Abstract
Dynamic inactivation in Kv4 A-type K(+) current plays a critical role in regulating neuronal excitability by shaping action potential waveform and duration. Multifunctional auxiliary KChIP1-4 subunits, which share a high homology in their C-terminal core regions, exhibit distinctive modulation of inactivation and surface expression of pore-forming Kv4 subunits. However, the structural differences that underlie the functional diversity of Kv channel-interacting proteins (KChIPs) remain undetermined. Here we have described the crystal structure of KChIP4a at 3.0A resolution, which shows distinct N-terminal alpha-helices that differentiate it from other KChIPs. Biochemical experiments showed that competitive binding of the Kv4.3 N-terminal peptide to the hydrophobic groove of the core of KChIP4a causes the release of the KChIP4a N terminus that suppresses the inactivation of Kv4.3 channels. Electrophysiology experiments confirmed that the first N-terminal alpha-helix peptide (residues 1-34) of KChIP4a, either by itself or fused to N-terminal truncated Kv4.3, can confer slow inactivation. We propose that N-terminal binding of Kv4.3 to the core of KChIP4a mobilizes the KChIP4a N terminus, which serves as the slow inactivation gate.
Highlights
A-type inactivation, a rapid process of channel closing, leads to the reduction or elimination of potassium currents that can be dynamically regulated by a variety of intracellular factors
We propose that N-terminal binding of Kv4.3 to the core of KChIP4a mobilizes the KChIP4a N terminus, which serves as the slow inactivation gate
It is of interest that KChIP4a can be functionally converted to KChIP1 by truncation of a K-channel inactivation suppressor (KIS) domain that resides in the N terminus of KChIP4a
Summary
Molecular Biology and Materials—Constructs of full-length Mouse KChIP4a (229 amino acids, GenBankTM accession number AF453243) and the N terminus of human Kv4.3 containing amino acids 6 –145 were cloned by PCR into pET28a and pMAL-c2 vectors, respectively. The purified protein was loaded on a Superdex 200 HR 10/30 column (Amersham Biosciences), and SEC was performed on an Amersham Biosciences FPLC system using a buffer (containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 3 mM dithiothreitol) with a flow rate of 0.5 ml/min. The remaining resin was eluted with buffer containing 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 250 mM imidazole, and proteins were concentrated and further fractionated by SEC-FPLC. Crystallization and Data Collection—Crystals of KChIP4a were obtained at room temperature by hanging drop vapor diffusion in which the well solution consisted of 1.6 –1.8 M ammonium sulfate, 4% (v/v) isopropanol, and 0.1 M dithiothreitol, and crystals were flash-frozen in mother liquor supplemented with 20% (v/v) glycerol. X-ray Coordinates—The PDB ID code obtained for KChIP4a is 3DD4, and the atomic coordinates have been deposited in the Protein Data Bank
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