Figure 3

The emergence of a slow component of intracellular Mg²⁺ unblock in mutant E224 and E299 channels.

(a) The Kd of intracellular Mg²⁺ block in E224G, E224Q and E299S mutant channels is plotted against voltage on a semi–logarithmic scale (the same approach as that shown in Fig. 1c). The changes in Kd of intracellular Mg²⁺ block around 0 mV are evidently much smaller with specific of E224G, E224Q and E299S mutations. (b) The patches which expressed E299S, E224G and E224A mutant channels were first held from 0 mV and stepped twice to −100 mV^12,13,27. The sample sweeps show a “slow tail” when the patch was repolarized from a positive voltage of +100 mV to a negative voltage of −100 mV in the presence but not in the absence of intracellular Mg²⁺, signaling Mg²⁺ block from the channel. The development of the slow tail in the second −100 mV pulse thus is used as a measure of intracellular Mg²⁺ binding to this slow unblocking site at +100 mV (dashed lines, see also part c). The dashed lines indicate the zero current level. Note that when the currents are turning from outward to inward, there is a very fast phase of development of inward current before the slow tail phase (insert figure). (c) Cumulative results were obtained with the same protocol described in part b for the E299S, E224G and E224A mutant channels (n = 4–7). tau _{on, 1} and tau _{on, 2} denote the time constants of the growing courses of slow tails and the time constants of the decay of outward currents in the presence of 100 μM intracellular Mg²⁺ in part b respectively. (d) The reciprocals of the time constants of development of slow tail currents (on rate at +100 mV) are plotted against the decay time constant of the slow tail (off rate at −100 mV) for each of the E224 mutant (E224A, E224C and E224G) and E299 mutant (E299A, E299S and E299C) channels. The lines are linear regressions demonstrating the strong linear correlation between the two parameters.