NMR-Derived Dynamic Aspects of N-Type Inactivation of a Kv Channel Suggest a Transient Interaction with the T1 Domain

Abstract

Some eukaryotic Kv channels contain an N-terminal inactivation peptide (IP), which mediates a fast inactivation process that limits channel function during membrane depolarization and thus shapes the action potential. We obtained sequence-specific NMR assignments for the polypeptide backbone of a tetrameric N-terminal fragment (amino acids 1-181) of the Aplysia Kv1.1 channel.Additional NMR measurements show that the T1 domain has the same globular structure in solution as previously determined by crystallography, and that the IP (residues 1-20) and the Linker (residues 21-65) are in a flexibly disordered, predominantly extended conformation. A potential contact site between the T1 domain and the flexible tail (residues 1-65) has been identified based upon chemical shift changes of individual T1 domain amino acids, which maps to the T1 surface near the interface between adjacent subunits. Paramagnetic perturbation experiments further indicate that in the ensemble of solution conformers, there is at least a small population of species with the IP localized in close proximity to the proposed interacting residues of the T1 tetramer. Electrophysiological measurements show that all three mutations in this pocket we tested slow the rate of inactivation and speed up recovery, as predicted from the preinactivation site model. These results suggest that specific, short-lived transient interactions between the T1 domain and the IP or the Linker segment may play a role in defining the regulatory kinetics of fast channel inactivation.Cytoplasmic domains of voltage-gated K + (Kv) channels regulate channel assembly, trafficking and function, and control cytoplasmic access to the ion conduction pathway (1-7). Inactivation of Kv channels is a critical biological timing mechanism that limits the duration of channel activity in response to depolarization, as well as suppressing further channel activity for a period of time following depolarization. Thus, the ability to inactivate is an essential property of a variety of channels involved in shaping and regulating the excitability of the cell (2). A particular form of rapid inactivation of Kv channels, called Ntype inactivation, is mediated by an inactivation peptide (IP) segment of approximately 20 amino acids located at the N-terminal end of the α-subunits or of certain associating β-subunits. Results from experiments with free synthetic IP's, which restore inactivation to IPfree Kv channels (3,8,9), support the idea that a single IP can function by physically Because of the presence of the bulky tetrameric T1 assembly located between the IP and the central IP-binding site (12)(13)(14), and since the central cavity of the T1 domain is not part of the ion conduction pathway (15), it has been hypothesized that there must exist a pathway that allows the IP to move around the intervening T1 domain to reach its target site within the transmembrane (TM) pore of the channel, and that the lateral "side-windows" formed between T1 and the TM domains most likely provide this route (...

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