Abstract Our previous study showed that KChIP3 (Kv Channel Interacting Protein 3) probably was a physiological targete protein of beta-bungarotoxin (beta-Bgt) as evidenced by yeast two-hybrid system. Thus, extensive efforts are carried out to explore the molecular interaction between KChIP3 and beta-Bgt in the present study. KChIPs are potassium (Kv) channel-interacting proteins that bind to the 1~90 amino acid of N-terminus of Kv4 alpha-subunits and regulate the ion current density, shift the voltage dependence of activation and speed their recovery from inactivation. beta-Bgt, a presynaptic neurotoxin purified from Bungarus multicinctus venom, consists of A chain and B chain which cross-linked by an interchain disulfide bond. The results of pull-down assay revealed that, in contrast to other KChIP proteins, KChIP3 bound with beta-Bgt. Moreover, it was found that the B chain of beta-Bgt was a functional subunit in the binding with KChIP3, and the binding of KChIP3 to beta-Bgt showed a Ca2+-dependent manner. Removal of the third and the fourth EF-hand regions of KChIP3 abolished its interaction with beta-Bgt. Noticeably, the binding of beta-Bgt with KChIP3 did not influence the interaction between KChIP3 and Kv4. In the meantime, rat brain KChIP3 could be isolated using a beta-Bgt-Sepharose column. These observations suggest that KChIP3 is an intra-cellular target recognized by beta-Bgt. Accidently, it was found that direct protein-protein interaction between Taiwan cobra cardiotoxin3 (CTX3) and potassium channel-interacting proteins (KChIPs) was investigated. It was found that KChIPs bound with CTX3, in which KChIP1 and CTX3 formed a 1:1 complex as evidenced by the results of chemical crosslinking. Pull-down assay revealed that the intact EF-hand 3 and 4 of KChIP1 was critical for CTX3-binding. Whereas, all mutated KChIP3 were able to bind with CTX3. In contrast to the interaction between KChIP1 and KvN, the binding of CTX3 to KChIP1 showed a Ca2+-independent manner. Fluorescence measurement revealed that CTX3 affected the binding of ANS to Ca2+-bound KChIP1, but not Ca2+-free KChIP1. Alternatively, KChIP1 simultaneously bound with KvN and CTX3, and the interaction between KChIP1 and KvN was enhanced by CTX3. In terms of the fact that KChIPs regulate the electrophysiological properties of Kv K+ channel, the potentiality of beta-bgt and CTX for this biomedical application could be considered.

Abstract KChIPs are Kv channel-interacting proteins that bind to the cytoplasmic N-terminus of Kv4

Downstream regulatory antagonist modulator (DREAM) is a calcium sensing protein that co-assembles with KV4 potassium channels to regulate ion currents as well as with DNA in the nucleus, where it regulates gene expression. The interaction of DREAM with A-type KV4 channels and DNA has been shown to regulate neuronal signaling, pain sensing, and memory retention. The role of DREAM in modulation of pain, onset of Alzheimer’s disease, and cardiac pacemaking has set this protein as a novel therapeutic target. Moreover, previous results have shown a Ca2+ dependent interaction between DREAM and KV4/DNA involving surface contacts at the N-terminus of DREAM. However, the mechanisms by which Ca2+ binding at the C-terminus of DREAM induces structural changes at the C- and N-terminus remain unknown. Here, we present the use of biophysics and biochemistry techniques in order to map the interactions of DREAM and numerous small synthetic ligands as well as KV channels. We further demonstrate that a highly conserved network of aromatic residues spanning the C- and N-terminus domains control protein dynamics and the pathways of signal transduction on DREAM. Using molecular dynamics simulations, site directed mutagenesis, and fluorescence spectroscopy we provide strong evidence in support of a highly dynamic mechanism of signal transduction and regulation. A set of aromatic amino acids including Trp169, Phe171, Tyr174, Phe218, Phe235, Phe219, and Phe252 are identified to form a dynamic network involved in propagation of Ca2+ induced structural changes. These amino acids form a hydrophobic network connecting the N- and C-terminus domains of DREAM and are well conserved in other neuronal calcium sensors. In addition, we show evidence in support of a mechanism in which Ca2+ signals are propagated towards the N-terminus and ultimately lead to the rearrangement of the inactive EF-hand 1. The observed structural motions provide a novel mechanism involved in control of the calcium dependent KV4 and DNA binding. Altogether, we provide the first mechanism of intramolecular and intermolecular signal transduction in a Ca2+ binding protein of the neuronal calcium sensor family.