The N-Terminal Extension of KChIP3 is Responsible for KChIP3-Calmodulin Complex Formation

Abstract

Potassium channel interacting proteins (KChIPs), belong to the family of neuronal calcium sensors that are expressed in brain, lung and heart tissue. KChIPs bind to Kv4 channels and regulate channel trafficking, membrane association, and current kinetics. Among them, KChIP-3, also known as Downstream Regulatory Element Antagonistic Modulator (DREAM) and calsenilin, interacts with other intracellular partners (presenilin, calmodulin, and DNA) and was implied in Alzheimer's disease and pain sensing, although a molecular mechanism of KChIP-3 interactions with distinct intracellular partners remains unknown. The objective of this study is to provide a molecular insight into the mechanism of KChIP-3 interactions with calmodulin and determine the role of the N-terminus of KChIP-3 in the formation of calmodulin:KChIP-3 complex. Full length KChIP-3 (residue 1-256) and ΔN KChIP-3 (residue 65-256) were over-expressed in E. coli and purified according to an established protocol. In addition, a peptide that corresponds to residues 29-44 in KChIP-3 N-terminus was synthesized. The equilibrium dissociation constants and rotational correlation times for calmodulin: KChIP-3 complexes were determined using fluorescence anisotropy. Molecular dynamic simulations were implemented to provide an insight into the potential molecular structure of the calmodulin:KChIP-3 protein complex. Calmodulin interaction with KChIP-3 shows a Kd=3.3 µM whereas the peptide analogous to KChIP-3(29-44) binds with Kd=150 nM, deletion of 64 residues from the KChIP-3 N-terminus abolishes the complex formation. The interactions between KChIP-3 and calmodulin are regulated by the presence of Ca2+. A rotational correlation time of 35ns was determined for the KChIP-3:calmodulin complex, in agreement with a heterodimer of ellipsoidal shape. These results show that interactions between calmodulin and KChIP-3 are controlled by the intracellular calcium concentration and that the N-terminal extension in KChIP-3 provides a binding interface for calmodulin.