Guo D , Ramu Y , Klem AM , Lu Z .

GM55560 NIGMS NIH HHS , HL03814 NHLBI NIH HHS , R01 GM055560 NIGMS NIH HHS

	Figure 1. . Alignment of the M2 sequence among three Kir channels, and KcsA structural model. (A) Alignment (Ho et al., 1993; Kubo et al., 1993a; Schrempf et al., 1995; Doyle et al., 1998) of the M2 sequences of KcsA, ROMK1 and IRK1. Substituting aspartate reduces the ratio of outward to inward currents negligibly or modestly at yellow residues, but dramatically at red residues. (B) KcsA structure (Zhou et al., 2001b) with residues highlighted as in A. (C) Same structure as in B but with residues highlighted as for the ROMK1 sequence in A (position equivalence based on the sequence alignment shown in A).
	Figure 2. . Effects of substituting aspartate in M2 of ROMK1 on inward rectification. (A) Voltage pulse protocol. (B-F) Currents of ROMK1 and four mutants containing Asp at various positions in M2. (G) Absolute ratio of steady-state currents (mean ± SEM; n = 5–12) at 80 and at −80 mV for wild-type and Asp-substituted channels, plotted against the M2 sequence.
	Figure 3. . Effects of substituting aspartate in M2 of KcsA-IRK1 on inward rectification. (A–D) Currents of original KcsA-IRK1 and mutants containing Asp at three representative positions. (E) Absolute ratio of steady-state currents (mean ± SEM; n = 5–10) at 80 and at −80 mV for the original and the Asp-substituted KcsA-IRK1 constructs.
	Figure 4. . Blocker structures, channel models, and blocking reaction scheme. (A) Chemical structures of mono-Cn, bis-Cn, SPD, and SPM. (B and C) Models for voltage dependence of channel block by alkylamines. Model B has four K+ ions in the inner pore, and the binding of a blocking amine “pushes” the K+ ions to sites 1 and 3 from sites 2 and 4 in the narrow outer pore (gray versus black dots). The dotted circle at the internal end of the pore represents a blocking QA. Model C has five K+ ions in the inner pore, and the binding of an amine does not affect the binding of K+ ions in the outer pore. The arrowheads midway through the vertical lines that represent the alkyl chains symbolize the fact that these lines represent a series of alkylamines of increasing chain length. (D) One-step steady-state model for channel block by an amine molecule. The total number of K+ ions in a conducting channel (Ch) is m + n. Binding of an intracellular amine (AMint) to the channel pore displaces n K+ ions to the extracellular solution (nK+ext), and vice versa. The voltage-dependent equilibrium constant (Keq) is defined in the text.
	Figure 5. . Block of IRK1 and its D172N mutant by alkylamines. Data in each column were collected from a separate oocyte. (A–D) Voltage protocols. (E–P) Wild-type and mutant currents without (E–H) and with bis-C4 (I and J), mono-C4 (K and L), bis-C9 (M and N), or mono-C9 (O and P).
	Figure 6. . Quantitative analyses of steady-state alkylamine block of wild-type and D172N mutant IRK1 channels. (A) Natural logarithm of Kd(0 mV) (determined as before [Guo and Lu, 2000a]; mean ± SEM; n = 6–10; all error bars within the symbols) for mono- and bis-alkyl-amines in wild-type and mutant channels, plotted against the number of methylene groups in the alkyl chain. The three lines are linear fits to the data, yielding (top to bottom) slopes of 0.68, 0.71, and 0.71, and Y-intercepts at “zero alkyl chain length” of −1.54, −1.96, and −4.93. The fourth (dotted) curve was drawn by hand. (B) Zobs (determined as in Guo and Lu, 2000a) of wild-type and mutant channel block by alkyl-mono- and bis-amines plotted against chain length; mean ± SEM; n = 6–10; connected by straight lines. (C) An illustration of thermodynamic cycles (Hidalgo and MacKinnon, 1995). The four corners are Kds (0 mV) of wild-type and mutant channels for mono- or bis-C2 and for a given mono- or bis-Cn. Ω = (wtKdC2 × mtKdCn)/(mtKdC2 × wtKdCn), where Kds (all at 0 mV) are taken from A. (D) Ω values, computed as in C, plotted against chain length. Bis-C11 is not available commercially.
	Figure 7. . Block of wild-type and double mutant (E224G + E299S) IRK1 channels by a short and a long alkyl-bis-amine. Currents of wild-type (A, C, and E) and mutant (B, D, and F) channels recorded in the absence (A and B) and presence of bis-C4 (C and D) or bis-C9 (E and F).
	Figure 8. . Thermodynamic analysis of block of wild-type and double mutant (E224G + E299S) IRK1 channels by a short and a long alkyl-bis-amine. The four corners of the thermodynamic cycle are Kds (0 mV) (mean ± SEM; n = 5–8) of wild-type and mutant channels for bis-C4 and bis-C9. The numbers around the cycle are the ratios of adjacent Kds. Ω = (wtKdbis-C4 × E224G+E299SKdbis-C9)/(E224G+E299SKdbis-C4 × wtKdbis-C9).
	Figure 9. . Kinetics of voltage jump-induced IRK1 current relaxations in the presence of bis- and mono-amines. (A and B) Current traces at three concentrations of bis-C9 (A) or mono-C9 (B) elicited by stepping membrane voltage from 0 to 70 mV. The curves superimposed on the current transients are single exponential fits. (C and D) Reciprocals of the time constants (1/τ; mean ± SEM, n = 5), obtained from the fits shown in A and B, are plotted against bis-C9 (C) and mono-C9 (D) concentrations and fitted with straight lines. (E and F) Natural logarithms of the slopes (k1 in G) of the linear fits shown in C and D are plotted against membrane voltage and fitted with straight lines: ln k1 = ln k1(0 mV) + z1VF/RT. (G) A kinetic model with one open (O) and two blocked states (B1 and B2).
	Figure 10. . Characteristics of rate constants (k1 in Fig. 9 G) for block of IRK1 by alkyl-bis- and mono-amines of varying chain length. The values (mean ± SEM; n = 5) were obtained from linear fits as illustrated for alkyl-bis- and mono-C9 in Fig. 9, E and F. (A) Rate constants extrapolated to 0 mV, k1(0 mV), for IRK1 block by bis-amines and mono-amines are plotted against alkyl chain length. (B) Observed valence of rate constants (z1) for IRK1 block by bis-amines and mono-amines, plotted against alkyl chain length.
	Figure 11. . Block of the wild-type and a mutant IRK1 by quaternary ammoniums. Currents of the wild-type and an IRK1 construct containing ROMK1's M2 sequence recorded in the absence (A, E) and presence of tetramethylammonium (TMA; B and F), tetraethylammonium (TEA; C and G), or tetrapropylammonium (TPrA; D and H).
	Figure 12. . Quantitative analyses of block of wild-type and mutant ROMK1 and IRK1 channels by quaternary ammoniums. (A and B) Kd(0 mV) and Zobs (mean ± SEM; n = 5–7; determined as before, see Guo and Lu, 2001) for block of wild-type and mutant IRK1 and ROMK1 channels by TMA, TEA, TPrA, tetrabutylammonium (TBA), and tetrapentylammonium (TPeA).

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