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Figure 1. Internal TEA is less effective in the presence of external TEA. (Top) Shaker channel currents at several voltages before, during, and after recovery from application of 2 mM internal TEA with no TEA in the external solution. (Bottom) Currents from another patch before, during, and after recovery from application of 2 mM internal TEA with 100 mM TEA in the external solution. Currents elicited with 40-ms steps to voltages of −30, −10, +10, and +30 mV.
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Figure 2. Channel block by internal TEA in the absence and presence of external TEA. Fraction block of Shaker channel current at the indicated internal TEA concentrations in the absence (▪) and presence (○) of 100 mM external TEA. Currents measured at 0 mV. (Solid lines) Fits of to the data with the indicated dissociation constants. Mean values of three measurements except at 5 mM TEAi in the absence of TEAo (two observations) and single measurements at 0.1 and 0.25 mM TEAi at 100 mM TEAo. For remaining data, standard error bars are omitted if smaller than symbol. (Inset) Channel block by external TEA. Block of Shaker current at a potential of 0 mV at the indicated concentrations of external TEA. Mean values from four to five measurements with SEM limits. (Line) Best fit of to the data with the indicated dissociation constant.
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Figure 4. Lack of interaction between internal and external TEA in the presence of Rb+ ions. (Top) Shaker channel currents at several voltages before, during, and after recovery from application of 2 mM internal TEA with no TEA in the external solution. (Bottom) Currents from another patch before, during, and after recovery from application of 2 mM internal TEA with 100 mM TEA in the external solution. Currents elicited with 40-ms steps to voltages of −30, −10, +10, and +30 mV. Currents measured in K+-free, Rb+ solutions as described in methods.
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Figure 3. Interaction between external and internal TEA. Ordinate is the apparent dissociation constant for block by internal TEA at the indicated concentrations of external TEA. Error limits illustrated (when larger than symbol) are the estimated errors from the fitting routine (see methods). The dotted line is the expectation for no interaction between internal and external TEA. The dashed line is the expectation for competitive inhibition (). Solid line is the best fit of to the data with a value of Q of 4.6.
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Figure 8. Interaction between external gallamine and internal TEA. (A) Block of Shaker channels (at 0 mV) by the indicated internal TEA concentrations in the absence (•) and presence (□) of 5 mM external gallamine. Lines are fits of to the data with the indicated Kd values. n = 2–5; duplicate values plotted individually, mean values (n = 3–5) with SEM limits only if larger than symbols. (B) Ordinate is the apparent dissociation constant for block by internal TEA at the indicated concentrations of external gallamine. Error limits illustrated (when larger than symbol) are the estimated errors from the fitting routine (see methods). Solid line is the best fit of to the data with a Q value of 2.0. The dashed lines illustrate the predicted effects of an external trivalent, divalent, and monovalent antagonist (see text for details).
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Figure 5. Channel block by internal TEA in the absence and presence of external TEA in Rb+ solutions. Fraction block of Shaker channel current by the indicated internal TEA concentrations in the absence (▪) and presence (○) of 100 mM external TEA. Currents measured at 0 mV. (Dotted line) Fit of to the data in the absence of external TEA with a Kd value of 0.4 mM. (Solid line) Fit of to the data in the presence of 100 mM external TEA with a Kd value of 0.41 mM. Mean values of three to four measurements. All standard error limits were smaller than the symbols. (Inset) Channel block by external TEA in the presence of Rb+ ions. Block of Shaker current at a potential of 0 mV at the indicated concentrations of external TEA. Mean values from three to four measurements. All standard error limits were smaller than the symbols. (Line) Best fit of to the data with the indicated dissociation constant.
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Figure 6. Lack of interaction between external and internal TEA in the presence of Rb+ ions. Ordinate is the apparent dissociation constant for block by internal TEA at the indicated concentrations of external TEA in K+ (▪, from Fig. 3) and Rb+ (○) solutions. Error limits illustrated (when larger than symbol) are the estimated errors from the fitting routine (see methods). Solid line from Fig. 3. Dotted line represents a constant apparent Kd value of 0.4 mM.
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Figure 7. External gallamine block of Shaker K channels. (A) Block of Shaker channels (at 0 mV) by the indicated external gallamine concentration. (Solid line) Fit of to the data with the indicated value of the dissociation constant. Mean and SEM limits from three to five measurements, except as indicated. (Inset) Space-filling model of gallamine. (B) Voltage dependence of channel block by external gallamine. Block of wild-type (○) and T449Y (▪) channels by 0.25 mM external gallamine at the indicated membrane voltages. Lines are fits of to these data with the indicated values of zδ.
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Figure 9. Interaction between external TEA and internal K+ ions. (Inset) Currents (at 0 mV) recorded with 135 mM (left) and 20 mM (right) internal K+ in the absence (larger current) and presence of 25 mM external TEA. (Main figure) Channel block (at 0 mV) at the indicated concentrations of external TEA in 135 mM (•) and 20 mM (□) internal K+. Block by 25 mM TEA was measured (▴) with 20 mM internal K+ and a nominally 0 K+ external solution (see methods).
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Figure 10. Internal Na+ ions and block by external TEA. (Left) Inset shows Shaker currents (at 0 mV) recorded in the absence (larger) and presence of 5 mM external TEA with an internal solution without Na+ ions. The main figure shows the Shaker channel current–voltage relation recorded before (▪), during (○), and after (•) recovery from application of 5 mM external TEA. Internal solution with 20 mM K+ and NMDG (see methods). (Right) Same as left, but data was recorded in the presence of 50 mM internal Na+ ions (and 20 mM K+).
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Figure 11. Simulation of apparent antagonism between external and internal TEA. (A, left ordinate) Rate constant representation (RCR, see methods) for (top) K+ (solid line) and Rb+ (dashed line) permeation of the model pore and for TEA block of the pore (bottom). (Right ordinate) Transition energies for the model using a frequency factor of 3 × 1011 s−1. The barrier heights would be three RT larger if the Eyring frequency factor (5.8 × 1012) were used. See text for details. The external solution is on the left side of the energy profiles. (B) Simulation of the apparent Kd for block by internal TEA as a function of the external TEA concentration with the K+ and Rb+ rate constants from A. Solid lines are fits of with the indicated values of Q for simulations with K+ (▪) and Rb+ (○) rate constants. In these simulations, external and internal ion concentrations were 10 and 150 mM, respectively. The size of the simulated current at 0 mV with the K+ ion rate constants was near 1 pA.
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