mM K+, 5 mM EDTA, and 10 mM phosphate (pH 7.6) besides the tested chemicals, whereas the extracellular solution contained 100 mM K+, 0.3 mM Ca2+, 1 mM Mg2+, 10 mM phosphate (pH 7.6). The currents are corrected for background current. Dotted lines identify the zero current level. (DâF) Normalized I-V curves, corresponding to AâC, which were constructed from the currents determined at the end of each test voltage-pulse. Current at each voltage is normalized (except for its signs) to that at â100 mV. Each data point represents the mean (± SEM) of currents recorded from 5â7 patches.
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Figure 2. . HEPES, HEP, and piperazine concentration dependence of channel block. The fraction of unblocked currents in the presence of three representative concentrations of HEPES (A), HEP (B), or piperazine (C) is plotted against membrane voltage. The theoretical curves are fits of the Woodhull equation, which give Kd(0 mV) = 2.47 ± 0.02 M (mean ± SEM, n = 6) and Z = 1.02 ± 0.02 for HEPES, Kd(0 mV) = 2.97 ± 0.16 mM (n = 6) and Z = 1.09 ± 0.03 for HEP, and Kd(0 mV) = 0.27 ± 0.04 mM (n = 8) and Z = 1.08 ± 0.02 for piperazine.
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Figure 4. . IRK1 currents in the presence of three metal ion chelators. (A) Current records collected from the same patch in the presence of intracellular EDTA, EGTA, or CDTA, each at 5 mM. Phosphate was used to buffer pH. (B) Normalized I-V curves in the presence of the metal ion chelators. All data points are mean ± SEM (n = 5).
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Figure 5. . Effects of EDTA concentration on IRK1 currents. All traces were recorded from the same patch, with intracellular EDTA concentrations as indicated. The corresponding I-V curves are shown in Fig. 6. Solution pH was buffered with phosphate.
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Figure 6. . Effects of EDTA concentration on the I-V curves of IRK1 channels. Normalized I-V curves in the presence of various concentrations of EDTA. For clarity, I-V curves with 0.1â5 mM intracellular EDTA (A) are plotted separately from those with 5â30 mM EDTA (B). All data points are mean ± SEM (n = 4â6). (C) Normalized current at 80 mV, taken from the I-V curves in A and B, is plotted against the concentration of EDTA. The data represented by the circles (mean ± SEM) were determined experimentally, whereas those by triangles were calculated using Eq. 1, as described in discussion.
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Figure 7. . Comparisons of channel block by âEDTAâ with block by Mg2+ or ethylenediamine. (A and C) Currents with 0.1 mM and 30 mM EDTA normalized to that with 5 mM EDTA are plotted against membrane voltage, respectively. (B and D) The fraction of unblocked currents in the presence of Mg2+ or ethylenediamine (ED), respectively, is plotted against membrane voltage. All data points are mean ± SEM (n = 5). All curves are fits of the equation I/Io = 1/(1 + [blocker]/Kd), where Kd = Kd(0 mV)eâZFV/RT. For A and B, the two nearly superimposed curves for each data set are fits either all data points (continuous curves) or all but the rightmost three data points (dashed curves). For C or D, the dashed curve through each dataset is a fit to all but the rightmost three data points.
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Figure 8. . Comparisons of the effects on IRK1 currents of intracellular EDTA and ethylenediamine (structures shown at top). (A and B) Currents were recorded from the same membrane patch with, respectively, 30 mM EDTA and 0.1 μM ethylenediamine. (C and D) Normalized I-V curves; each data point represents the mean (± SEM; n = 5) of currents.
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Figure 9. . Effects of intracellular pH on the currents of wild-type and D172N mutant IRK1 channels. Currents were recorded at various intracellular pH, with extracellular pH = 7.6 throughout. For each channel type, all currents were obtained from the same inside-out patch.
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Figure 10. . Effects of intracellular pH on the I-V curves of wild-type and mutant IRK1 channels. (A and B) I-V curves of wild-type and D172N mutant IRK1 channels at various intracellular pH, determined from the current records, as shown (and including those) in Fig. 9, except for those at pH 7.6, which were taken from those as shown in Fig. 4 A. (C and D) Currents through IRK1 and D172N channels, normalized to those at pH 8.5, are plotted against membrane voltage. All data points are mean ± SEM (n = 5).
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Figure 11. . Variable degree and rate of removal of endogenous blockers by perfusion. Current traces shown in A and B were recorded from two separate patches excised from two oocytes injected with different amounts of cRNA (higher in A than in B). The recordings were made at the indicated times following the start of perfusion.
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Figure 12. . Relaxation of inward currents caused by extracellular divalent cations. Currents were recorded with the voltage protocol shown at the top. Intra- and extracellular solutions were buffered with 10 mM phosphate. The extracellular solution contained 0.3 mM Ca2+ and 1 mM Mg2+ in A, but 5 mM EDTA and no added Ca2+ or Mg2+ in B. (C) The ratio of currents at the end and the beginning of voltage pulses (Iend/Ibgn) is plotted against membrane voltage. The data corresponding to A and B are labeled by letters a and b, respectively. All data points are mean ± SEM (n = 5).
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Figure 13. . K+ dependence of the inward current relaxation in the presence of extracellular HEPES. All currents were elicited with the voltage protocol shown in Fig. 12, with 5 mM EDTA and no added divalent cations present in the extracellular solution. Extracellular solutions were buffered with 10 mM HEPES (A and B) or phosphate (C and D), and contained either 100 mM K+ (A and C) or 20 mM K+ (B and D). (E) The ratio of currents at the end and the beginning of voltage pulses (Iend/Ibgn) is plotted against membrane voltage. The data corresponding to AâD are labeled aâd, respectively. All data points are mean ± SEM (n = 5).
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Figure 14. . K+ dependence of inward current relaxation in the presence of extracellular HEP or piperazine. Intra- and extracellular solutions were buffered with 10 mM phosphate. The extracellular solution contained 3 μM HEP (A and B) or 0.3 μM piperazine (C and D), and either 100 mM K+ (A and C) or 20 mM K+ (B and D). (E) The ratio of currents at the end and the beginning of voltage pulses (Iend/Ibgn) is plotted against membrane voltage. The data corresponding to AâD are labeled aâd, respectively. All data points are mean ± SEM (n = 5).
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