Fujiwara Y, Kubo Y.

	Figure 1. Structure of the cytoplasmic pore of Kir2.1. (A) Schematic drawing of the structure of Kir based on the crystal structures of Kir2.1, Kir3.1, and KirBac1.1 (Nishida and MacKinnon, 2002; Kuo et al., 2003; Pegan et al., 2005); note that the pore has both transmembrane and cytoplasmic regions. (B) Alignment of the amino acid sequences of Kir family proteins that form the wall of the cytoplasmic pore and the surrounding region. The original reports used for the alignment are as follows: Kir 2.1 (Kubo et al., 1993a), Kir1.1 (Ho et al., 1993), Kir 3.1 (Kubo et al., 1993b), Kir 4.1 (Bond et al., 1994; Takumi et al., 1995), Kir5.1 (Bond et al., 1994), Kir 6.2 (Inagaki et al., 1995). (C) Amino acids on the wall of the cytoplasmic pore of Kir2.1. Negatively charged residues (E224, D255, D259, and E299) are colored red, positively charged residues (R228 and R260) are colored blue, and other residues are colored yellow.
	Figure 2. Comparison of macroscopic currents through WT Kir2.1 and mutants. (A) Macroscopic currents recorded using two-electrode voltage clamp with Xenopus oocytes in 10 mM K+o. The holding potential was −50 mV; step pulses from +50 to −160 mV were applied in 10-mV decrements. (B) Current–voltage (I-V) relationships for the data in A; values were measured 5 or 100 ms after the onset of each step pulse. (C and D) Comparison of the intensities of the inward rectification of currents through WT Kir2.1 and the mutants. The ratios of the current amplitudes measured 100 ms after the onset of step pulses to +50 and −100 mV were calculated as an index of the rectification intensity. Bars depict means ± SEM (n = 5–8) in C and means ± SEM (n = 3–5) in D. Mean values were compared statistically using Tukey's test (***, P < 0.001; **, P < 0.01; *, P < 0.05). (E) Representative G-V relationships for the macroscopic currents in which the indicated mutants were compared.
	Figure 3. Comparison of the hyperpolarization-evoked activation kinetics in WT Kir2.1 and mutants. (A) Inward currents recorded using two-electrode voltage clamp with Xenopus oocytes at the indicated membrane voltages in 10 mM K+o. (B) The activation phases were fitted with a single exponential function, and the time constants of the fittings are plotted. The symbols used are as shown in the figure. Bars depict means ± SEM (n = 6–8). (C) Same as in B; in this case, data obtained from E224Q, E224Q/R228K, E224Q/R228Q, E224Q/R228K, or E224Q/R260K were compared. Bars depict means ± SEM (n = 3–4).
	Figure 4. Comparison of the outward tail currents through WT Kir2.1 and mutants. All channels were expressed in Xenopus oocytes. (A) Outward currents recorded using two-electrode voltage clamp with Xenopus oocytes at +50 mV after depolarization from various potentials ranging from +50 to −160 mV. K+o was 10 mM. (B) The data obtained with E224Q, D259N, E299Q, and E224Q/E299Q were fitted with a two exponential function, while those obtained with E224Q/D259N were fitted with a single exponential function; the time constants are plotted. The bottom axis indicates the voltage of the preceding step pulses. The symbols used are as indicated in the figure. Representative data were plotted for each channel. (C) Same as in B; in this case, data obtained with E224Q, E224Q/R228K, E224Q/R228Q, E224Q/R228K, or E224Q/R260K were compared.
	Figure 5. Comparison of the susceptibility of macroscopic currents through WT Kir2.1 and mutants to blockade by intracellular Mg2+. (A) Macroscopic currents recorded in the presence of the indicated concentrations of Mg2+i from excised patches of HEK293T cells expressing WT Kir2.1 or mutants. The concentrations of Mg2+i are shown on the top; note the difference in the concentration ranges used for WT and E224Q. K+o in the pipette was 20 mM, and K+i in the bath was 140 mM. The calculated EK was −49 mV. The holding potential was −50 mV, and step pulses from +70 to −120 mV were applied in 10-mV decrements. (B) Normalized dose–block relationships derived from data in A; values were measured 500 ms after the onset of each step pulse. (C) Relationships between the Kd and voltage for Mg2+i blockade of outward K+ currents. Data obtained from three patches were plotted for each channel, and each set was fitted with a line. (D) Relationships between the voltage and the mean of the fitted lines in C, and the dashed lines indicate SEMs. (E) Zd values of each channel calculated from the slopes of the lines in C. Mean values were compared statistically between WT and the mutants using Tukey's test (***, P < 0.001; *, P < 0.05; NS, P > 0.05); statistical comparison of the data obtained from E224Q and E224Q/R228Q is also shown. (F) Values of the Hill coefficient for each channel. Bars depict means ± SEM (n = 3). The symbols used are as indicated in the figure.
	Figure 6. Comparison of the susceptibility of macroscopic currents through WT Kir2.1 and mutants to blockade by intracellular spermine. (A) Macroscopic currents recorded in the presence of the indicated concentrations of spermine from excised patches of HEK293 T cells expressing WT Kir2.1 or the indicated mutant. Note the difference in the concentration ranges used for WT and mutants, as well as the difference in the time scales. K+o and K+i were 20 mM and 140 mM, respectively. The holding potential was −50 mV, and step pulses from +70 to −120 mV were applied in 10-mV decrements. (B) Normalized dose–block relationships; values were measured 800 ms (in WT and D259N) or 3,000 ms (in E224Q and E224Q/R228Q) after the onset of each step pulse. (C) Relationships between Kd and voltage for spermine blockade of outward K+ currents. Data obtained from three patches were plotted for each channel. The entire dataset could not be fitted with a straight line, but data from each patch was fitted with a straight line for the voltage range from −70 to −10 mV. (D) Relationships between voltage and the mean Kd value in C. (E) Zd values of each channel calculated from the slopes of the lines in C. Mean values were compared statistically between WT and the mutants using Tukey's test (***, P < 0.001; *, P < 0.05; NS, P > 0.05); statistical comparison of the data obtained from E224Q and E224Q/R228Q is also shown. (F) Values of the Hill coefficient of each channel. Bars depict means ± SEM (n = 3). The symbols used are as indicated in the figure.
	Figure 7. Comparison of single-channel currents through WT Kir2.1 and mutants. (A) Representative single-channel currents recorded from Xenopus oocytes using the cell-attached patch configuration. The K+o in the pipette and in the bath were 140 mM, and Mg2+ in the solution was chelated by EDTA. The holding potential was −120 mV. The dashed lines indicate the zero current level after subtracting the leak current. The traces are shown on an expanded time scale on the left, and event histograms are shown on the right. (B) Relationship between single-channel current amplitude and holding potential. Symbols are as shown in the figure. Bars depict means ± SEM (n = 3–4). (C) Single-channel conductance calculated from the slopes of the plots in B; plotted are means ± SEM (n = 3–4).
	Figure 8. Comparison of the intrinsic inward rectification of WT Kir2.1 and the indicated mutants. (A) Macroscopic currents recorded in the absence of intracellular blockers from excised patches of HEK293T cells expressing WT or the indicated mutant. Representative current traces were recorded in solution containing 140 mM K+ on both sides of the patch. The holding potential was 0 mV; step pulses from +100 to −100 mV were applied in 10-mV decrements. (B) Mean I-V relationships derived from the accumulated data; values were measured 2 ms after the onset of each step pulse. Bars depict means ± SEM (n = 3–4). The symbols used are as indicated in the figure. (C) The ratios of the current amplitudes measured 2 ms after the onset of step pulses to +100 and −100 mV were calculated as an index of rectification intensity. Two different holding potentials were used for recording and analysis: 0 mV (solid bars) and −100 mV (open bars). Bars depict means ± SEM (n = 3–4). Mean values were compared statistically between WT and the mutants using Tukey's test (***, P < 0.001; *, P < 0.05; NS, P > 0.05); statistical comparison of E224Q and E224Q/R228Q is also shown. (D and E) Contribution of the negatively charged amino acid residue at the outer mouth of the pore to the intrinsic inward rectification of Kir2.1. (D) Representative macroscopic currents through E125Q and E125K recorded as in A. (E) Mean I-V relationships analyzed as in B. Bars depict means ± SEM (n = 3–4). The symbols used are as indicated in the figure.
	Figure 9. Comparison of the inward rectification properties of macroscopic currents through WT Kir2.1 and the indicated mutants of the H226 residue. (A) Macroscopic currents recorded using two-electrode voltage clamp with Xenopus oocytes and the intensities of the inward rectification of them were analyzed as in Fig 2. Bars depict means ± SEM (n = 4–5), and the values were compared statistically between WT and the mutants using Tukey's test (**, P < 0.01; P > 0.05). (B) Comparison of the susceptibility of macroscopic currents through WT Kir2.1 and H226K under the indicated intracellular pH to blockade by intracellular Mg2+. Macroscopic currents recorded in the presence of the indicated concentrations of Mg2+i from excised patches of HEK293T cells as in Fig. 5. The concentrations of Mg2+i are shown in the figure. The relationships between the Kd and voltage for Mg2+i blockade of outward K+ currents are shown on the right. The solid lines with symbols indicate the means of the fitted lines with data obtained from four patches for H226K (pH 7.2) and WT (pH 5.8), and the dashed lines indicate SEMs. For reference, the data of WT (pH 7.2) shown in Fig. 5 were also plotted with SEM. (C) Comparison of the intrinsic inward rectification of WT and H226K as in Fig. 8. Bars depict means ± SEM (n = 4) of the normalized current at each voltage. The symbols used are as indicated in the figure.
	Figure 10. Schematic drawings illustrating the mechanism of the voltage-dependent blockade of Kir2.1. (A) Model of the long pore of the permeation pathway based on the structures of Kir2.1 and KirBac1.1. Negatively and positively charged amino acid residues are respectively indicated with red and blue sticks; the selectivity filter is indicated with yellow sticks. K+ ions and a spermine are indicated with space fillings. Dotted circles depict the charged rings with the diameters shown. (B) Schematic drawings explaining the function of the cytoplasmic pore in WT Kir2.1 (left) and a neutralized mutant (right). The filled circles indicate K+ ions, and the Bs indicate blocker molecules. On the left, the negatively charged cytoplasmic pore (obliquely lined) electrostatically gathers cations, such as K+ and blockers, and as a result the blocking area in the transmembrane are smoothly furnished with many K+, enabling high affinity, strongly voltage-dependent blockade with adequate outward K+ conductance. The bottom drawings illustrate the I-V relationships with and without the blockers in WT Kir2.1 and a neutralized mutant. With the WT channel, in which the negativity of the pore is intact, the outward current is steeply blocked with a significant K+ efflux at membrane potentials slightly above EK. This characteristic may facilitate generation of the cardiac action potential.

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