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Figure 1. . Single-channel conductance and ROMK2/IRK1 chimeras. (A) ROMK2/IRK1 chimeras. Parts of ROMK2 are white, and parts of IRK1 are shaded. Chimera C13 has the entire COOH terminus, probably including the last part of the second transmembrane domain (M2), from IRK1, and the rest of the channel from ROMK2. Other chimeras have progressively smaller portions of the COOH terminus from IRK1. (B) Inward current traces measured in the cell-attached configuration with a cell potential of −100 mV with respect to the pipette. The dotted lines indicate the closed state, with upward deflections indicating channel openings. (C) I–V relationships. Data represent means ± SEM for at least three different patches. A minimum of 15 transitions at each voltage were measured to obtain values for each patch. Slope conductances measured between −60 and −140 mV were ROMK2, 43 pS; C13, 31 pS; C132, 18 pS; C133, 37 pS; C133a, 38 pS; C134, 51 pS; and C135, 46 pS.
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Figure 2. . Single-channel conductance of the N240R mutation. (A) Alignment of the amino acid sequences of the rat Kir channel family, showing sequence differences at positions N240. (B) Inward current traces for the mutant N240R measured in the cell-attached configuration with cell potentials of −40 to −140 mV. Dotted lines indicate the closed state and upward deflections indicate channel openings. (C) I–V relationships for N240R, N240K, and N240E, with data from ROMK2 and C13 shown for comparison. Data represent means ± SEM for at least three different patches. A minimum of 15 transitions at each voltage were measured to obtain values for each patch. The slope conductances measured between −60 and −140 mV were ROMK2, 43 pS; C13, 31 pS; and N240R, 31 pS.
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Figure 3. . Effects of triple point mutations S286AA287M C289T (ROMK3M) on single-channel conductance of ROMK2. (A) Alignment of sequences of the Kir family in the region between residues 279 and 294 of ROMK2. (B) I–V relationships of ROMK2, C133 (in which the region in A is derived from IRK1), and the triple mutation S286AA287MC289T (ROMK3M) in which three of the residues of ROMK2 are mutated to the corresponding residues of IRK1, and the combined ROMK3M + N240R mutant. For comparison the I–V relationships of ROMK2 and C133 from Fig. 1 are superimposed. Slope conductances measured between −40 and −140 mV were ROMK2, 43 pS; C133, 37 pS; ROMK3M, 37 pS; ROMK3M N240R, 10 pS; C132, 18 pS.
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Figure 4. . Kinetic properties of ROMK2 and ROMK2N240R. Currents were recorded from cell-attached patches containing only one channel at a cell potential of −100 mV. Above, closed-time histograms were fit with two exponentials. For ROMK2, the exponentials had time constants of 1.5 ms (77% of closures) and 70 ms (23%). For ROMK2N240R, the exponentials had time constants of 1.7 ms (87%) and 61 ms (13%). Below, open-time histograms were fit with one exponential distribution. Time constants were 20 ms for ROMK2 and 25 ms for N240R.
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Figure 5. . Macroscopic current–voltage relationships for ROMK2 and N240R. Above, current traces for an uninjected (control) oocyte. Steady-state currents were <200 nA. Middle, current traces measured with two-electrode voltage clamp in oocytes expressing ROMK2 and N240R. The extracellular K concentration was 110 mM. The membrane voltage was held at 0 mV and stepped to values between −120 and +80 mV for 50 ms. Below, steady-state I–V relationships.
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Figure 6. . pH-dependence of macroscopic currents. Oocytes were bathed in solutions containing 55 mM Kacetate. The pH of the extracellular solution was reduced in steps, and the currents were measured after a new steady-state was achieved in 10–15 min. Inward conductance was normalized to maximal values measured at pH 7.8. Data points represent means ± SEM for four oocytes. Solid lines represent best fits to a Hill equation with pKa = 7.20 and n = 4.1 (ROMK) and pKa = 7.19 and n = 4.9 (N240R).
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Figure 7. . The N240R mutation does not affect Ba2+ block. Oocytes were superfused with solutions containing 110 mM KCl with and without added Ba2+. Whole-cell currents were measured using the two-electrode voltage-clamp at voltages between 0 and −140 mV. (A) Typical current traces for ROMK2 and ROMK2 N240R with 0, 0.5, and 10 mM Ba2+. (B) Currents at a test potential of −100 mV were normalized to their value in the absence of Ba2+ and plotted vs. Ba2+ concentration. Lines represent fits to a simple titration curve with Ki values of 0.62 mM (ROMK2), 0.56 mM (ROMK2 N240R), 0.25 mM (C7), and 0.25 mM (C7 N240R).
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Figure 8. . Effects of the N240R mutation on a channel with a higher conductance of the selectivity filter. (A) Single-channel traces for C7 chimera and C7 N240R with a holding potential of −80 mV. (B) I–V relationships for C7 and N240R. Single-channel conductances were C7, 58 pS, and C7 N240R, 36 pS.
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Figure 9. . Effect of charge at position 240 on single-channel conductance. Conductances measured between −40 and −140 mV were ROMK2, 43 pS; C13, 31 pS; N240R, 31 pS; N240K, 37pS; and N240E, 45pS.
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Figure 10. . Tl+ conductance and selectivity. Above, single-channel currents recorded in cell-attached patches with 110 mM Tl+ in the pipette and a holding potential of −60 mV. Below: I-V relationships for K+ and Tl+ conductance in wild-type ROMK2 and N240R mutant channels. The single-channel conductances for Tl+ were 18 pS in both channels. The Tl+:K+ conductance ratios were 0.51 for ROMK2 and 0.78 for N240R.
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Figure 11. . Model of the effect of N to R substitution in the cytoplasmic pore based on the crystal structure of GIRK1. (A) A side view of the crystal structure of the cytoplasmic domain of GIRK1-Kir3.1 (pdb:1N9P) is shown from the side with two subunits removed for clarity. Residue Q261, corresponding to residue R260 in IRK1-Kir2.1 and N240 in ROMK2-Kir1.1 is highlighted using space-filling mode along with a potassium ion in the same plane and centered along the pore axis. (B) Models of the GIRK1 cytoplasmic domain in which residue Q261 has been mutated to asparagine (left) or arginine (right) are shown here. A cytoplasmic view of the pore-lining residues 223–230, 253–264, and 298–311 is represented with cartoon rendering except for residue 261, which is highlighted using space-filling mode. A potassium ion is shown along the central axis and in the plane of residue 261.
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