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Figure 1. . (A) Shaker K253C subunit showing location of target cysteine in the S1âS2 extracellular loop. (B) Structures and extended lengths (measured from the quaternary nitrogen to the distal olefinic carbon of the maleimide) of the two maleimidoâQA compounds used.
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Figure 7. . Fits of data using Eq. 20. (A) Gly5TEA data of Fig. 6 were fitted to Eq. 20 using the following parameters: Bf = 50 μM, Bt = 2 mM, Kd = 1.47 mM. The intrinsic maleimide reaction rate constant k, and the bound maleimide concentration Mt were varied to best simultaneously fit the three datasets, with values of k = 26 Mâ1sâ1 and Mt = 10 mM yielding the best fits (by eye). (B) Fit of Gly3TEA data of Fig. 2 C using Eq. 20. The same value for k was used. Bt was adjusted to reflect the lower effective concentration of QA headgroup near the pore; a value of 0.24 mM was calculated using the 45% fraction of blocked current with a Kd for reversible Gly3TEA block of 1.23 mM (Table I). A value for Mt of 1.25 mM was used to maintain a ratio of Mt to Bt identical to that used for the Gly5TEA data.
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Figure 2. . Effects of tethered maleimidoâQAs on Shaker K253C channels. (A) Gly5TEA. Current traces in response to 75-ms pulses to 60 mV from a holding potential of â90 mV. Dotted line represents zero current. The top trace is before wash-in of 50 μM Gly5TEA; subsequent traces are at 5, 10, 20, 40, 160, and 1,400 s of exposure. (B) Gly3TEA. Currents obtained as above. Top trace is before wash-in of 50 μM Gly3TEA; subsequent traces are at 45, 90, 180, 360, and 1,450 s of exposure. (C) Kinetics of tethered block. Isochronal (at 74 ms) currents from A and B were normalized to their values before maleimidoâQA exposure and plotted against time. Arrows indicate exposure periods. Circles, Gly5TEA. Time points are every 5 s. Time course was fitted (solid line) by a double-exponential function with time constants of 25 s (76%) and 268 s (24%). This experiment was performed eight times with mean fast and slow time constants (± SEM) of 28.1 ± 1.6 s and 263 ± 20 s, respectively. Triangles, Gly3TEA. Time points are every 15 s. Time course was fitted by a single-exponential function (solid line) with a time constant of 223 s (mean ± SEM was 227 ± 25 s, n = 7).
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Figure 3. . Effect of Gly5TEA concentration on tethering kinetics. (A) The first 750 s of data are shown to illuminate the differences in kinetics. Solid lines represent fits as described below. Circles, 50 μM Gly5TEA. The data are those of Fig. 2 C. Triangles, 25 μM. Time points are every 8 s. The fit is to a double-exponential function with time constants of 52 s (76%) and 398 s (24%). Squares, 10 μM Gly5TEA. Time points are every 15 s. Fit is to a double-exponential function with time constants of 112 s (63%) and 741 s (37%). Inset. Full time course of 10 μM Gly5TEA exposure. (B) Reciprocals of the time constants are proportional to [Gly5TEA]. Experiments were performed using 10 μM (n = 5), 25 μM (n = 6), and 50 μM (n = 8) concentrations of Gly5TEA and fits are to double-exponential functions as above. At each concentration, reciprocals of the fast time constants (circles) were averaged and plotted against concentration; the same was done for the slow time constants (squares). Error bars represent standard errors. Lines are linear least square fits constrained to go through the origin.
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Figure 4. . MaleimidoâQAs react in two ways. Cartoon depicting a cysteine-bearing channel that reacts with a bound maleimidoâQA blocker via an affinity-label effect (bottom path) or with a compound free in solution (top path). Shaded circles represent volumes swept out by the compounds when bound.
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Figure 5. . Kinetics of single-cysteineâcontaining channels using a ball-tagging approach. Subunits containing both a K253C mutation and the N-type inactivation sequence were mixed with subunits containing neither, and resultant channels were exposed to 50 μM Gly5TEA. (A) Current traces in response to 100 ms pulses to +60 mV from a holding potential of â90 mV. Dotted line represents zero current. The top trace is before wash-in of 50 μM Gly5TEA; subsequent traces are at 30, 90, 210, and 630 s of exposure. (B) Inactivating fraction of current (peak minus steady-state) was normalized to that before Gly5TEA exposure and plotted against time. Time points are every 10s. Time course was fitted by a single-exponential function (solid line) with time constant of 108 s (mean ± SEM was 97 ± 3 s, n = 7).
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Figure 6. . Effect of TEA on Gly5TEA tethering kinetics. 50 μM Gly5TEA was used for all experiments. Currents were scaled to a 0â1 range of inhibition. Circles, no TEA (same data as Fig. 2). Triangles, 0.2 mM TEA (occupancy of 0.5). Squares, 1.25 mM (occupancy of 0.86). Occupancies were calculated using a measured Kd for TEA block of 0.2 mM. The time courses shown are representative of experiments repeated at least six times at each TEA concentration.
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SCHEME I. All possible states and reaction pathways for a tetrameric channel bearing four equivalent cysteine targets. The M at the end of the curvy line represents an unreacted maleimide group and an encircled plus sign represents an ammonium group. k, Bt, Bf, Kd, Mt are as defined in the text. The vertical double arrows represent rapid equilibria between blocked and unblocked states. Horizontal single-headed arrows represent irreversible tethering reactions in which channels react with free maleimidoâQAs; downward-pointing single-headed arrows represent reactions with bound blockers. For a compound exhibiting a large affinity label effect, the reaction represented by the thicker downward and rightward-pointing arrow will dominate the tethering kinetics.
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SCHEME II.
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Figure .
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