Subthreshold activation of Kv4.2 channels reduces dendritic excitability, but Kv4.2 inactivation, even by moderate depolarization, leads to a local increase in excitability and may cause long term synaptic strengthening. The mechanism underlying the "silent" low-voltage Kv4.2 inactivation, which may occur from closed-states, is unknown. We examine if uncoupling of the voltage-sensor from the gate may underly Kv4.2 closed-state inactivation. We performed an alanine-scanning mutagenesis within the distal S6 and the S4-S5 linker. Kv4.2 mutants were functionally characterized under two-electrode voltage-clamp in Xenopus oocytes. Time constants of inactivation at low voltages (between -90 and -50 mV) and the respective steady-state levels of inactivation provided the kinetic parameters k_on and k_off from which we calculated K_d, the affinity for the closed-inactivated state. In some constructs the affinity for the closed-inactivated state was higher (e.g. V404A: ln K_d/K_d,wt = -2.79 kT), in others it was lower than in wild-type

(e.g. N408A: K_d/K_d,wt = +2.04 kT). Double-mutant cycle analysis with E323A in the S4-S5 linker yielded lnW-values as high as 5.0 (E323A,V404A) suggesting that residues involved in voltage-dependent opening of the gate also play a role in closed-state inactivation of Kv4.2 channels.

Supported by the Deutsche Forschungsgemeinschaft (BA 2055/1)