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Intra- and Intersubunit Dynamic Binding in Kv4.2 Channel Closed-State Inactivation.

ABSTRACT: We studied the kinetics and structural determinants of closed-state inactivation (CSI) in Kv4.2 channels, considering a multistep process and the possibility that both intra- and intersubunit dynamic binding (i.e., loss and restoration of physical contact) may occur between the S4-S5 linker, including the initial S5 segment (S4S5), and the S6 gate. We expressed Kv4.2 channels in Xenopus oocytes and measured the onset of low-voltage inactivation under two-electrode voltage clamp. Indicative of a transitory state, the onset kinetics were best described by a double-exponential function. To examine the involvement of individual S4S5 and S6 amino acid residues in dynamic binding, we studied S4S5 and S6 single alanine mutants and corresponding double mutants. Both transitory and steady-state inactivation were modified by these mutations, and we quantified the mutational effects based on apparent affinities for the respective inactivated states. Double-mutant cycle analyses revealed strong functional coupling of the S6 residues V404 and I412 to all tested S4S5 residues. To examine whether dynamic S4S5/S6 binding occurs within individual ?-subunits or between neighboring ?-subunits, we performed a double-mutant cycle analysis with Kv4.2 tandem-dimer constructs. The constructs carried either an S4S5/S6 double mutation in the first ?-subunit and no mutation in the second (concatenated) ?-subunit or an S4S5 point mutation in the first ?-subunit and an S6 point mutation in the second ?-subunit. Our results support the notion that CSI in Kv4.2 channels is a multistep process that involves dynamic binding both within individual ?-subunits and between neighboring ?-subunits.

SUBMITTER: Wollberg J

PROVIDER: S-EPMC4805869 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Intra- and Intersubunit Dynamic Binding in Kv4.2 Channel Closed-State Inactivation.

Wollberg Jessica J Bähring Robert R

Biophysical journal 20160101 1

We studied the kinetics and structural determinants of closed-state inactivation (CSI) in Kv4.2 channels, considering a multistep process and the possibility that both intra- and intersubunit dynamic binding (i.e., loss and restoration of physical contact) may occur between the S4-S5 linker, including the initial S5 segment (S4S5), and the S6 gate. We expressed Kv4.2 channels in Xenopus oocytes and measured the onset of low-voltage inactivation under two-electrode voltage clamp. Indicative of a ...[more]

PMID: 26745419

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Project description:Kv4 channels mediate the somatodendritic A-type K+ current (I(SA)) in neurons. The availability of functional Kv4 channels is dynamically regulated by the membrane potential such that subthreshold depolarizations render Kv4 channels unavailable. The underlying process involves inactivation from closed states along the main activation pathway. Although classical inactivation mechanisms such as N- and P/C-type inactivation have been excluded, a clear understanding of closed-state inactivation in Kv4 channels has remained elusive. This is in part due to the lack of crucial information about the interactions between gating charge (Q) movement, activation, and inactivation. To overcome this limitation, we engineered a charybdotoxin (CTX)-sensitive Kv4.2 channel, which enabled us to obtain the first measurements of Kv4.2 gating currents after blocking K+ conduction with CTX (Dougherty and Covarrubias. 2006J. Gen. Physiol. 128:745-753). Here, we exploited this approach further to investigate the mechanism that links closed-state inactivation to slow Q-immobilization in Kv4 channels. The main observations revealed profound Q-immobilization at steady-state over a range of hyperpolarized voltages (-110 to -75 mV). Depolarization in this range moves <5% of the observable Q associated with activation and is insufficient to open the channels significantly. The kinetics and voltage dependence of Q-immobilization and ionic current inactivation between -153 and -47 mV are similar and independent of the channel's proximal N-terminal region (residues 2-40). A coupled state diagram of closed-state inactivation with a quasi-absorbing inactivated state explained the results from ionic and gating current experiments globally. We conclude that Q-immobilization and closed-state inactivation at hyperpolarized voltages are two manifestations of the same process in Kv4.2 channels, and propose that inactivation in the absence of N- and P/C-type mechanisms involves desensitization to voltage resulting from a slow conformational change of the voltage sensors, which renders the channel's main activation gate reluctant to open.

Dataset Information

Intra- and Intersubunit Dynamic Binding in Kv4.2 Channel Closed-State Inactivation.

Publications

Intra- and Intersubunit Dynamic Binding in Kv4.2 Channel Closed-State Inactivation.

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