N-type Inactivation Features of Kv4.2 Channel Gating

Abstract

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Delta40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K+. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Delta40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Delta40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.

The Stoichiometry and Biophysical Properties of the Kv4 Potassium Channel Complex with K+ Channel-interacting Protein (KChIP) Subunits Are Variable, Depending on the Relative Expression Level

“…2C and 4E). According to a previous study, an N terminus truncation mutant of Kv4.2 (Kv4.2⌬40) shows slower inactivation than WT Kv4.2, and the inactivation of Kv4.2⌬40 is accelerated by an additional Kv4.2 N terminus peptide; therefore, it was concluded that the inactivation of Kv4.2 is produced by occluding the channel pore with the N terminus (24). Because binding of KChIP to the N terminus slows down the inactivation kinetics of Kv4.2, free N terminus without KChIP binding may be required for the fast inactivation.…”

“…First, KChIP promotes the surface expression of Kv4 and increases the current amplitude of Kv4 (22,23). Second, KChIP binding slows the inactivation of Kv4 by restricting the movement of the N terminus, which may occlude the ion channel pore (24). Third, KChIP accelerates the recovery from inactivation of Kv4 (2,25,26).…”

Kv channel-interacting proteins as neuronal and non-neuronal calcium sensors

“…It is possible that Kv4.3/KChIP2 complex formation occurs in a different fashion depending on whether Ca 2+ is bound to KChIP2 or not. Thus, in the absence of Ca 2+ KChIP2 binding to the Kv4.3 N-terminus may not be strong enough to suppress N-type inactivation [77], while KChIP2 binding to the Kv4.3 T1-domain may still be sufficiently strong to modulate other aspects of inactivation [71,78]. Such a modulatory rather than decisive role of EF-hand-mediated Ca 2+ binding in Kv4/KChIP complex formation was also supported by the finding that binding of wild-type KChIP2c to a 90 amino acid NTF of Kv4.2 is enhanced, whereas the binding of KChIP2cΔEF mutants to that fragment, although possible, is not enhanced by Ca 2+ [35,37].…”

KChIP3 Rescues the Functional Expression of Shal Channel Tetramerization Mutants

“…Kv4 channel inactivation has been dissected in numerous biophysical studies into three separate processes: open state, closed state, and slow inactivation (15,20,21). Open state inactivation utilizes a pore block produced by the Kv4 cytoplasmic N terminus and is lost by deletion of the Kv4 N terminus or binding of KChIP proteins to the N terminus (16,22). Closed state inactivation is a separate process from open state inactivation.…”

“…The N terminus of Kv4.2 is clearly involved in inactivation gating, since deletion of this region produces slowed inactivation. Moreover, experiments on the functional properties of the N terminus show that it can act as a pore-binding peptide (15)(16)(17). Therefore, the gating changes produced by KChIPs might depend on the binding and sequestering of the Kv4 N terminus, causing an alteration to a complex set of allosteric interactions that involve other structural elements of the channel (18).…”