Dynamic Coupling of Voltage Sensor and Gate Involved in Closed-State Inactivation of Kv4.2 Channels

Abstract

Voltage-gated potassium channels related to the Shal-gene of Drosophila (Kv4 channels) mediate a subthreshold-activating current (ISA) which controls dendritic excitation and the backpropagation of action potentials in neurons. Kv4 channels also exhibit a prominent low-voltage-induced closed-state inactivation, but the underlying mechanism is poorly understood. We examined a structural model, in which uncoupling of the voltage sensor from the cytoplasmic gate mediates inactivation in Kv4.2 channels. Support for such a model comes from our finding that chimeric swapping of S4-S5 linker and distal S6 sequences between N-terminally truncated Kv4.2Δ2-40 and ShakerIR channels slowed inactivation of the former and induced a form of fast macroscopic inactivation in the latter under two-electrode voltage-clamp in Xenopus oocytes. We performed a Kv4.2 alanine scanning-mutagenesis in the S4-S5 linker, the initial part of S5, and the distal part of S6 and functionally characterized these mutants. In a large fraction of the mutants (> 80%) normal channel function was preserved, but the mutations influenced the likelihood of the channel to enter the closed-inactivated state. Depending on the site of mutation, the onset kinetics of low-voltage inactivation and/or the kinetics of recovery from inactivation were accelerated or slowed and the voltage dependence of steady-state inactivation was shifted positive or negative. In some mutants these inactivation parameters remained unaffected. Double-mutant cycle analysis based on kinetic and steady-state parameters of low-voltage inactivation revealed that residues known to be critical for voltage-dependent gate-opening, including Glu 323 and Val 404, are also critical for Kv4.2 closed-state inactivation. Selective redox modulation of corresponding double-cysteine mutants by dithiothreitol (DTT) tert-butyl hydroperoxide (tbHO2) supported the idea that these residues are involved in a dynamic coupling, which mediates both transient activation and closed-state inactivation in Kv4.2 channels.