Role of N-terminal domain and accessory subunits in controlling deactivation-inactivation coupling of Kv4.2 channels.

We examined the relationship between deactivation and inactivation in Kv4.2 channels. In particular, we were interested in the role of a Kv4.2 N-terminal domain and accessory subunits in controlling macroscopic gating kinetics and asked if the effects of N-terminal deletion and accessory subunit coexpression conform to a kinetic coupling of deactivation and inactivation. We expressed Kv4.2 wild-type channels and N-terminal deletion mutants in the absence and presence of Kv channel interacting proteins (KChIPs) and dipeptidyl aminopeptidase-like proteins (DPPs) in human embryonic kidney 293 cells. Kv4.2-mediated A-type currents at positive and deactivation tail currents at negative membrane potentials were recorded under whole-cell voltage-clamp and analyzed by multi-exponential fitting. The observed changes in Kv4.2 macroscopic inactivation kinetics caused by N-terminal deletion, accessory subunit coexpression, or a combination of the two maneuvers were compared with respective changes in deactivation kinetics. Extensive correlation analyses indicated that modulatory effects on deactivation closely parallel respective effects on inactivation, including both onset and recovery kinetics. Searching for the structural determinants, which control deactivation and inactivation, we found that in a Kv4.2 Delta 2-10 N-terminal deletion mutant both the initial rapid phase of macroscopic inactivation and tail current deactivation were slowed. On the other hand, the intermediate and slow phase of A-type current decay, recovery from inactivation, and tail current decay kinetics were accelerated in Kv4.2 Delta 2-10 by KChIP2 and DPPX. Thus, a Kv4.2 N-terminal domain, which may control both inactivation and deactivation, is not necessary for active modulation of current kinetics by accessory subunits. Our results further suggest distinct mechanisms for Kv4.2 gating modulation by KChIPs and DPPs.

Voltage sensor inactivation in potassium channels.

In voltage-gated potassium (Kv) channels membrane depolarization causes movement of a voltage sensor domain. This conformational change of the protein is transmitted to the pore domain and eventually leads to pore opening. However, the voltage sensor domain may interact with two distinct gates in the pore domain: the activation gate (A-gate), involving the cytoplasmic S6 bundle crossing, and the pore gate (P-gate), located externally in the selectivity filter. How the voltage sensor moves and how tightly it interacts with these two gates on its way to adopt a relaxed conformation when the membrane is depolarized may critically determine the mode of Kv channel inactivation. In certain Kv channels, voltage sensor movement leads to a tight interaction with the P-gate, which may cause conformational changes that render the selectivity filter non-conductive ("P/C-type inactivation"). Other Kv channels may preferably undergo inactivation from pre-open closed-states during voltage sensor movement, because the voltage sensor temporarily uncouples from the A-gate. For this behavior, known as "preferential" closed-state inactivation, we introduce the term "A/C-type inactivation". Mechanistically, P/C- and A/C-type inactivation represent two forms of "voltage sensor inactivation."

Dynamic coupling of voltage sensor and gate involved in closed-state inactivation of kv4.2 channels.

Voltage-gated potassium channels related to the Shal gene of Drosophila (Kv4 channels) mediate a subthreshold-activating current (I(SA)) that 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 molecular mechanism is poorly understood. Here, we examined a structural model in which dynamic coupling between the voltage sensors and the cytoplasmic gate underlies inactivation in Kv4.2 channels. We performed an alanine-scanning mutagenesis in the S4-S5 linker, the initial part of S5, and the distal part of S6 and functionally characterized the mutants under two-electrode voltage clamp in Xenopus oocytes. 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, low-voltage inactivation kinetics were slowed or accelerated, and the voltage dependence of steady-state inactivation was shifted positive or negative. Still, 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 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.