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Figure 2. Voltage dependence of deactivation rate. Deactivation time constants were obtained by fitting the tail currents to monoexponential decay functions and were plotted semilogarithmically versus test potential: Kv2.1 (ââfilled circles), 5â²S5/Kv2.1 (open circles), Kv3.1 (ââfilled squares), or 5â²S5/Kv3.1 (open squares). The first 0.3 ms of the tail current was ignored during curve fitting to avoid limitations imposed by the clamp settling time. Each symbol represents average data from 5â10 membrane patches.
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Figure 3. Effects of S5 mutations on single channel tail currents. Single-channel currents in Kv3.1 (A), 5â²S5/Kv3.1 (B), Kv2.1 (C), and 5â²S5/Kv2.1 (D) were recorded from cell-attached patches and the ensemble averages of 300â500 traces (except 5â²S5, 128 traces). The patch membrane was pulsed to +40 mV for 200 ms and then returned to the holding potential of â80 mV. Pipette solution containing 60 mM NaCl and 60 mM KCl, and oocytes were bathed in a depolarizing isotonic KCl solution.
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Figure 4. Cumulative first latency distributions. First latency distributions were obtained at +40 mV by patch-clamp single channel recording in the cell-attached mode. (A) Shows representative records in Kv3.1, 5â²S5/Kv3.1, Kv2.1, and 5â²S5/Kv2.1 channels. Pipette solution was normal Ringer containing 120 mM NaCl, and 2.5 mM KCl. Oocytes were bathed in a depolarizing isotonic KCl solution. (B) Shows the cumulative distribution of first latencies (from the representative experiments in A) at test potential +40 mV: Kv3.1 (open squares), 5â²S5/Kv3.1 (ââfilled squares), Kv2.1 (fâilled circles), and 5â²S5/Kv2.1 (open circles), respectively. All data were obtained from single channel patches except 5â²S5/Kv2.1 (3â8 channels/patch estimated from the maximum number of overlapping open channel events). These data were corrected (Aldrich et al., 1983) by taking the Nth root of the cumulative distribution (expressed as a survivor function, where N = 4, the estimated number of channels/patch in this experiment). As discussed in the text, because of the low Po of this mutant channel, the correction underestimates the slowing effect. (C) Compares the first latency distributions for Kv2.1 and 5â²S5 obtained from the kinetic model described in Fig. 6.
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Figure 6. Numeric simulation of macroscopic currents in Kv2.1 and 5â²S5/Kv2.1 using a sequential model. The effect of the mutation was assumed to be a destabilization of the open state caused by a sevenfold acceleration of the first closing step, O4â C3, and a 50-fold slowing of the last opening step, C3â O4. A and B show simulated tail currents, comparable to the actual records illustrated in Fig. 1. C and D show the predicted gating currents and Po-V relationships for the two channels. In D the smooth curves represent fits to single Boltzmann distributions with midpoint potentials (and slope factors) of â6 (11) and 68 (31.5) mV, respectively in Kv2.1 and 5â²S5/Kv2.1. As indicated by the broken lines a better fit of the 5â²S5/Kv2.1 data was obtained by using the sum of two Boltzmann distributions.
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Figure 5. Voltage dependence of single channel open time in Kv2.1. Mean open times were estimated from the time constant obtained by fitting a single exponential to the open time histogram. Each histogram was constructed from >100 events in a patch at a given test potential, and each plotted point gives the time constant obtained from a single patch. Data were obtained in cell-attached patches exposed extracellularly to high [K+]o solution (either 120 or 60 mM). The solid line was fit according to the equation:\documentclass[10pt]{article}
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\begin{equation*}{\tau}({\mathit{V\hspace{.167em}}})\;=\;{\tau}_{0}\;{\cdot}\;exp({\mathit{z}}\;{\cdot}\;0.0395{\mathit{V\hspace{.167em}}}),\end{equation*}\end{document} where z = equivalent valence, 0.22 eâ and Ï0 = 6.4 ms.
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Figure 7. Gating currents measurements. Nonlinear capacitative currents in Kv3.1 (A), Kv2.1 (B), and 5â²S5/Kv2.1 (C) were recorded in inside-out patches in the absence of permeant cations (NMG and TEA, respectively, were substituted in the internal and external solutions). The traces illustrate typical recordings obtained at test pulse potentials of â60 to +60 mV (20-mV increments) for Kv3.1, or â80 to +40 mV (20-mV increments) for Kv2.1 and 5â²S5/Kv2.1 were applied from a holding potential of â90 mV. Linear components of the leak and capacitative currents were subtracted on-line using a P/â4 protocol (subtraction holding potential = â100 mV). No signal averaging was used. In A and B the first 0.3 ms of the ON and OFF transients are blanked because of imperfect subtraction.
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Figure 8. Mutations in 5â²S5 selectively affect OFF gating current kinetics. A shows a semilogarithmic plot of the time constant of the major component of Ion decay in Kv2.1 (âfilled circles), 5â²S5/Kv2.1 (open squares), and Kv3.1 (open triangles) versus test potential. Time constants were obtained by fitting the decay phase of the on-gating current to a biexponential function and at each potential the time constant of the component that accounted for >90% of the decay was selected. Each symbol represents the average of 5â10 patches for each channel. B compares the time course of Ioff decay in peak-normalized Ioff records obtained from Kv3.1 (solid line), 5â²S5/Kv2.1 (broken line), and Kv2.1 (dotted line) at a return potential of â90 mV after conditioning steps to produce near maximum activation (+60 mV for Kv3.1, 5â²S5/Kv2.1 and +40 mV for Kv2.1). The arrowheads mark the time constants obtained by fitting the traces to monoexponential functions. The average time constants obtained from 5 to 10 membrane patches for each channel was 0.75 ± 0.15, 0.80 ± 0.51, and 8.10 ± 1.39 ms, respectively, in Kv3.1, 5â²S5/Kv2.1, and Kv2.1.
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Figure 9. Voltage dependence of steady-state activation. Gating charge (A) was obtained from the time integral of Ion and was normalized to the maximal gating charge (Qâmax). Normalized charge was plotted versus test potential (Q-V) for Kv2.1 (âfilled circles), 5â²S5/Kv2.1 (open circles), and Kv3.1 (âfilled squares). The conductance-voltage (G-V) relationship (B) was obtained by normalizing ionic conductances at each test potential by the maximal conductance. Normalized conductance was plotted for 5â²S5/Kv3.1 (open squares), Kv2.1 (âfilled circles), Kv3.1 (âfilled squares), and 5â²S5/Kv2.1 (open circles). Both Q-V and G-V data were fit to single Boltzmann functions (smooth curves). Data were pooled from >5 experiments (patches or oocytes, respectively, in A and B). For Kv2.1 the midpoint (V0.5) potentials were 10 and â25 mV, respectively for the G-V and Q-V curves, whereas V0.5 values for Kv3.1 were 20 and 0.9 mV. The midpoint of the Q-V curve for the 5â²S5/Kv2.1 chimera was nearly identical to that of Kv2.1 (V0.5 = â22 mV), but the G-V curve (B; V0.5 = 22 mV) was shifted toward that of Kv3.1. The valence z for the gating charge (obtained from the slope of the Q-V curve) was the same in both Kv2.1 and the 5â²S5/Kv2.1 chimera.
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