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Figure 1. Molecular docking of AA to Kv4.2 homology model. The model was built based on Kv4.2 homology with Kv1.2 and KcSA (see Methods). In this and other figures, all images of the protein surface are color-coded by amino acid side chain property; gray = polar; green = hydrophobic, blue = basic, red = acidic. (A) An intracellular view of the homotetramer. (B) A lateral view of the channel with seven overlapping but unique docking outputs for AA located near the internal surface of the channel. (C) A zoomed-in view of the predicted hydrophobic AA binding pocket. The same seven AA conformations as in (B) are shown for closer inspection. Note that for each docking result, the nonpolar region of AA is embedded in the hydrophobic cavity (green) and the polar part of AA is oriented toward two positively charged residues (blue) lining the pocket. (D) Identification of the residues predicted to comprise the AA binding pocket. Note that four pockets are predicted, each made of residues from two adjacent subunits.
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Figure 2. Residues V261, G314 and K318 are important to the inhibitory effects of AA on Kv4.2. (A) Peak current inhibition by 10 μM AA of wild-type and mutant Kv4.2 channels. K+ currents were recorded with voltage steps to +40 mV from a holding potential of â90 mV. The bar graphs show the mean current intensity after 10 min of exposure to 10 μM AA, relative to the intensity before application of AA. Numbers represent the number of cells tested for each mutant; * indicates p < 0.01. Dotted line designates the mean wild-type effect. N.E. = no expression. (B) Mutated residues located on or in close proximity of the S4-S5 linker and their relative importance in AA binding based on results in (A). The radii of the filled circles are proportional to the relative K+ current amplitude after treatment with AA. The inset shows the six transmembrane α-helices of a single Kv4.2 subunit. Residues 256 and 259 are predicted to be outside the proposed AA binding pocket.
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Figure 3. Conductance-voltage plots for wild-type and three mutant channels. Peak conductances were calculated from G = I/(Vm-Vh) with Vh = -100 mV and were normalized to the maximal conductance (Gmax) for each cell and plotted vs. the step membrane potential (Vm). The conductance-voltage relationships are plotted in the absence (open symbols) and presence (closed symbols) of 10 μM AA for (A) wild-type (WT) Kv4.2 (n = 14), (B) V261F (n = 8), (C) K318Q (n = 8), K318E (n = 3â6), and (D) V261F/K318Q (n = 6). Solid lines are the best fits of the data to a first order Boltzmann function. In each panel, the black curve is without AA and the gray curve is with AA. The fitted parameters for the slope factor (k) and V1/2 values for the curve without AA and the curve with AA were, respectively: (A) 9.5, â20.3 mV; 12.1, â13.4 mV, (B) 9.2, â20.8 mV; 10.9, â17.1 mV, (C) K318Q: 10.3, â15.6 mV; 11.3, â8.6 mV, K318E: 7.9, â30.7, 7.1, â25.2, (D) 7.3, â26.4 mV; 8.4, â19.1 mV. All fitted curves had a coefficient of determination (R2) ⥠0.998.
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Figure 4. Concentration-dependence of the current inhibition by AA. (A) Representative current traces elicited by voltage steps from â90 mV to +40 mV before and following the application of 1 to 300 μM AA until saturation for wild-type (WT) channels and one mutant construct. Arrows designate peak current amplitude for each test concentration. Horizontal and vertical scale bars represent 25 ms and 1 μA, respectively. (B) Concentration-response curves for AA inhibition of peak Kv4 currents for WT and mutant channels (8â19 cells/point). Data were fitted to a Hill equation with the Hill slope (nH) = 1. All fitted curves had a coefficient of determination (R2) ⥠0.991. Fitted Kd values were: WT, 21.1 μM; V261F, 37.7 μM; K318Q, 57.4 μM; V261F/K318Q, 45.7 μM.
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Figure 5. Effects of AA on WT and mutant Kv4 current inactivation. The first two columns show representative Kv4 currents evoked by voltage steps from â90 mV to â40, â20, 0, +20, +40 and +60 mV for wild-type (WT) and selected mutant channels in the absence (first column) and after inhibition by 10 μM AA (second column). The third column displays the current with 10 μM AA (red) superimposed on the current recorded in the absence of AA (black) at +40 mV; inset boxes show the peak of the AA-inhibited current scaled to its control to clarify the effects on the time course of current decay. Horizontal and vertical scale bars represent 25 ms and 0.5 μA, respectively. The scale bars do not apply to the insets.
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Figure 6. Concentration-dependence of the kinetic modulation by AA. (A) Representative current traces elicited by voltage steps from â90 mV to +40 mV before (black trace) and following the application of 10 μM AA (red trace) for wild-type (WT) channels and one mutant construct. The peak current in the presence of AA was normalized to the pre-AA current in order to measure the effects on current decay. Dashed lines show how t1/2 was measured (time of half-inactivation, measured at +40 mV and normalized to the value measured in the absence of AA). (B) Concentration-response curves for AA-dependent modulation, measured as the fractional reduction in t1/2 Data for 6â12 cells each were fitted to a Hill equation with the Hill slope (nH) = 1; all fitted curves had a coefficient of determination (R2) ⥠0.986. Fitted Kd values were: WT, 23.7 μM; V261F, 64.0 μM; K318Q, 60.7 μM; V261F/K318Q, 43.5 μM.
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Figure 7. AA docking specificity. (A) Kv4.2 wild-type (WT) channel model with AA docked; residues of interest are noted. (B) Zoomed-in view of WT channel model predicting the molecular distances of hydrogen bonds with AA. (CâE) Virtual mutants and docked AA. (F) Representative time course of peak outward K+ current amplitudes before and during application of AA (10 μM) for WT Kv4.2 (blue; +40 mV test potential) and Kv1.2/2.1 (red; +110 mV test potential. Inset: Docking result showing the lack of a plausible AA binding site on the Kv1.2/Kv2.1 chimera (PDB: 2R9R63). The same docking and search parameters were used in attempts to dock AA to WT or mutant Kv4.2 channels and the Kv1.2/Kv2.1 chimera.
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