Values are mean … The model’s rate constants were used to calculate single-channel properties to determine whether the slowing of the current decay observed for R1448H can arise from longer open times or an increased number of openings.

The estimated mean open times were up to 4-fold longer for R1448H than for WT. Cooling increased the mean open time of both R1448H and Inhibitors,research,lifescience,medical WT afatinib cancer channels (Fig. 8 top). The bell-shaped curves showed promotion information open-time maxima between -50 and 0 mV. To the left of the maximum, the mean open time was dominated by the rate beta2 and to the right of the maximum by alpha6. This means that Na+ channels open and close several times before they finally enter the inactivated state. Importantly the calculated number of openings was ~20% greater for R1448H than for WT (Fig. 8 bottom). Cooling reduced the number of re-openings for both WT and R1448H. In summary the slowed decay of whole-cell currents (Fig. 1) is due to an increase in open times which are further increased by cooling. The rate constants Inhibitors,research,lifescience,medical and the transition probabilities showed Inhibitors,research,lifescience,medical that the increased number of R1448H openings is due to re-openings from the closed state C4 and not from the inactivated states. As the mutant channel shows the minimum of the energy landscape for I3, the channels reach this state by the C4– I2 pathway instead of by IT. Mutant channels go

along the O→C4→I2→I3 pathway. Figure 8. Temperature and voltage dependence of open times and number of openings. Temperature and voltage dependence of the mean open time (top) and the number of openings before inactivation (bottom) was calculated for indicated Inhibitors,research,lifescience,medical voltages for WT (left) and R1448H … Discussion Our whole-cell data confirms previous studies

in so far as R1448H slows open-state inactivation and shifts steadystate rapid inactivation to more negative potentials (3, 20- 22) Inhibitors,research,lifescience,medical and that the seemingly temperature sensitivity in paramyotonia is a result of channel kinetics which are already Drug_discovery slowed in the warmth and undergo a normal slowing with cooling (23, 24). Therefore, we assume that the required changes made to our model to best fit the data are not the result of our specific measurement or our set-up but rather reveal generally valid states and transitions. The required introduction of the transient inactivated state IT into our model suggests that open-state inactivation may result from a two-step process. The two inactivation phases become more obvious at low temperatures whereas they cannot be temporally resolved at higher temperatures. A biphasic inactivation process is actually in agreement with the classical HH model and with previous single channel measurements (3). We interpret the two phases to be linked to deactivation and inactivation.