Figure 4

The rate of C-type inactivation of full-length Shaker is dictated by the dwell time of the long N-terminus in the inner pore cavity.

(A). Current and fluorescence decay rates plotted in Figures 2 and 4 are re-plotted against each other to compare inactivation rates in full length Shaker (y-axis) and Shaker-IR (x-axis) with the equivalent I470 mutation in the presence of either 3 mM (squares) or 99 mM extracellular potassium (triangles). The black lines represent regression line fits for I470 and the mutant constructs. (B). Ionic currents measured in response to a two pulse protocol used to calculate the off-rates of the N-terminus from its blocking site in 3 mM (top) and 99 mM K+ (bottom). Following a pulse to +60 mV, cells were returned to –100 mV for an increasing period, then re-pulsed to +60 mV. The amplitudes of the second peaks were fit to a single exponential function (dashed lines). (C). Recovery rates from N-type inactivation were calculated from single exponential fits from panel B using Equation 2 and are plotted for the I470 and the four studied mutants. I470F rates were too slow to be visible on the y-axis time scale (n = 6 for I470; n = 5 for I470V, I470C and I470F; n = 4 for I470L). (D). Regression lines are fit to data from each construct from panel A (see inset). The slopes of these curves are then plotted against rates calculated in panel C for each equivalent I470 mutation in the presence of either 3 mM (squares) or 99 mM extracellular potassium (triangles).