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Figure 1. The gating charge transfer center (Tao et al., 2010). (A) The membrane topology of one subunit of the tetrameric Shaker K+ channel is shown. The approximate positions of conserved charged residues and I287, F290, F324, and A359 in the voltage sensor domain are indicated. Conserved charged residues are labeled using the following generic nomenclature: E0, E247 in S1; E1, E283 in S2; E2, E293 in S2; D3, D316 in S3; R1, R362 in S4; R2, R365 in S4; R3, R368 in S4; R4, R371 in S4; K5, K374 in S4. The S2, S3, and S4 transmembrane segments are shown in yellow, red, and blue, respectively, for comparison to B. (B) The charge transfer center consists of F290 (orange) and E2 in S2 (yellow/red) and D3 in S3 (yellow/red), and is occupied by K5 in S4 (gray/blue) in the Kv1.2/Kv2.1 paddle chimera x-ray structure (Long et al., 2007). Also shown are I287 in S2 and F324 in S3 (green), which correspond to residues that form a naturally occurring binding site for extracellular divalent cations in eag (Silverman et al., 2000; Lin et al., 2010). I287 and F324 are located extracellular to the charge transfer center. Ribbons representing the backbone atoms of S2, S3, and S4 are shown in yellow, red, and blue, respectively. Backbone atoms and the indicated side chains were extracted from the 2r9r x-ray structure and labeled according to the Shaker sequence (Long et al., 2007). In the chimera, the F324 position is occupied by a tyrosine residue, which was mutated in silico using PyMOL (v1.3; The PyMOL Molecular Graphics System; Schrödinger LLC). The figure was made using PyMOL.
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Figure 2. Extracellular Zn2+ slows activation in I287H+F324H channels. (A) Currents were evoked by depolarizing from −80 to +60 mV in the absence (black) or presence (red) of 1 µM Zn2+. Traces were fitted with single-exponential functions (dashed green lines) to provide activation time constants (τact). (B) τact values measured at test voltages ranging from +10 to +90 mV in the absence (black squares) or presence (red circles) of 1 µM Zn2+ differed significantly (§, P < 0.0005) at all voltages. Data are shown as mean ± SEM (n = 13). At +60 mV, τact values were 66 ± 4 ms and 8.6 ± 0.6 ms with and without 1 µM Zn2+, respectively. (C) Values of τact at +60 mV were plotted versus Zn2+ concentration and fitted with a rectangular hyperbola (black line) to determine [Zn2+]1/2, which was 0.12 µM (n = 6–13). (D) The box plot shows τact values measured at +60 mV in the indicated concentrations of Zn2+ for I287H+F324H, the Shaker-IR parent channel, and the I287H and F324H single-mutant channels. Mean values of τact that differed significantly from no Zn2+ are indicated: *, P < 0.05; ‡, P < 0.005; §, P < 0.0005 (n = 2–13).
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Figure 3. Extracellular Zn2+ delays pore opening in I287H+F324H channels. (A) Representative current traces, evoked by depolarizing from −80 to +40 mV in the absence (black) or presence (red) of 1 µM Zn2+, are shown on an expanded scale to illustrate the effect of Zn2+ on the delay before pore opening. Delays were determined by extrapolating fitted single-exponential functions (dashed green lines) to the zero current level (dashed gray line) (Perozo et al., 1994; Lin et al., 2010). (B) The delay before pore opening was measured at test voltages ranging from +10 to +80 mV in the absence (black squares) or presence (red circles) of 1 µM Zn2+. Delay values differed significantly in the presence and absence of Zn2+, as indicated (*, P < 0.05; ‡, P < 0.005). Values are shown as mean ± SEM (n = 4–10). At +40 mV, delays measured with and without Zn2+ were 3.4 ± 0.6 ms and 0.6 ± 0.2 ms, respectively. (C) Zn2+ shifts the dependence of the delay on prepulse potential in the depolarized direction. Delay values have been plotted on an inverted scale as a function of prepulse potential. Delays were measured in the absence (black squares) or presence (red circles) of 1 µM Zn2+ by stepping from the holding potential of −80 mV to prepulse voltages ranging from −130 to 25 mV for 20 ms before depolarizing to +60 mV. Delay data were fitted with single Boltzmann functions (solid lines). Values of V1/2 and slope were −39 ± 1 mV and 18 ± 1 mV, and −14 ± 3 mV and 12 ± 2 mV, respectively, in the absence and presence of Zn2+ (n = 6–8). Normalized conductance values, measured as a function of test potential in the absence (open black squares) or presence (open red circles) of Zn2+, are shown on the same axes. Conductance data fitted with single Boltzmann functions (solid lines) with (red) or without (black) Zn2+ did not differ significantly. Values of V1/2 and slope were 44 ± 1 mV and 17 ± 1 mV, and 43 ± 1 mV and 18 ± 1 mV, respectively, in the absence and presence of Zn2+ (n = 5). For clarity, error bars have been omitted from the figure.
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Figure 4. Zn2+ induces a slow component of activation in I287H+R1H channels. (A) Currents were evoked by depolarizing from −80 to +60 mV in the absence (black) or presence (red) of 50 µM Zn2+. (B) Current trace obtained at +60 mV in the presence of Zn2+ (red) has been fitted with one (blue line) or the sum of two (black line) exponential functions.
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Figure 5. A subset of I287H+R1H voltage sensors binds Zn2+ at −80 mV. (A) The box plot shows τact values measured at +60 mV in the indicated concentrations of Zn2+ for I287H+R1H and the R1H single-mutant channel. I287H+R1H current traces were fitted with a single-exponential function in the absence of Zn2+ (black box, τact) and the sum of two exponential functions in the presence of Zn2+ (blue boxes, τfast; red boxes, τslow). R1H current traces were fitted with a single-exponential function in the presence and absence of Zn2+ (black boxes, τact). Mean values of τact, τfast, or τslow that differed significantly from no Zn2+ are indicated: *, P < 0.05; †, P < 0.01; ‡, P < 0.005; §, P < 0.0005 (n = 3–13). (B) The normalized amplitude of the slow component of activation measured at +60 mV in I287H+R1H channels has been plotted versus Zn2+ concentration (n = 4–7). The data were fitted with a rectangular hyperbola (black line) to obtain values for [Zn2+]1/2 and Aslow,max, which were 9.4 µM and 0.56, respectively. (C) Aslow,max was measured as a function of prepulse voltage in the presence of a saturating Zn2+ concentration (200 µM; n = 4–7). The membrane was stepped from −80 mV to prepulse voltages ranging from −140 to −40 mV for 1 s before depolarizing to +80 mV.
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Figure 6. Extracellular Zn2+ slows activation and delays pore opening in I287H+A359H channels. (A) At subsaturating concentrations, Zn2+ induced a slow component of activation in I287H+A359H channels. The membrane was depolarized from −80 to +60 mV for 2 s. Representative current traces recorded in the presence or absence of Zn2+ (black, 0 µM; red, 1 µM; green, 10 µM) were scaled to the same amplitude, overlaid, and fitted with one (no Zn2+) or the sum of two (+Zn2+) exponential functions (black lines). (B) The box plot shows τact values measured at +60 mV in the indicated concentrations of Zn2+ for I287H+A359H and the A359H single-mutant channel. I287H+A359H current traces were fitted with the sum of two exponential functions (blue boxes, τfast; red boxes, τslow), except at 50 µM Zn2+, where one component was sufficient (black box, τact). A359H current traces were fitted with a single-exponential function (black boxes, τact). Mean values of τact, τfast, or τslow that differed significantly from no Zn2+ are indicated: ‡, P < 0.005; §, P < 0.0005 (n = 3–25). (C) The normalized amplitude of the slow component of activation measured at +60 mV has been plotted versus Zn2+ concentration (n = 3–10). The data were fitted with a rectangular hyperbola (black line) to obtain values for [Zn2+]1/2 and Aslow,max, which were 1.2 µM and 1.0, respectively. (D) Zn2+ increases the delay before pore opening. Representative current traces, evoked at +60 mV in the absence (black) or presence (red) of a saturating concentration (50 µM) of Zn2+, were fitted with single-exponential functions (green). The fitted functions were extrapolated to the zero current level (dashed line) to estimate the delay (Perozo et al., 1994; Lin et al., 2010). (E) The box plot shows the delay before pore opening measured in the absence (black box) or presence (red box) of 50 µM Zn2+. Mean values obtained at +60 mV were 496 ± 50 ms and 0.8 ± 0.1 ms, with and without Zn2+, respectively, and differed significantly (§, P < 0.0005; n = 5–9).
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Figure 7. Effects of Zn2+ on channels containing I287H paired with histidine mutations in the S3–S4 loop. (A; left) I127H+I360H double mutant. (Middle) I360H single mutant. (B; left) I127H+L361H double mutant. (Middle) L361H single mutant. Currents were evoked by pulsing from −80 to +60 mV in the absence (black) or presence of 10 µM Zn2+ (red). Representative current traces have been scaled to the same amplitude and overlaid. At right, the box plots show τact values measured at +60 mV in the indicated concentrations of Zn2+ for (A) I287H+I360H and the I360H single-mutant channel or (B) I287H+L361H and the L361H single-mutant channel. Double-mutant current traces were fitted with the sum of two exponential functions (blue boxes, τfast; red boxes, τslow). Single-mutant current traces were fitted with a single-exponential function (black boxes, τact). Mean values of τact, τfast, or τslow that differed significantly from no Zn2+ are indicated: *, P < 0.05; †, P < 0.01; ‡, P < 0.005; §, P < 0.0005 (n = 3–10).
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Figure 8. Zn2+ slows activation in I287H+A359H+F290W+R1K+K5R channels. (A) Currents were evoked by depolarizing to +60 mV in the absence or presence of Zn2+: black, 0 µM; red, 1 µM; green, 10 µM. Traces have been scaled to the same amplitude and overlaid. Currents were fitted with one exponential component (black lines) to obtain values for τact. (B) The box plot shows values of τact measured at +60 mV in I287H+A359H+F290W+R1K+K5R or the F290W+R1K+K5R control channel as a function of Zn2+ concentration. Mean values of τact that differed significantly from no Zn2+ are indicated: #, P < 0.001; §, P < 0.0005 (n = 6–15).
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Figure 9. R1 in S4 occupies the gating charge transfer center in the resting state. (A) Cartoon of the resting conformation shows an arginine representing R1 in the gating charge transfer center. (A and B) Ribbons representing the backbone atoms of S2, S3, and S4 are shown in yellow, red, and blue, respectively. Backbone atoms and side chains corresponding to I287, F290, E2, D3, and F324 (mutated in silico from tryptophan) in Shaker were extracted from the Kv1.2/Kv2.1 paddle chimera x-ray structure (2r9r) (Long et al., 2007). (B) Cartoon of the penultimate closed state shows arginines representing R1 in the vicinity of I287 and R2 in the gating charge transfer center. Other side chains and colored ribbons are the same as in A. The figure was made with PyMOL (v1.3).
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