Characterization of Compound-Specific, Concentration-Independent Biophysical Properties of Sodium Channel Inhibitor Mechanism of Action Using Automated Patch-Clamp Electrophysiology

Figures

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  FIGURE 1. The sequence of analysis to obtain effective inhibitor potency plots. The process is illustrated in an example of an experiment, where four concentrations of riluzole were applied to a cell ensemble (10, 30, 100, and 300 µM). (A) Schematic picture of the voltage protocol, which was repeated at 1 Hz frequency. The color of pulses match the color of amplitude plots in panel (B), the color of concentration-inhibition curves in panel (C), left column, and the color of open circles in EIP plots in panel (D). The number of pulses, and the three main sections of the protocol: SDO, RFI, and SSI, are indicated. (B) Left column: Amplitude plots throughout the four riluzole applications, for the three main sections. Grid size: 1 nA (vertical), 100 s (horizontal). Arrowheads show the time points, from where data were collected to construct plots in the right column. Right column: SDO, RFI, and SSI plots for four different concentrations of riluzole, and four sets of control data before drug application. The color of curves match the color of arrowheads in the left column. (C) Middle column: 3D plots of drug/control ratios for each of the four concentrations (front axis), and for each of the…

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  FIGURE 2. Summary of EIP plots for the four compounds, and the significance of micro-dynamics. (A) The micro-dynamics (EIP plots for SDO and RFI), and voltage-dependence of EIP are compared for n = 6 measurements for each of the four compounds. Thin lines show data from individual measurements, thick dashed lines show the geometric mean. For SSI data the calculated KR and KI values are also shown on the right side of the figures (thin crosses for individual measurements, and thick crosses for the geometric mean). (B) One example of how micro-dynamics is translated into frequency-dependent inhibition. The activity pattern of a simulated neuron is shown in the background, SDO-EIP and RFI-EIP plots are shown under the activity pattern, these are identical to the ones shown in dashed lines in panel (A), upper and middle row, but on a linear time scale. Horizontal grid: 10 ms. SDO-EIP plots were aligned with the upstroke of the first action potential because state-dependent onset is triggered by depolarization. RFI-EIP plots were aligned with the repolarization because hyperpolarization triggers recovery from inhibition. The intensity of red coloring illustrates how EIP is expected to change…

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  FIGURE 3. Construction of EIP plots for lidocaine. The figure illustrates how EIP plots are constructed on the example of a single measurement of the effect of three concentrations of lidocaine (100, 300, and 1,000 µM). (A) Peak amplitudes of currents evoked by the 17-pulse protocol were grouped into three sections: SDO (upper row), RFI (middle row), and SSI (lower row). Grid size: 5 nA (vertical), 100 s (horizontal). (B) Amplitudes in control sections and during lidocaine perfusion were plotted against conditioning pulse duration (SDO), interpulse interval duration (RFI), and membrane potential (SSI). (C) Drug/control ratios were calculated for all three sections. (D) The same drug/control ratios were used to construct concentration-inhibition plots, as it was explained in Figure 1 (thick lines), which were then fitted by the Hill equation (thin lines). (E) The resulting IC50 (EIP) values are plotted against the same abscissae as in the (B) and (C) columns: conditioning pulse duration (SDO), interpulse interval duration (RFI), and membrane potential (SSI).

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  FIGURE 4. Construction of EIP plots for benzocaine. The figure illustrates how EIP plots are constructed on the example of a single measurement of the effect of three concentrations of benzocaine (100, 300, and 1,000 µM). (A) Peak amplitudes of currents evoked by the 17-pulse protocol were grouped into three sections: SDO (upper row), RFI (middle row), and SSI (lower row). Grid size: 5 nA (vertical), 100 s (horizontal). (B) Amplitudes in control sections and during benzocaine perfusion were plotted against conditioning pulse duration (SDO), interpulse duration (RFI), and membrane potential (SSI). (C) Drug/control ratios were calculated for all three sections. (D) The same drug/control ratios were used to construct concentration-inhibition plots (thick lines), which were then fitted by the Hill equation (thin lines). (E) The resulting IC50 (EIP) values are plotted against the same abscissae as in the (B) and (C) columns.

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  FIGURE 5. Construction of EIP plots for bupivacaine. The figure illustrates how EIP plots are constructed on the example of a single measurement of the effect of three concentrations of bupivacaine (10, 30, and 100 µM). (A) Peak amplitudes of currents evoked by the 17-pulse protocol were grouped into three sections: SDO (upper row), RFI (middle row), and SSI (lower row). Grid size: 5 nA (vertical), 100 s (horizontal). (B) Amplitudes in control sections and during bupivacaine perfusion were plotted against conditioning pulse duration (SDO), interpulse duration (RFI), and membrane potential (SSI). (C) Drug/control ratios were calculated for all three sections. (D) The same drug/control ratios were used to construct concentration-inhibition plots (thick lines), which were then fitted by the Hill equation (thin lines). (E) The resulting IC50 (EIP) values are plotted against the same abscissae as in the (B) and (C) columns.

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  FIGURE 6. Construction of EIP plots for TTX. The figure illustrates how EIP plots are constructed on the example of a single measurement of the effect of three concentrations of TTX (30, 100, and 300 nM). (A) Peak amplitudes of currents evoked by the 17-pulse protocol were grouped into three sections: SDO (upper row), RFI (middle row), and SSI (lower row). Grid size: 4 nA (vertical), 100 s (horizontal). (B) Amplitudes in control sections and during TTX perfusion were plotted against conditioning pulse duration (SDO), interpulse duration (RFI), and membrane potential (SSI). (C) Drug/control ratios were calculated for all three sections. (D) The same drug/control ratios were used to construct concentration-inhibition plots (thick lines), which were then fitted by the Hill equation (thin lines). (E) The resulting IC50 (EIP) values are plotted against the same abscissae as in the (B) and (C) columns. Data from the other five cell ensembles are shown in thin black lines. For the SSI plots the calculated KR and KI values are also shown on the right side of the figure (thin crosses for individual measurements, and thick crosses for the geometric mean).