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Figure 2. Measurement of the rate of MTS-ET modification of F1304C in the presence of lidocaine. The experimental protocol shown in A consists of a series of 20-ms exposures of excised inside-out patches to 8 μM MTS-ET with test pulses between each exposure. 1.0 mM lidocaine was present at all times. In B, selected traces from the modification experiment described in A are superimposed. To determine the degree of modification after each trace, the value of the macroscopic current between 3 and 3.5 ms after depolarization was averaged. Rates were determined by fitting the degree of modification of each trace as a function of cumulative exposure time with a monoexponential containing a maximum value after complete modification, a nonzero initial value before modification, and a time constant as free parameters. C shows these averages, normalized to the difference between the maximum value and the initial value, plotted against cumulative exposure time, with the normalized curve fit superimposed.
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Figure 3. Effect of tonic block on accessibility of site 1304. Tonic block of F1304C (â¢) was evaluated by dividing the peak current obtained from infrequent depolarizations to â20 from â120 mV in the presence of 0.1, 0.5, 1.0, 2.0, 4.0, or 8.0 mM lidocaine by the peak current in the absence of drug (n = 4 for each point). These data were fit to a binding curve with a hill coefficient of 1.0, giving a Kd of 1.9 mM. Also shown in the graph is the modification rate of F1304C with 8 μM MTS-ET at â120 mV in the presence of 0.5 (n = 4), 1.0 (n = 10), 2.0 (n = 8), or 4.0 (n = 10) mM lidocaine divided by the rate measured without lidocaine present (n = 4) (â). The rates were measured using the protocol shown in Fig. 2 A. A fit to the modification rate data gives a Ka of 6.6 mM.
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Figure 4. Effect of lidocaine on the steady state availability curve of F1304C. hâ curves were recorded with 200-ms prepulses, followed by a test pulse to â20 mV. For each patch, the hâ curve was measured in the absence and presence of 1.0 mM lidocaine (n = 5) in rapid succession. In other experiments without lidocaine, we did not observe significant left shifts in gating after patch excision on the time scale of these experiments. Data from each curve was fit with a Boltzmann with maximal value (Imax), half-maximal voltage (V1/2), slope (k), and nonzero plateau (c) as free parameters. The graph shows data normalized to the maximum value for curves measured in the absence of lidocaine, and then averaged across trials. Without lidocaine, V1/2 = â78.9 ± 1.9 mV, k = 6.2 ± 0.7 mV, and c = 0.11 ± 0.02. In 1.0 mM lidocaine, V1/2 = â89.7 ± 1.8 mV, k = 7.1 ± 0.4 mV, c = 0.08 ± 0.02, and Imax in 1.0 mM lidocaine was 78 ± 6% of Imax in the absence of lidocaine. This represents a hyperpolarizing shift in the apparent hâ curve by 10.8 ± 2.6 mV.
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Figure 5. Effect of lidocaine on the voltage dependence of site 1304 accessibility. The rate of MTS-ET modification of F1304C was measured at a variety of voltages, as shown in A. Excised, inside-out patches underwent a series of 300-ms depolarizations: 200 ms to achieve steady state fast inactivation, followed by 50 ms for exposure to 8.0 μM MTS-ET, and a final 50-ms period after exposure to insure complete washout of MTS-ET before repolarization. After each 300-ms depolarization, the patch was maintained at â120 mV for enough time to insure complete recovery (2â8 s, depending on the conditioning voltage) before assaying macroscopic current with a test pulse to â20 mV. Modification rates are shown in B when the experiments were conducted in the absence (â¢) and presence (â) of 1.0 mM lidocaine. (On average, n = 6, n = at least 3 for each point.)
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Figure 6. Lidocaine slows the repriming of Na+ channels after brief depolarizations. A two-pulse recovery protocol was used to assess the rate of repriming of F1304C channels after a 20-ms depolarization to 0 mV. The peak current measured during the test pulse was divided by the peak current measured during the conditioning pulse, and plotted as a function of the recovery period. In the absence of lidocaine, the current recovered almost completely within 10 ms, while in the presence of 1.0 mM lidocaine, the time constant of recovery is roughly 100-fold slower. The shaded area indicates the duration of MTS-ET exposure relative to Na+ channel repriming for the protocol shown in Fig. 7 A.
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Figure 7. Lidocaine does not slow the return of site 1304 accessibility after brief depolarizations. In the protocol diagrammed in A, 20-ms exposures to 8 μM MTS-ET were timed to occur 7.5 ms after 20-ms depolarizations to 0 mV. The macroscopic current was assayed between each exposure, and modification rates were measured as shown in Fig. 2. The 7.5 ms reflects the experimentally measured delay between the voltage command to the piezoelectric stack (which occurred exactly at the end of the conditioning pulse) and the commencement of solution exchange (for details, see Vedantham and Cannon, 1998). B shows the average of several modification experiments conducted with or without 1.0 mM lidocaine. The solid lines are exponentials whose time constants are the mean values of the time constants obtained from fits to data from individual experiments. The dashed line reflects the curve that would be expected if the accessibility of site 1304 paralleled the degree of Na+ channel availability in 1.0 mM lidocaine during the exposure. The fraction of current recovered in the presence of 1.0 mM lidocaine after 8-, 10-, and 20-ms recovery times was averaged (corresponding to the shaded area in Fig. 6), giving 0.2962. An accessibility of 0.30 predicts a rate of 2.1 sâ1 at 8 μM MTS-ET (predicted rate = 0.30 à (Rmax â Rmin) + Rmin), or 0.26 μmolâ1 sâ1.
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Figure 8. A model for lidocaine action. In A, a section of the activation pathway for sodium channels is shown, in which each noninactivated state (Cn) is connected to an inactivated state (In). The length of the vertical arrows between inactivated and noninactivated states indicate the degree to which the equilibrium favors inactivated channels: the longer the arrow, the greater the fraction of inactivated channels. Thus, depolarization causes rightward movement and increases the fraction of inactivated channels. In B, a set of states is added to the model that incorporate lidocaine binding. The arrows that move between unbound (Cn or In) and lidocaine-bound states (CnL or InL) indicate the degree to which the equilibrium favors lidocaine binding: the longer the arrow, the greater the fraction of lidocaine-bound channels. Thus, as for the case of inactivation, depolarization favors lidocaine block as well as inactivation. The model implies that addition of lidocaine causes a rightward shift in the distribution of channels owing to coupling between activation and lidocaine binding, while the vertical equilibria experience no such coupling (explaining why recovery from fast inactivation is not altered in lidocaine-bound channels). The rightward movement of the distribution will tend to increase the fraction of channels that are inactivated, thereby causing a shift in the hâ curve. The slowing of repriming is a result of the slow dissociation of lidocaine from the CnL states.
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