Total chemical synthesis and biophysical characterization of the minimal isoform of the KChIP2 potassium channel regulatory subunit.

The potassium channel accessory subunit KChIP2 associates with Kv4.2 channels in the cardiac myocyte and is involved in the regulation of the transient outward current (I(to)) during the early phase of repolarization of the action potential. As a first step to biophysically probe the mechanism of KChIP2, we have chemically synthesized its minimal isoform, KChIP2d, using Boc chemistry solid phase peptide synthesis in conjunction with native chemical ligation. The synthetic KChIP2d protein is primarily alpha-helical as predicted and becomes more structured upon binding calcium as assessed by (1)H-NMR and CD spectroscopy. Synthetic KChIP2d is in a monomer-dimer equilibrium in solution, and there is evidence for two monomer binding sites on an N-terminal peptide of Kv4.2. Planned future studies include the incorporation of fluorescent and spin labeled probes in KChIP2d to yield structural information in parallel with electrophysiologic studies to elucidate KChIP2d's mechanism of action.