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Figure 2. (A) Raw current traces of wild-type, Y401C, W402C, and E403C channels recorded in oocytes following depolarization to −10 mV from a holding potential of −120 mV with (broken line) and without (solid line) 100 μM extracellular Cd2+. Clearly, the amount of current reduction is much greater for the mutant channels (Y401C, W402C, and E403C) than for the wild-type channels. The currents amplitudes have been scaled for ease of comparison. Peak currents in μA were: 3.8 (WT), 3.5 (Y401C), 3.5 (W402C), and 2.0 (E403C). (B) Plots of the fraction of peak current remaining as a function of the extracellular [Cd2+] for the same channels shown in A. Curve fits allow estimation of the dissociation constants (i.e., KD) for Cd2+ binding to the channel pore. (C) Summary of the Cd2+ block in single-cysteine mutants. The ratio of the estimated KD for Cd2+ block of current in single-cysteine mutants divided by the wild-type channels (i.e., KD,mut/KD,WT) for control conditions (filled bars) and following the application of 1.0 mM methane-thiosulfate-ethylammonium (MTSEA) to oxidize the inserted free sulfhydryls (open bars). For each mutant, except W756C, KD,mut was significantly reduced (p < 0.001) more than 3-fold, which was abolished by the application of MTSEA.
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Figure 3. (A) Na+ current tracings of Y401C, E758C, and Y401C/E758C mutant channels in response to depolarization to −10 mV from a holding potential of −120 mV before (solid traces) and after (broken traces) the addition of 100 μM Cd2+. Na+ currents for Y401C/E758C channels are shown before and after the addition of DTT. The currents have been scaled for ease of comparison. The current amplitudes in the absence of Cd2+ were (μA): 3.5 (Y401C), 2.7 (E758C), 2.2 (Y401C/E758C −DTT), and 4.6 (Y401C/ E758C + DTT). (B) Dose-response curves of the normalized peak Na+ current as a function of the [Cd2+]. The channels become about 1,200-fold more sensitive to Cd2+ (KD changes from 1,353 ± 382 μM to 1.1 ± 0.2 μM) following the addition of 2 mM DTT.
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Figure 4. Ratios of the predicted dissociation constant, KD,pre, to the experimentally observed dissociation constant, KD,ob for all the double mutants studied before (filled bars) and after (open bars) reduction by DTT. KD,pre is estimated from the dissociation constants recorded for the single-cysteine mutant channels assuming independent binding of Cd2+ to the inserted cysteines in double-cysteine mutants (see methods, Eq. 1). For the mutants with ratios of KD,ob/KD,pre around 1, we assume that the two inserted cysteines are binding Cd2+ independently. Values of KD,ob/KD,pre statistically different from 1 are identified by asterisks (*). The mutant channels with KD,ob/KD,pre below 0.37 (i.e., with stabilization energies of 1 kT) are identified by open triangles.
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Figure 5. Single-channel recordings of Y401C, E758C, and reduced Y401C/E758C mutant channels following fenvalerate application at −80 mV. (A) Currents recorded in Y401C, E758C, and Y401C/E758C channels in the presence of 10 μM fenvalerate which is added to maintain the channels in the open state for tens to hundreds of milliseconds. (B) Currents recorded in Y401C and reduced Y401C/ E758C mutants channels with 5 μM extracellular Cd2+ and in E758C channels with 400 μM Cd2+. Cd2+ caused full closures of the Y401C and Y401C/E758C channels while blocking E758C channels to a sub-conductance level (closed level indicated by the broken line). In the presence of 5 μM Cd2+ the double-mutant channel Y401C/E758C displayed bursts of short-lived blocking events separated by long-lived blocking events not seen in either single-mutant. These long-lived blockages likely represent the “trapping” of the Cd2+ ion as a result of simultaneous interactions with the two free sulfhydryl groups. (C) The mean block-time histograms are shown for the channels illustrated in A for Y401C, E758C, and Y401C/E758C channels. The blocked-time histograms could be adequately fit using a mono-exponential equation for Y401C and E758C channels, while a bi-exponential function was required for Y401C/E758C channels. Note: the time axes are different in the different panels. See text for further details. (D) The mean unblocked-time (i.e., open-time) histogram is shown for the same channels illustrated in B. For Y401C, E758C, and Y401C/E758C channels the unblocked-time histogram could be adequately fit using a mono-exponential function.
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Figure 6. Effect of cross-linking on conductance and selectivity properties of double-cysteine mutants. (A) Raw current traces following depolarization to −10 mV from a holding potential of −120 mV for E403C/D1532C channels expressed in oocytes before (□) and after (▪) reduction with DTT. Note the nearly twofold increase in current after reduction at this voltage. (B) The corresponding current-voltage relationships for E403C/D1532C. In this particular mutant, there is little change in the channel's conductance (161 μS before and 156 μS after DTT), estimated from the slope of the current-voltage curve at voltages above 0 mV, but the reversal potential is shifted by 19 mV to the right following reduction with DTT. (C) Raw traces for E403C/ A1529C before (○) and after (•) the application of DTT. The current increased more than sixfold following reduction with DTT. (D) The current-voltage relationship for E403C/A1529C mutants shows a fivefold increase in slope at voltages above 0 mV (22 μS before and 97 μS after DTT), whereas the reversal potential is only slightly shifted rightward (7 mV) by reduction with DTT.
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