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Figure 1. C10-TEA blocks CNG channels. Leak-subtracted currents in response to 1 mM cGMP in the absence (A) and in the presence (B–F) of increasing concentrations of C10-TEA. All these current traces were acquired from the same inside-out patch and in response to voltage jumps from −80 to +80 mV in steps of 20 mV, from a holding potential of 0 mV.
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Figure 2. General properties of C10-TEA blockade. (A) Normalized dose–response at +80 mV. The solid line represents the fit to a Hill equation (see text; Eq. 1). The best-fit parameter values for KD and n were 21.5 ± 0.6 μM and 1.19 ± 0.04, respectively. (B) Voltage dependence of C10-TEA blockade. KD values at each voltage were estimated by Eq. 2. The solid line represents the fit to a Woodhull-type model (Eq. 3). The best-fit parameter value for δ is −0.45.
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Figure 3. The tail of C10-TEA is a major contributor to the binding affinity to CNG channels. Leak-subtracted currents from five consecutive traces in the presence of 1 mM cGMP to a voltage step of +80 mV, from a holding potential of 0 mV. All traces were obtained from the same inside-out patch. The first trace from the left was acquired in the absence of any blocker, the second in the presence of 50 μM C10-TEA, the third with 50 μM C10-TMA, the fourth with 5 mM TEA, and finally the experiment was bracketed with control conditions. Between traces, the patch was perfused with a solution devoid of cGMP and blocker for at least 30 s. Leak currents were acquired during these periods and they showed no changes throughout the experiment.
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Figure 4. General properties of blockade by alkyl-TEA derivatives. (A) Control conditions. Leak-subtracted currents in the presence of 1 mM cGMP acquired in response to voltage jumps from −80 to +80 mV in steps of 20 mV, from a holding potential of 0 mV. (B) Leak subtraction currents from the same patch and under the same conditions as A, but in the presence of QA derivatives with successively smaller alkyl chains. (C) Voltage dependence of blockade by QA derivatives. KD values at each voltage were estimated by Eq. 2. Solid lines represent Woodhull model fits (Eq. 3). The best-fit parameter values for δ and KD were −0.44 ± 0.01, 32 ± 2 μM (C10-TEA; open circles); −0.44 ± 0.02, 605 ± 7 μM (C8-TEA; filled circles); −0.53 ± 0.02, 6144 ± 21 μM (C6-TEA; open squares); −0.45 ± 0.02, 14642 ± 35 μM (C4-TEA; filled squares).
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Figure 5. Binding energy decreases as the length of the alkyl tail is shortened. The binding energy of each blocker was calculated from the values of KD0 by assuming an arbitrary standard concentration of 1 M (ΔG = −RT ln(1M/KD0)). Binding energies are plotted in RT units. The line represents a fit of the binding energies for C6-, C8-, and C10-TEA to a straight line. The best-fit value for the slope is −1.22 ± 0.03, which means an increase of binding energy of ∼1.2 RT for each additional methylene group. Except for C8- and C10-TEA, all standard errors are smaller than the size of the symbols.
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Figure 6. C10-TEA binds within the permeation pathway: the effect of external Mg2+ on intracellular C10-TEA blockade. Voltage dependence of intracellular C10-TEA blockade assessed in two different patches, either in the absence (open circles) or in the presence of 50 μM (filled squares) external Mg2+.
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Figure 7. C10-TEA blockade at the single channel level. (A) Single channel recordings at +70 mV with solutions containing 2 mM cGMP. (B) Single channel recordings from the same channel in the presence of 2 mM cGMP and 5 μM of C10-TEA. Dashed lines represent the current level when the channel is closed. (C) Amplitude histogram from 20 s of recording in the presence of 2 mM cGMP. The solid line represents a two-component Gaussian fit. The best-fit parameter value for the amplitude for the open state was 2.209 ± 0.002 pA, comprising an area of 95%. (D) Amplitude histogram from 40 s of recording in the presence of 2 mM cGMP and 5 μM of C10-TEA. The solid line represents a two-component Gaussian fit. The best-fit parameter value for the amplitude for the open state was 2.317 ± 0.002 pA, contributing to an area of 74%.
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Figure 8. C10-TEA binds to an open channel. (A and B) Open dwell-time distributions at +70 mV under saturating (2 mM) cGMP concentrations in the absence and presence of 25 μM of C10-TEA. The solid lines represent double-exponential fits to the data. The best-fit parameter values were τ1 = 0.7 ms, A1 = 0.07, τ2 = 12.2 ms, and A2 = 0.93 in control conditions and τ1 = 0.6 ms, A1 = 0.19, τ2 = 3.7 ms, and A2 = 0.81 in the presence of the blocker. (C) C10-TEA dose response of the apparent mean open time of the slow component (τ2). In each concentration, the effect of C10-TEA was normalized to the value of τ2 in the absence of the blocker. The solid line represents a Hill equation fit with a best-fit parameter value for the midpoint of 16 ± 4 μM.
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Figure 9. C10-TEA binds to closed channels: effects of agonists. (A) Leak-subtracted current traces to +80 mV in a patch where blockade by 5 μM C10-TEA was assessed under saturating (left) and subsaturating (right) concentrations of cGMP. (B) Leak-subtracted current traces to +80 mV in a patch where blockade by 15 μM C10-TEA was evaluated under saturating (16 mM) concentrations of cGMP (left), cIMP (middle), and cAMP (right). Each set of traces was normalized to a value of 1 in the absence of the blocker, therefore the ordinate scale lacks units.
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Figure 10. C10-TEA binds to closed channels: steady-state cGMP activation. Open circles show the dose response for cGMP in the absence of C10-TEA. The solid line represents a fit of Scheme 2 to the dose response data. Parameter K was fixed at 3500 M−1 (Fodor et al., 1997a), and L and Imax were allowed to vary. The best-fit parameter values for L and Imax were 16 ± 1 and 2533 ± 45 pA, respectively. Filled circles illustrate the dose response for cGMP in the presence of 10 μM C10-TEA. The solid line symbolizes a fit of the model shown in text to the dose response data. In this fit, L and Imax were fixed to the values obtained in the absence of the blocker. The best-fit parameter values for KDc and KDo were 3 ± 0.3 and 21 ± 2 μM, respectively.
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Figure 11. MTS reagents of comparable sizes to QA blockers can access position V391C in closed CNGA1 channels. (A) Alignment of the intracellular side of the S6 segment of Shaker Kv and CNGA1 channels. In red is highlighted the position where chemical modification was assessed. The bottom part shows the S6 helices from the X-ray crystal structure of Kv1.2 (Long et al., 2005). On the left is shown a side view of the S6 helices and on the right is shown an intracellular view. Cβ and Cγ atoms of V474 are represented in red. (B) The experimental protocol consisted of 2-s reagent application at 0 mV in the presence (open state) or absence (closed state) of 2 mM cGMP. A brief 50-ms depolarization to +80 mV in the presence of 2 mM cGMP was applied to monitor the cGMP-activated current (blue step). Leak currents were acquired by a similar pulse to +80 mV but in the absence of cGMP. This protocol was applied every 15 s. (C) Modification of 391C CNGA1 channels by 500 μM MTS-PtrEA in the open (open circles) and in the closed states (filled circles). Circles symbolize leak-subtracted currents normalized to the average maximal current from the eight records acquired previous to MTS-PtrEA treatment (arrow). The decrease in cGMP-dependent currents were fitted to single exponentials (solid lines). The best-fitted parameter values for τ were 5.7 ± 0.3 and 27 ± 1 s for the open and closed state, respectively. (D) Modification rates in the open (open circles) and closed states (filled circles) for 391C CNGA1 channels by three different MTS reagents. Structural models of each reagent are shown. Symbols represent the mean of at least three experiments.
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Figure 12. C10-TEA slows Cd2+ inhibition of 391C CNGA1 channels in the closed state. (A) The experimental protocol consisted of 2-s treatment with 6 μM Cd2+ ions with solutions lacking divalent chelators. Immediately after treatment, the patch was perfused with a solution containing 200 μM EDTA to quickly chelate any Cd2+ contamination. In addition, when the effect of the presence of C10-TEA was assessed, the Cd2+-C10-TEA treatment was preceded and followed by 2-s perfusion with solutions containing C10-TEA in the absence of Cd2+. (B) Filled circles correspond to an experiment in which 6 μM Cd2+ treatments in the closed state were initiated at the arrow. The solid line through symbols represents a single exponential fit. The best-fit parameter value for τ was 2.64 ± 0.08 s. Filled squares symbolize an experiment in which 6 μM Cd2+ was applied in the presence of 200 μM C10-TEA. In this case, the best-fit parameter value for τ was 18 ± 2 s.
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