The effect of saturating concentrations of lidocaine on ionic and gating currents of the sodium channel. (A) Development of ionic current block in the presence of 10 mM lidocaine in wild-type sodium channels. The ionic currents were elicited by pulsing to −10 mV for 20 ms with a prepulse to −120 mV for 50 ms. Red trace shows the currents obtained before the addition of lidocaine. A family of 100 traces was recorded by pulsing at 10 Hz 5 min after lidocaine application (in black). Ionic current blockade was nearly complete after the first pulse. These recording were obtained with 23 mM sodium in the external solution. (B) Fractional ionic current block of wild-type and mutant sodium channels elicited by repetitively pulsing to −10 mV (10 Hz frequency) after the addition of 10 mM lidocaine. The currents were normalized to the ones obtained before the addition of lidocaine. (C) Effect of 10 mM lidocaine on the steady-state Q-V relationships of wild-type and mutant channels. The dark lines and symbols show the data in control (with TTX and no lidocaine), while the red lines and symbols show the data after lidocaine application. Filled circles represent the mean ± SE of at least four independent experiments, and lines represent the best fits of the averaged data to a Boltzmann function. Measured gating charges at each potential were normalized to the maximum charge determined for each oocyte without lidocaine.

The effect of lidocaine on an alternate site in the S4-DIII of the sodium channel. (A) Effect of 10 mM lidocaine on voltage-dependent fluorescence changes from TMR-labeled S1113C before (top) and after (bottom) lidocaine application. Inset shows the pulse protocol. The y axis in the scale bar represents percent fluorescence change (ΔF/F). The traces were obtained by averaging 10 trials per test potential with an interval of 1 s between pulses. (B) F-V relationships from TMR-labeled S1113C mutants before (black) and after (red) a 10-mM lidocaine application. The averaged fluorescence intensity in the last 3 ms of the voltage pulse was measured (marked with dashed lines in A). These fluorescence intensity changes were normalized to the maximum fluorescence change. Circles represent the means ± SE of five independent experiments, and the line represents the best fit of the averaged data to a single Boltzmann function.

The effect of saturating concentrations of QX-314 on the voltage-dependent fluorescence of labeled sodium channel domains. (A) Time-dependent fluorescence changes from TMR-labeled S216C, S660C, L1115C, and S1436C channels are shown before (left) and after (right) the application of 3 mM QX-314. 10 min after the application of QX-314, oocytes were pulsed repetitively until ionic current block was complete (typically 200 pulses). Traces were obtained by averaging 10 trials per test potential with an interval of 1 s between pulses. The y axis in the scale bar represents percent fluorescence change (ΔF/F). L1115C fluorescence traces were inverted for comparison. (B) Effect of 3 mM QX-314 on the steady-state F-V relationship of S216C, S660C, L1115C, and S1436C channels. Black and red traces represent the data before and after QX-314 application, respectively. Symbols represent the mean ± SE of at least five independent experiments, and lines represent the best fits of the averaged data to a single Boltzmann function. Steady-state fluorescence intensities at each potential were normalized to the maximum changes in fluorescence in each oocyte.

Simulations of F-V relationships in a simplified model system to consider two possible perturbations by the local anesthetic. Simulated F-V curves tracking the voltage sensor movement (R→A transition) in the two subunits of a hypothetical channel (Subunit X [red] and Subunit Y [blue]). (Left) The effect of a perturbation that stabilizes the open state (K₄ = 100) of the subunit Y while the coupling remains intact (n = 10) on the F-V curves of the two subunits (Case I). (Right) The effect of perturbations stabilizing the open state (K₄ = 100) of the subunit Y and disrupting the interdomain interaction (n = 1) on the F-V curves of the two subunits (Case II). Both panels show that the F-V curves before (solid lines) and after perturbation (dashed lines). All parameters are listed in Table IV.

A schematic of the sodium channel and chemical structures of local anesthetics. (A) A schematic diagram of the α-subunit of the sodium channel. Positions of the substituted cysteines at S216 (domain I [DI]), S660 (domain II [DII]), L1115 (domain III [DIII]), and S1436 (domain IV [DIV]) are depicted as colored stars. (B) Chemical structure of lidocaine and QX-314.

The effect of lidocaine on the on the voltage-dependent fluorescence of labeled sodium channel domains. (A) Time-dependent fluorescence changes from TMR-labeled S216C, S660C, L1115C, and S1436C channels before (left) and after (right) 10 mM lidocaine application. Inset shows the pulse protocol. Each trace was obtained by averaging 10 trials with an interval of 1 s between pulses. The y axis in the scale bar represents percent fluorescence change (ΔF/F). L1115C fluorescence traces were inverted for comparison. (B) Steady-state F-V relationship of TMR-labeled S216C, S660C, L1115C, and S1436C channels before (black) and after (red) a 10-mM lidocaine application. Circles represent the means ± SE of at least five independent experiments, whereas the lines represent the best fits of the averaged data to a single Boltzmann function.

The effect of saturating concentrations of QX-314 on ionic currents of the sodium channel. Use-dependent block of outward potassium currents through the sodium channel obtained by repetitively pulsing in the presence of 3 mM QX-314. Red trace represents the current in the absence of QX-314. 10 min after the application of 3 mM QX-314 to the internal solution, ionic currents (black) were recorded by repetitive pulses (10 Hz frequency) to −10 mV for 20 ms after a −120-mV prepulse for 50 ms.

Correlations between use-dependent block of ionic currents and voltage-dependent fluorescence of S4-DIII. (A) A comparison of the development of use-dependent block of ionic current and shift in the voltage-dependent fluorescence of S4-DIII (L1115C). Shift in the voltage dependence of fluorescence manifests as a decrease in fluorescence amplitude when pulsed from −120 to +50 mV. Ionic currents and fluorescence signals were obtained by pulsing repetitively (10 Hz frequency) 10 min after the addition of QX-314. Black symbols represent ionic currents, which were normalized to the current obtained before QX-314 application. Red symbols represent fluorescence intensity, which was normalized to the fluorescence signals before the addition of QX-314. Blue symbols represent fluorescence intensity without QX-314 application obtained in an independent experiment. Large deviations in fluorescence intensities occurred when the capacitor in the amplifier headstage discharged in the middle of a trace. Those datasets were eliminated from the final plots. (B) A comparison of use-dependent block in the L1115C and L1115C/F1579A double-mutant sodium channel. Use-dependent block of outward potassium currents was elicited by repetitively pulsing in the presence of 3 mM QX-314. Black symbols represent the current in the absence of QX-314 in the L1115C/F1579A channel. 10 min after the application of 3 mM QX-314 to the internal solution, ionic currents of L1115C/F1579A channel (red symbols) were recorded by repetitive pulses (10 Hz frequency) to −10 mV for 20 ms after a −120-mV prepulse for 50 ms. Blue symbols represent the current in the presence of QX-314 in the L1115C channel, obtained with the same protocol pulse. (C) Effect of QX-314 on the voltage-dependent fluorescence of the L1115C/F1579A mutant channel. F-V relationships from TMR-labeled L1115C/F1579A mutants before (black) and after (red) a 3-mM QX-314 application (after 100 depolarizing pulses at 10 Hz frequency). These fluorescence intensity changes were normalized to the maximum fluorescence change. Circles represent the means ± SE of four independent experiments, and the line represents the best fit of the averaged data to a single Boltzmann function.

A schematic depicting a preliminary model of sodium channel modulation by local anesthetics. Local anesthetic binding to the open and inactivated state primarily stabilizes the pore segments of domain III and to some extent of domain IV in the open state. Because the movement of pore helices (blue circles) is coupled to the voltage sensor (pink ellipsoids), local anesthetic binding also stabilizes the voltage sensors of domains III and IV in the activated state. The interdomain interaction is mediated by the residues near the inner helix bundle crossing. In this model, as in the potassium channel, these residues interact in the closed state of the channel. This interaction is disrupted if the local anesthetic acts as a wedge and prevents the S6 segments of domains III and IV from fully closing.