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Figure 1. Inhibition of Kir2.1 channels by intracellular spermine and spermidine. (A) Current traces recorded from an inside-out giant patch in control and in the presence of the indicated concentrations of spermidine (spd). Currents were recorded from a holding potential of −80 mV, followed by test pulses (400 ms) in the range of −100 to +100 mV in 10-mV increments. (B) Normalized steady-state I–Vm relationships in the presence of spermine (a) and spermidine (b). Currents were normalized to that obtained at −100 mV in control. (C) Normalized G–Vm relationships shown on a semilogarithmic scale for the indicated [spermine]i (a) and [spermidine]i (b). Black lines show fits to Eq. 1. Fitting parameters are listed in Table S1. (D) Normalized G–Vm relationships obtained at 0.01 µM [spermine]i (a) and 0.1 µM [spermidine]i (b) were fit to Eq. 1. Red and green lines are the principal (high-affinity block) and minor (low-affinity block) Boltzmann components, respectively. (E) Voltage dependence of dissociation constants for high- and low-affinity block. Straight lines are fits with Eq. S2. For spermine block, Kd(0) = 0.27 µM and z = 6.7 for high-affinity block; Kd(0) = 43 µM and z = 3.0 for low-affinity block. For spermidine block, Kd(0) = 4.8 µM and z = 5.9 for high-affinity block; Kd(0) = 782 µM and z = 3.3 for low-affinity block. |
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Figure 2. Inhibition of single-channel currents by 0.1 µM [spermidine]i. (A) Single-channel recording obtained at +40 mV in the presence of 150 mM of symmetrical [K+]. (B) Single-channel currents recorded with voltage steps from a holding potential of 0 mV in control at the indicated Vm. Segment of 500-ms recordings are shown. (C) Current traces recorded at 0.1 µM [spermidine]i. (D) Amplitude histograms of substates recorded under control conditions and in the presence of 0.1 µM [spermidine]i at +40 mV. (E) i–Vm relationships for the fully open state and the O–S and S–B transitions. (F) The voltage dependence of mean single-channel conductance (open symbols) and chord conductance (closed symbols) in the presence of 0.1 µM [spermidine]i. Black lines show fits to Eq. 1, with A1 = 0.79, A2 = 0.16, Vh1 = +20.8 mV, and Vh2 = +65.0 mV, in terms of the mean γ–Vm relationship, and with A1 = 0.83, A2 = 0.154, Vh1 = +10.6 mV, and Vh2 = +50.4 mV, in terms of averaged G–Vm relationships, in the presence of 0.1 µM [spermidine]i. The z1 and z2 values were set to 5 and 2.8, respectively. n = 2–7. Error bars are not shown when smaller than the symbol. |
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Figure 3. Voltage dependence of the kinetics of spermidine block. (A) Histograms for open times at +30 mV (top) and +40 mV (bottom). (B) Histograms for the substate times at the indicated voltage. (C) Histograms for the blocked times at the indicated voltage. Gray bars are data obtained in the presence of 0.1 µM [spermidine]i, and red bars are those in the control condition. |
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Figure 4. Concentration dependence of the kinetics of spermidine block. (A) Single-channel currents recorded in the presence of 0.03, 0.1, and 0.3 µM [spermidine]i at +50 mV. (B) Histograms for the substate and blocked times at 0.03 and 0.3 µM [spermidine]i. (C) Dose dependence of association and dissociation rates. |
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Figure 5. Substate induced by 0.1 µM [spermidine]i was selective for K ions. Single-channel currents recorded in the presence of 20 mM [K+]o and the indicated intracellular ions. Similar results were found in six other patches. |
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Figure 6. The most frequent observed endogenous channels were not cation selective. Single-channel recordings in the presence of 20 mM [K+]o and the indicated intracellular solution. Similar observations were made in five other patches. |
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Figure 7. Inhibition of D172N currents by intracellular spermidine. (A) Currents in D172N mutants were recorded using inside-out giant patches under control conditions and in the presence of the indicated concentrations of spermidine (spd). Currents were recorded using the protocol described in Fig. 1 A. (B) Normalized G–Vm relationships shown on a semilogarithmic scale. Black lines show fits to a single Boltzmann equation, with z = 2.0/Vh = +66 mV (0.1 µM [spermidine]i), z = 2.6/Vh = +44.8 mV (1 µM [spermidine]i), and z = 2.3/Vh = +25.4 mV (10 µM [spermidine]i). (C) Single-channel currents recorded under control conditions at the indicated Vm values. (D) i–Vm relationships for the conductive states were ohmic, both under control conditions (O–C) and at 0.1 µM [spermidine]i (O–B). n = 3. Error bars are not shown when smaller than the symbols. (E) Mean γ–Vm and normalized G–Vm relationships under control conditions and in the presence of 0.1 µM [spermidine]i. The black line shows the single Boltzmann relationship fitted to single-channel data, with z = 2.9/Vh = +74.3 mV. |
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Figure 8. Voltage dependence of spermidine block in the D172N mutant. (A) Single-channel currents recorded in the presence of 0.1 µM [spermidine]i at +40, +50, and +70 mV. (B) Histograms for the substate and blocked times. (C) Voltage dependence of dissociation constants for the wild-type Kir2.1 (closed symbols) and the D172N mutant (open symbols). |
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Figure 9. Concentration dependence of the kinetics of spermidine block in the D172N mutant. (A) Single-channel currents recorded in the presence of 0.03, 0.1, and 0.3 µM [spermidine]i at +50 mV in the D172N mutant. (B) Histograms for the substate and blocked times. (C) Dose dependence of association and dissociation rates. |
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Figure 10. The O–S–B Model for the regulation of outward Kir2.1 currents by high-affinity and low-affinity polyamine blocks. (A) The dependence of chord conductance and currents on driving force (Vm–EK). Red line indicates the conductance sensitive to high-affinity block, and blue dash denotes low-affinity block of conductance. The blue-shaded area indicates the voltage range where the slope of I–Vm relationship is positive, and the purple-shaded area indicates the voltage range with negative slope. (B) Cartoons of the channels in C, O, S, and B states and the corresponding single-channel currents. Red dots indicate K ions. (C) Steady-state I–Vm relationships in the presence of only high-affinity block by spermine at the indicated concentrations are shown in the top panel. Currents = G/Gmax × Vm, and G/Gmax values were calculated from Eq. S6, with A1, A2, z1, and Vh1 listed in Table S2. Steady-state I–Vm relationships in the presence of both high- and low-affinity block by spermine are shown in the bottom panel. G/Gmax values were calculated from Eq. S3, with A, A2, z1, z2, Vh1, and Vh2 listed in Table S2. |
