Figure 5

Similar voltage dependence of the intracellular Mg²⁺ unblocking kinetics in the WT, E224G and M183W mutant channels.

(a) The two–pulse protocol is basically similar to that in Fig. 3b, except that the voltage across the patch membrane was first depolarized from the holding potential −100 mV to +100mV for ~50 ms and then stepped to different test voltages between −100 and 0 mV for ~300 ms in 10 mV increment for M183W single and E224G/M183W double mutant channels. Representative current traces were recorded in control or in internal 100 μM Mg²⁺. The slow tail is present only in the presence of intracellular Mg²⁺ at appropriate membrane potentials. (b) The reverses of the relaxation time constants of the slow tail currents are plotted against voltage in semi–logarithmic scale for the WT and single as well as corresponding double mutant channels. The negative pulse in the same protocol was set to −20 ~ −100 mV to examine the voltage dependence of Mg²⁺ unblock from the binding site. The E224G mutant channel data are taken from Fig. 4c for comparison (the blue line). The off rate₍₀₎ and Zδ are ~4300 s⁻¹and 0.48 for the WT channel, ~370 s⁻¹ and 0.56 for the E224G, ~2800 s⁻¹ and 0.46 for the M183W, ~230 s⁻¹ and 0.62 for the E224G/M183W mutant channels, respectively.