Figure 3.

Effects of 2,6-dimethylphenol on fast inactivated channels assessed by shifts in the steady-state availability curve. (A (upper panel)) Steady-state availability curves assessed by a two-pulse protocol in the absence (control, circles) and presence of 500 μM 2,6-dimethylphenol (triangles) in the skeletal muscle isoform. Each symbol represents the mean fractional current (n=3 for each concentration) elicited by a 4 ms test pulse to 0 mV, following a 50 ms inactivating prepulse from −150 mV to the indicated prepulse potential. Currents were normalized to maximum value (in each series at −150 mV prepotential). Solid lines represent the best Boltzmann fit (Eq. 2) to the data yielding the membrane potential at half-maximum channel availability (V_0.5) and the slope factor k. Error bars are standard deviations. In the presence of 500 μM 2,6-dimethylphenol, currents were normalized either to the maximum in the presence of the drug (filled symbols) or to the maximum in the controls (empty symbols). Vertical arrows illustrate the increase in the peak current suppression induced by 500 μM 2,6-dimethylphenol at more depolarized holding potentials versus hyperpolarized holding potentials. This reduction in channel availability at depolarized prepotentials resulted in a voltage shift in the midpoints of the availability curve (ΔV_0.5), indicated by the horizontal arrows. (B,C (lower panel)) Concentration-dependence of drug-induced negative shifts in the midpoints (ΔV_0.5 [mV], mean±s.d.) of the steady-state availability plots relative to the starting values in both isoforms. As the diluent ethanol applied at a concentration corresponding to a drug concentration of 1000 μM induced a small shift of +5 mV, i.e. into the opposite direction in comparison with the 2,6-dimethylphenol-induced shifts, the data were not corrected for the ethanol shifts. The solid line is the least-squares fit to Equation 3. The IC₅₀ at −150 mV holding potential entered as fit parameter was 481 μM for the neuronal and 558 μM for the skeletal muscle isoform, respectively, the K_d values estimated for the inactivated sodium channels were 28 μM for the neuronal and 25 μM for the skeletal muscle isoform, respectively.