FIGURE 10. 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.