Fig. 5. Block of the inactivation-deficient mutant Nav1.4-WCW by buprenorphine. (A ) Representative current traces of Nav1.4-WCW in the presence of increasing concentrations of buprenorphine (V^h= −140 mV). Currents were activated by 50 ms-long test pulses from −140 mV to 50 mV in intervals of 30 s. (B ) Concentration-dependent block of Nav1.4-WCW by buprenorphine. After that, the block by each concentration had reached steady-state after 90–160 s, open-channel (filled symbols ) block was determined at the end of the 50 ms-long persistent current, and block of resting (open symbols ) channels at the peak current amplitude. Currents were normalized with respect to the amplitude in control solution and plotted against the concentration of buprenorphine. Solid lines  are fits of the data with the Hill equation. (C ) The decaying phase of the persistent Na⁺currents was fitted with a single exponential function and the resulting time constants were inverted and plotted against the concentrations of buprenorphine. The plot was fitted with a linear regression (y = k^off + k^on  x), which revealed an on-rate (k^on ) of 10.5 μM^{−1 −s}and an off-rate (k^off ) of 33.7^−s. A K^D -value of 3.2 μM was calculated by the equation K^D = k^off /k^on . Bupr. = buprenorphine.