Chemical formulae of the drugs and inhibition of neuronal Na⁺ currents by diclofenac. (A) Chemical formulae of carbamazepine and diclofenac. Both drugs contain the diphenyl structural motif (see Table I for more detailed comparison of the structural features). (B) Na⁺ currents in control, 10 μM, or 30 μM diclofenac were recorded in a neuron. The cell was held at −120 mV and then stepped to 0 mV to elicit Na⁺ currents. 10–30 μM diclofenac produces only equivocal inhibition of the Na⁺ current. The dotted line indicates the zero current level. (C) The same experiment was repeated in the same cell as in B except that the holding potential was changed to −70 mV. The control current is much smaller than that in B but is scaled to the same size for a better comparison (note the difference in the vertical scale bars). In contrast to the findings in part B, 10–30 μM diclofenac has a pronounced and dose-dependent inhibitory effect on the Na⁺ current. The dotted line indicates the zero current level.

Shift of the inactivation curve of neuronal Na⁺ channels by diclofenac. (A) The inactivation curves are documented in the absence or presence of different concentrations of diclofenac. The neuron was held at −120 mV and stepped to the indicated inactivating prepulses for 9 s. The neuronal Na⁺ currents were then measured at 0 mV right after the 9-s prepulses. The available fraction is defined as the normalized current relative to the current elicited after a prepulse of −120 mV. The solid lines are the fits to each set of data with a Boltzmann function 1/(1 + exp((V − Vh)/k)), where the Vh values (in mV) are −73.7, −74.6, −81.4, −87.5, and −95.2 in 0 (control), 10, 30, 100, and 300 μM diclofenac, respectively. Because the slope of the fitting curves in different diclofenac concentrations always stay close to the slope in control, the above Vh values are obtained from the fits with a fixed k value of 5.4 (the mean of the k values in control). (B) The mean value of the shift of the inactivation curve by different concentrations of diclofenac. The shift (ΔV) is determined by the difference between the Vh values in control and in different concentrations of diclofenac. The mean ΔV values (in mV) are 3.7 ± 2.1 (n = 3), 5.8 ± 1.7 (n = 5), 12.1 ± 1.2 (n = 6), and 19.3 ± 0.9 (n = 12) for 10, 30, 100, and 300 μM diclofenac, respectively. (C) The mean ΔV values in B and a k value of 5.4 are used to calculate exp(ΔV/k), which is plotted against diclofenac concentrations to determine the dissociation constant of diclofenac binding to the inactivated channel. The solid line is the fit of the form (see Eq. 1 in the text): exp(ΔV/k) = [1 + (D/6.8)]/[1 + (D/880)], where a K_R value of 880 μM is taken from Fig. 7 D, and D is the diclofenac concentration in μM.

The effect of diclofenac on macroscopic neuronal Na⁺ currents. (A) The neuron was held at −120 mV and stepped to 0 mV to elicit Na⁺ currents. The external solution contains 150 mM NaCl, and the internal solution contains 150 mM CsCl (for details of the composition of the solutions, see materials and methods). 300 μM to 1 mM diclofenac mildly and dose dependently reduces the current peak as well as increases the time to peak without apparent acceleration of current decay. The dotted line indicates the zero current level. (B) The currents in A are superimposed and scaled to the same current size, showing that diclofenac shifts current peak rightwardly as well as slows current decay in a dose-dependent fashion. (C) The neuron was exposed to repeated pulses, which are stepped from −120 mV to 0 mV for 12 ms with an interpulse interval of 3 s to elicit a series of Na⁺ currents. The black horizontal bars indicate the external solutions (Tyrode's solution or Tyrode's solution with different concentrations of diclofenac) in which the neuron stayed. The time constant from a monoexponential fit to the macroscopic Na⁺ current decay is lengthened by diclofenac in a dose-dependent fashion. The diclofenac effect can be readily “washed out” when the external solution is switched back to the control Tyrode's solution. (D) Opposite effects of diclofenac and carbamazepine on the current decay rate. The decay rate is determined by the inverse of the time constant obtained from a monoexponential fit to the decay phase of the current. The relative decay rate is defined by the ratio between the decay rates in the presence of different drugs and in control, and are 0.86 ± 0.02 (n = 9, drug/control), 0.78 ± 0.01 (n = 11), 1.08 ± 0.02 (n = 6), 1.23 ± 0.03 (n = 9) for 300 μM diclofenac, 1 mM diclofenac, 100 μM carbamazepine, and 300 μM carbamazepine, respectively. The dashed line indicates the relative decay rate of 1.

To verify the effect of diclofenac on resting and open Na⁺ channels, we examined diclofenac action on the inactivation-deficient mutants (i.e., F1489Q mutant or F1489Q/F1651A double mutant RIIA Na⁺ channels expressed in oocytes). The F1489Q mutation is located at the intracellular loop connecting domains III and IV and presumably weakens binding of the inactivation “lid” to the open channel (West et al., 1992). Fig. 5 A shows that 300 μM and 1 mM diclofenac only mildly increase the peak of the inactivation-deficient F1489Q current elicited by a step depolarization to 0 mV from a holding potential of −120 mV. However, if the current is elicited by a less depolarized potential such as −20 mV, the peak current enhancement effect of diclofenac becomes much more evident (Fig. 5 B), making the currents as large as ∼1.5-fold of the original ones (Fig. 5 C). The following simplified Scheme 2 illustrates gating of the inactivation-deficient mutant channel. The inactivated state is disregarded in this scheme, where C, O, CD, and OD refer to the closed, open, drug-bound closed, and drug-bound open states, respectively, and V denotes the voltage-dependent gating processes.

Diclofenac enhancement and carbamazepine inhibition of the F1489Q/F1651A double mutant current. The experimental protocol is the same as that in Fig. 5. (A) The inactivation phase of the macroscopic current in the double mutant is more completely abolished (i.e., the current decays even slower in the double mutant) than in the F1489Q mutant. Also, diclofenac shows more enhancement of the peak current evoked by a pulse at 0 mV in the double mutant than in the F1489Q mutant (Fig. 5 A). In sharp contrast, carbamazepine evidently reduces the double mutant current. The dotted line indicates the zero current level. (B) Diclofenac has an even stronger enhancement effect on the double mutant current evoked by a pulse at −20 mV in the same oocyte as that in A. (C) Another oocyte expressing the F1489Q/F1651A double mutant channel was exposed to repeated pulses to −20 mV for 100 ms to elicit a series of mutant Na⁺ currents (sweeps) with a 4-s interpulse interval at −120 mV. The black horizontal bars indicate the external solutions (ND-96 only or ND-96 with different concentrations of diclofenac) in which the oocyte stayed. The double mutant current peak is greatly enhanced by diclofenac in a concentration-dependent fashion. The diclofenac effect can be readily “washed out” when the external solution is switched back to the control ND-96 solution. (D) Estimation of the affinity of diclofenac binding to the resting Na⁺ channel by analysis of the increase of the F1489Q/F1651A double mutant current. The F1489Q/F1651A double mutant peak current (the maximal inward current) elicited by a −20 mV pulse in different concentrations of diclofenac is normalized to that in the control (no diclofenac) condition to obtain the relative peak current. The enhancement effect on the currents is diclofenac concentration dependent and can be well described by a one-to-one binding curve of the form: relative peak current = (1 + 2.7*D/880)/(1 + D/880), where D is the concentration of diclofenac in μM (see text for more details).

Comparison of diclofenac enhancement of the F1489Q mutant current in external solutions containing high and low concentrations of Na⁺. (A) The oocyte expressing the F1489Q mutant channel was held at −120 mV in the ND-96 solution (containing 96 mM Na⁺) or in the ND-22 solution (containing only 22 mM Na⁺, see materials and methods) and stepped to −20 mV for 100 ms to elicit the Na⁺ currents. 1 mM diclofenac significantly enhances the F1489Q mutant current measured in the ND-96 solution, but has only a negligible effect on the current measured in the ND-22 solution. The dotted line indicates the zero current level. (B) The relative peak current in the ND-96 and in the ND-22 solutions is determined by the same way as that in Fig. 5 C. The dashed line indicates the relative peak current of 1. Diclofenac enhancement of the F1489Q mutant current is evidently weaker with the lower concentration of external Na⁺.

A schematic model describing one of the possible reasons why carbamazepine and diclofenac may bind to the same receptor area in the external pore mouth of the Na⁺ channel but produce different pore-blocking effects in the presence or absence of 150 mM external Na⁺. The diphenyl group in each drug is shown by two black squares, which in general have similar spatial orientation in these two drugs. However, the two phenyl groups are rigidly tied in carbamazepine but are more flexible (e.g., with more versatile torsion angles) in diclofenac (Table I). The shaded areas in the channel protein mark the location of the binding ligands for the diphenyl group. In the absence of external Na⁺, the two drugs both bind to the gray shaded areas in the external pore mouth to compromise the narrow part of the ion conduction pathway and block ion flow (left, 0 external Na⁺). When Na⁺ occupies the modulatory Na⁺ binding site in the external vestibule in 150 mM external Na⁺, local conformational changes happen with movement or reorientation of some potential binding ligands for the diphenyl group (right). A wider shaded area suitable for binding of the diphenyl group is thus formed in the external vestibule slightly farther away from the narrow part of the pore. With its more flexible diphenyl group, diclofenac can readily bind to this wider shaded area. The narrow part of the pore in the diclofenac-bound channel thus remains “patent” and permeable to Na⁺. In contrast, carbamazepine cannot effectively bind to the newly formed alternative receptive area because of its rigidly tied diphenyl group in the dibenzazepine tricyclic structure. Carbamazepine would therefore bind to the same area as that in the absence of Na⁺ and still blocks the pore. The modulatory Na⁺ binding site is probably located in the widened part of the external vestibule or even the external surface of the channel, so that its occupancy is correlated with only external but not internal Na⁺. On the other hand, the diclofenac binding site should be located at the junction of the narrow and widened parts of the external pore mouth (presumably the junction of the external vestibule and the single-file region of the pore), so that diclofenac could behave as an opportunistic pore blocker modulated by external Na⁺.

We characterize the voltage-dependent effect of diclofenac in more detail by examination of the inactivation curve, which describes the voltage-dependent steady-state distribution of the Na⁺ channel between the resting (R) and the inactivated (I) states (Scheme 1;Fig. 2).

Shift of the inactivation curve of neuronal Na⁺ channels by carbamazepine and diclofenac. (A) Simplified diagrams describing the possible action of the drugs on different Na⁺ channel gating states. R and I denote the resting and the inactivated states of the steady-state channel, respectively. D1 and D2 denote two different drugs. V denotes that the distribution between R and I states is voltage dependent. The one-site model is illustrated in the upper panel, where D1 and D2 share the same receptor site and thus the inactivated channel can only be bound with one drug molecule at a time. For the sake of simplicity, we did not show a scheme illustrating that the two different drugs bind to two different inactivated conformations allosterically incompatible with each other. However, the basic quantitative analysis (e.g., Eq. 3 in the text) would be the same whether binding of one drug precludes binding of the other directly or allosterically. The lower panel represents the gating scheme of the two-site model, where the two drugs have separate and unrelated binding sites and thus could doubly occupy the inactivated channel. (B) The experimental protocol and the fitting procedures (with a fixed k value of 5.4) are similar to the case in Fig. 2 A. The Vh values of the inactivation curves are −48.5, −64.7, −69.2, and −67.3 mV for control, 300 μM carbamazepine, 300 μM diclofenac, and 150 μM carbamazepine plus 150 μM diclofenac, respectively. (C) Shift of the inactivation curve in the presence of diclofenac and/or carbamazepine. The shift (ΔV) is calculated by the difference between the Vh values in control and in the presence of different drugs. The mean values of ΔV (in mV) are 19.3 ± 0.9 (n = 12), 13.9 ± 1.4 (n = 4), and 15.9 ± 1.5 (n = 9) for 300 μM diclofenac, 300 μM carbamazepine, and 150 μM carbamazepine plus 150 μM diclofenac, respectively. The data of 300 μM diclofenac are from the same cells measured in Fig. 2 B. The predicted shift in the presence of both drugs by the one-site model (the striped bar) is much closer to the experimental data (the black bar) than the prediction by the two-site model (the white bar) (see text for more details).

Diclofenac enhancement of the inactivation-deficient F1489Q mutant currents. (A) The oocyte expressing the F1489Q mutant channel was held at −120 mV and stepped to 0 mV to elicit currents. The control Na⁺ current recorded in ND-96 solution shows biphasic current decay (an initial fast phase followed by a slow phase of decay). 300 μM to 1 mM diclofenac only minimally or equivocally increases the peak current. The dotted line indicates the zero current level. (B) The same oocyte as that in A was stepped to −20 mV instead of 0 mV to elicit Na⁺ currents. The activation kinetics are consequently slower, but the enhancement effects of 300 μM and 1 mM diclofenac are stronger than that in A. (C) The F1489Q peak currents elicited by 0 (black bars) or −20 mV (gray bars) pulses in different concentrations of diclofenac are normalized to that in the control (no diclofenac) condition to obtain the relative peak current. The dashed line indicates the relative peak current of 1. The enhancement effect of diclofenac on peak currents is dependent on both the test pulse voltage and the diclofenac concentration.

Shift of the normalized conductance–voltage curve of the F1489Q mutant channel by diclofenac. (A) The normalized conductance–voltage curve of the F1489Q mutant channel in different concentrations of diclofenac. The oocyte expressing F1489Q mutant Na⁺ channels was held at −120 mV and stepped to different test voltages to elicit Na⁺ currents. The maximal conductance (G_max) is given by the slope of the linear regression fit to the data points between +10 and +40 mV in the peak current–voltage plot, and the reversal potential (V_rev, in mV) is estimated by extrapolating the foregoing linear regression to the zero current level. The normalized conductance is then determined by I_peak/((V − V_rev)*G_max), where I_peak and V are the peak current amplitude and the test voltage (in mV), respectively. The normalized conductance is plotted against the test voltage, and then fitted with a Boltzmann function of the form: 1/(1 + exp ((V − Vh)/4.8)), where the Vh values (in mV) are −13.7, −16.9, −17.8, and −19 in 0 (control), 100, 300, and 1000 μM diclofenac, respectively. Because the slope of the fitting curves in diclofenac always stays close to the slope in control, the Vh values are obtained from the fits with a fixed k value of 4.8 (the mean of the k values in control) to facilitate the following quantitative analysis. (B) Mean shift of the normalized conductance–voltage curve by different concentrations of diclofenac. The shift (ΔV) is determined by the difference between the Vh values in control and in different concentrations of diclofenac. The mean ΔV values (in mV) are 3.8 ± 1.1 (n = 3), 5.6 ± 1.1 (n = 3), and 8.4 ± 0.7 (n = 4) for 100, 300, and 1000 μM diclofenac, respectively. (C) The mean ΔV values in B and a k value of 4.8 are used to calculate exp(ΔV/k), which is plotted against diclofenac concentrations to determine the dissociation constant between diclofenac and the activated channel. The solid line is the fit of the form (see Eq. 5 in the text) exp(ΔV/k) = [1 + (D/88)]/[1 + (D/880)], where D is the diclofenac concentration in μM, and a K_R value of 880 μM is taken from Fig. 7 D.

Diclofenac inhibition of outward neuronal Na⁺ currents in 0 mM external and 150 mM internal Na⁺. (A) The cell was held at −120 mV and stepped to 0 mV to elicit Na⁺ currents. The external solution contains no Na⁺ ions but 150 mM Cs⁺, and the internal solution contains 150 mM Na⁺ (for details of the composition of the solutions, see materials and methods). 1 mM diclofenac has a stronger inhibitory effect on the current than 300 μM carbamazepine, and both drugs accelerate current decay (see B). The dotted line indicates the zero current level. (B) The currents in A are superimposed and scaled to the same current size. It is evident that 300 μM carbamazepine accelerates current decay, and 1 mM diclofenac is even stronger in doing so. (C) In 0–1000 μM diclofenac, the decay phase of the macroscopic outward Na⁺ currents in the absence of external Na⁺ is fitted with a monoexponential function. The inverse of the time constant in control is subtracted from the inverse of the time constant in diclofenac to give the macroscopic drug binding rate to the open channel. The line is a linear regression fit to the macroscopic binding rates in different concentrations of diclofenac with a slope of 4.1 × 10⁵ M⁻¹s⁻¹. (D) Comparison of the effects of diclofenac and carbamazepine on the macroscopic current decay rate in different ionic conditions. The neurons were held at −120 mV and stepped to 0 mV to elicit Na⁺ currents. The relative current decay rate is determined in the same way as that described in Fig. 4 D. The dashed line indicates the relative decay rate of 1. Diclofenac retards current decay in the presence of 150 mM external and 0 mM internal Na⁺ solutions but accelerates current decay in the presence of 0 mM external and 150 mM internal Na⁺ solutions, both in a diclofenac concentration–dependent fashion. On the other hand, carbamazepine always accelerates current decay irrespective of the Na⁺ concentration in the solutions.

Lack of significant effect of internal Na⁺ on diclofenac action and an estimate of the binding affinity of diclofenac to the resting neuronal Na⁺ channel. (A) Diclofenac action on macroscopic neuronal Na⁺ currents in the presence of symmetrical 150 mM Na⁺ (150 mM Na⁺ internal solution and Tyrode's solution containing 150 mM Na⁺ extracellularly). The neuron was held at −120 mV and stepped to −10 mV to elicit inward Na⁺ currents. The dotted line indicates the zero current level. The currents in the top panel are rescaled to the same peak level in the bottom panel. 1 mM diclofenac slightly but significantly reduces current peak and increases the time to current peak. However, it does not accelerate the macroscopic current decay. (B) Effect of diclofenac on the time to current peak in different solutions. The current is elicited by a test pulse of 0 or −10 mV from a holding potential of −120 mV in 150 mM external and 0 or 150 mM internal Na⁺ (Ex150/In0 and Ex150/In150, respectively), or in 0 mM external and 150 mM internal Na⁺ (Ex0/In150). The test pulse is given beside each bar. In Ex150/In150 solution, the current cannot be measured at 0 mV and thus only that measured at −10 mV is shown. The time to peak in the presence of 1 mM diclofenac is normalized to the time to peak in control to give the relative time to peak. The dashed line indicates the relative time to peak of 1. When the external solution contains 150 mM external Na⁺, 1 mM diclofenac always increases the time to peak whether the internal solution contains 0 or 150 mM Na⁺. In contrast, the time to peak is always unchanged by 1 mM diclofenac if the external solution contains no Na⁺ (0 mM external Na⁺). (C) Estimation of the binding affinity of diclofenac to the resting Na⁺ channel in the absence of external Na⁺. The neuronal Na⁺ current is evoked by a pulse to 0 mV from a holding potential of −120 mV in 0 mM external and 150 mM internal Na⁺. The peak current in the presence of diclofenac is subtracted from the peak current in control and then normalized to the control current to give the relative reduction of peak current, which is plotted against the diclofenac concentration. The line is a fit to the data of the form: relative reduction of peak current = (D/1483)/[1 + (D/1483)], where D denotes the concentration of diclofenac in μM.