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Figure 1. . Inward rectification of Kir2.1 current in cell-attached patch versus inside-out patch. (A) Currents were recorded from Xenopus oocytes injected with Kir2.1 mRNA in the cell-attached patch configuration (left) and following excision as an inside-out patch in the absence (middle) or presence of 100 μM spermine (right). (B) Currents recorded from a different patch in the absence (middle) and presence (right) of 100 μM spermidine. The voltage clamp protocol is shown in the left panel of B. The dotted lines indicate the zero current level. (C) I-V curves of the steady-state current at the end of the test pulses in A. (D) I-V curve of the steady-state current at the end of the test pulse in B. Note the similar shapes of the I-V curve for the cell-attached patch and the excised inside-out patch with 100 μM spermine or spermidine present.
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Figure 2. . Macroscopic I-V relationship for different concentration of spermine. (A) Representative current traces recorded from inside-out patch in the presence of 0 (control), 1, and 100 μM spermine. The dotted line indicates the zero current level. Voltage protocol is shown at the bottom. (B) Steady-state I-V curves for control (), and 1 μM (â), 10 μM (â¡), 100 μM (âµ) and 1 mM (â¿) spermine. (C) The ratio of the steady-state current (I) in the presence of spermine to that in its absence (Ictl) plotted against voltage, for the spermine concentrations in B (with corresponding symbols). (D) Comparison of the concentration dependence of the spermine block at â100 mV (â¢) and â20 mV (âª), respectively, from the experimental data in C. Solid lines are best fits to a Hill equation: I/Ictl = 1/(1 + ([spm]/Kd)H), where Kd is spermine concentration causing half-maximal block and H is the Hill coefficient. The values of H at â100 and â20 mV are as indicated.
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Figure 3. . Effect of spermine and TEA on Kir2.1 single channel currents, recorded from inside-out patches excised from COS7 cell transfected with Kir2.1 cDNA (holding potential of â60 mV). (A a) Application of 100 μM and 1 mM spermine reduced single channel amplitude. The continuous change of the single channel amplitude is particularly evident during the wash-out phase. The expanded traces in b, 1 and 2, are from the corresponding times in A a labeled 1, 2, and 3. (B) Blocking effect of TEA on single Kir2.1 channel currents. With 20 mM TEA, note the increase in open channel noise during the wash-in and washout of TEA (trace a). For 2 mM TEA (2 mM) at steady-state (trace b), the unitary amplitude is reduced but the open channel noise is increased. Zero current levels are indicated by horizontal arrows.
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Figure 4. . Two mechanisms of spermine block at different voltages. (A) Single channel recordings during a voltage ramp from â100 to 0 mV, showing the continuous I-V relationship of a single channel in the absence (control) and presence of 1 mM spermine (a). Currents were filtered at 1 kHz and sampled at 2 kHz. The ratio (i/ictl) of the two traces in (a) is shown as the noisy thin line in (b). (B) The single channel recordings at test potentials of â20, â40, and â60 mV in the absence and presence of 100 μM and 1 mM spermine (a). At â60 mV (right), the single channel conductance is reduced by spermine without affecting open channel noise. At â40 mV (middle) in the presence of 100 μM spermine, and at â20 mV (left) in the presence of 1 mM spermine, however, open channel noise is increased. The I-V relations are plotted in (b) and the ratio for 1 mM spermine is plotted in A b (open circles), showing good agreement with the ratio obtained from the voltage ramp.
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Figure 5. . Effects of ionic strength on wild-type and mutant Kir2.1 channel currents. Ionic strength was increased by replacing 140 mM sucrose with 70 mM NMG+ or 70mM Na+. (A and B) Macroscopic currents (a) of wild-type Kir2.1 channel were recorded with symmetrical 30 mM K in intracellular and extracellular solutions. The macroscopic I-V curves in the presence of sucrose vs. NMG+ or Na+ are shown in (b) in both panels. (C) Single channel recording from wild-type Kir2.1 under symmetrical 30 mM K+ conditions at holding potential of â80 mV. (D) Single channel recording of the double mutant channel E224G/E299S under the same condition at holding potential of â 160 mV. There were two channels in this patch.
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Figure 6. . Effect of neutralizing E224 and E299 on spermine block. (A) Representative macroscopic current recordings from giant inside-out patches excised from oocytes expressing wild-type Kir2.1 (WT), the mutant E224G, and the double mutant E224G/E299S, in the absence and presence of 100 μM spermine. (B) Ratio of the steady-state current in the presence of spermine to that in its absence (Ictl) vs. voltage for WT, E224G, and E224G/E299S. The smooth lines are fitted value to model 2 in Fig. 8 B. (C) Bar graph comparing the reduction in the macroscopic current amplitude at â80 mV by 100 μM spermine, in WT (n = 6), E224G (n = 4), and E224G/E299S (n = 4). **, P < 0.05 compared with WT, and #, P < 0.05 compared with E224G. (D) Single channel recording of the double mutant E224G/E299S in the absence (ctl) and presence of 1 mM spermine. The holding potential was at â80 mV. The left panel shows typical single channel current traces and right panel shows superimposed amplitude histograms.
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Figure 7. . Effect of neutralizing D172 on spermine block. (A) Representative macroscopic current recordings from oocytes expressing wild-type and D172N channels in the absence and presence of 100 μM spermine. (B) Ratio of the steady-state current in the presence of spermine (I) to that in its absence (Ictl) vs. voltage for WT (the same values as Fig. 6 B) and D172N. The smooth lines are fitted value to model 2 in Fig. 8 B. (C) Bar graph comparing the reduction in the currents at â80 mV by 100 μM spermine, in WT (n = 6), D172N (n = 3).
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Figure 8. . Proposed model for spermine block of Kir2.1 current. (A) Empirical method to obtain fractional reduction in single channel conductance due to charge-screening by spermine. A a shows superimposed I-V curves in the presence (spm) and absence (ctl) of spermine. A b shows the voltage dependence of the relative conductance (grel) obtained from the ratio of the single channel current amplitude in the presence and absence of spermine in a. See text for details. (B) Reaction schemes for models used to fit the results, with g representing the fractional conductance of open state(s) due to charge-screening by spermine. (C) Schematic representation of the inner vestibule of Kir2.1, cut open from its conical shape to form a sheet. Putative locations of D172, E224, and E299 as indicated, with examples of spermine molecules bound electrostatically at various depths.
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Figure 9. . Fitting model 2 to experimental data. (A) Family of steady-state I-V relationships in the presence of the spermine concentrations indicated. Points are experimental data taken from Fig. 2 C, and lines show the best fit of the model. The parameters determined from the fits are: g01 = 0.48, g02 = 0.28, Kd1 = 5.4 à 10â7 M; Kd2 = 1.48 à 10â5 M, Kd3 = 4.3 à 10â4, Z1 = 0, Z2 = 3.1, Z3 = 4.4. (B) Kinetics of Kir2.1 current block (a) and unblock (b) in the presence of 100 μM spermine using the voltage protocols shown. Points are the experimental data, with best fit of the model shown as superimposed lines. The forward (f) and backward (b) rate constants for the three transitions determined from fit in this patch are: k1f = 2.6 à 1012 Mâ1sâ1, k1b = 1.4 à 106 sâ1, k2f = 5.7 à 1012 Mâ1sâ1, k2b = 8.4 à 107 sâ1, k3f = 2.4 à 106 Mâ1sâ1 and k3b = 1.2 à 103 sâ1.
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