Abstract
The Kv4.2 transient voltage-dependent potassium current contributes to the morphology of the cardiac action potential as well as to neuronal excitability and firing frequency. Here we report profound effects of the Kv4.2 C terminus on the surface expression and activation gating properties of Kv4.2 that are modulated by the direct interaction between KChIP2, an auxiliary regulatory subunit, and the C terminus of Kv4.2. We show that increasingly large truncations of the C terminus of rat Kv4.2 (wild type) cause a progressive decrease of Kv4.2 current along with a shift in voltage-dependent activation that is closely correlated with negative charge deletion. Co-expression of more limited Kv4.2 C-terminal truncation mutants (T588 and T528) with KChIP2 results in a doubling of Kv4.2 protein expression and up to an 8-fold increase in Kv4.2 current amplitude. Pulsechase experiments show that co-expression with KChIP2 slows Kv4.2 wild type degradation 8-fold. Co-expression of KChIP2 with an intermediate-length C-terminal truncation mutant (T474) shifts Kv4.2 activation voltage dependence and enhances expression of Kv4.2 current. The largest truncation mutants (T417 and DeltaC) show an intracellular localization with no measurable currents and no response to KChIP2 co-expression. Co-immunoprecipitation and competitive glutathione S-transferase-binding assays indicate a direct interaction between KChIP2 and the Kv4.2 C terminus with a relative binding affinity comparable with that of the N terminus. Overall, these results suggest that the C-terminal domain of Kv4.2 plays a critical role in voltage-dependent activation and functional expression that is mediated by direct interaction between the Kv4.2 C terminus and KChIP2.
Highlights
We have shown that the Kv4.2 C terminus is a key determinant of a variety of important properties of the Kv4.2 channel, including voltage dependence, cell membrane expression, and KChIP2 interaction
Our results indicate that these functions require an intact C terminus
The intracellular retention phenotype of ⌬C may be due to an altered conformation of HA-tagged T474 and T528 in the presence of KChIP2 (B, bottom) were metabolically labeled for 1 h with [35S]methionine/cysteine and chased for the indicated time intervals
Summary
Preparation of cDNA Constructs—Full-length rat Kv4.2 WT3 was generated by PCR amplification using 5Ј and 3Ј primers with BglII and NotI recognition sequences, respectively. Truncation mutant constructs were made by amplifying Kv4.2 WT plasmid with the same forward primer as for Kv4.2 WT and a series of reverse primers containing a NotI recognition site and in-frame stop codon with a partial Kv4.2 WT sequence that extends from the distal N terminus to each selected position in the post-transmembrane region on the C terminus (positions 406, 417, 474, 528, and 588), thereby truncating the more distal portion of the C terminus, as shown in Fig. 1 (top) These were correspondingly named ⌬C (the T406 truncation of the entire Kv4.2 C terminus), T417, T474, T528, and T588. Equal amounts of protein were added to 50 l of 50% glutathione-SepharoseTM 4B bead slurry (Amersham Biosciences) in 500 l of cell lysis buffer (1 mM EDTA, 0.5% Nonidet P-40, protease inhibitors in PBS, pH 8.0) and incubated on a rotator at 4 °C for 4 h. Cells were lysed as described above, and equal amounts of protein were immunoprecipitated with anti-HA or anti-c-myc antibody, subjected to SDS-PAGE, and visualized by autoradiography followed by quantification with an FC8000 densitometer. A two- mutagenesis to noncharged residues caused a positive shift in the tailed p Ͻ 0.05 was considered to indicate statistical activation V1⁄2 from Ϫ17 Ϯ 4 mV (n ϭ 7) for Kv4.2 WT to Ϫ8 Ϯ 4 significance
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
