Functional implication with the metal-binding properties of KChIP1

To investigate the metal-binding properties of KChIP1, the interaction of KChIP1 and mutated KChIP1 with divalent cations (Mg(2+), Ca(2+), Sr(2+), and Ba(2+)) was explored by 8-anilinonaphthalene-1-sulfonate (ANS) fluorescence. It showed that KChIP1 possessed two types of Ca(2+)-binding sites, high-affinity and low-affinity Ca(2+)-binding sites. However, only low-affinity-binding site for Mg(2+), Sr(2+), and Ba(2+) was observed. The metal-binding properties of KChIP1 are not appreciably affected after removal of the N-terminal portion and EF-hand 1. Deleting the EF-hand 4 of KChIP1 abolishes its high-affinity Ca(2+)-binding site, but retains the intact low-affinity-binding site for metal ions. A decrease in the nonpolarity of ANS-binding site occurs with all mutants. However, the binding of ANS with KChIP1 is no longer observed after removal of EF-hands 3 and 4. Intermolecular interaction assessed by chemical cross-linking suggested that KChIP1 had a propensity to form dimer in the absence of metal ions, and a KChIP1 tetramer was pronouncedly produced in the presence of metal ions. Noticeably, the oligomerization state depends on the integrity of EF-hand 4. Taken together, our data suggest that EF-hand 4 is of structural importance as well as functional importance for fulfilling the physiological function of KChIP1.     

Protein-protein interactions of KChIP proteins and Kv4.2

To prove heteromeric assembly of KChIP proteins, the present study is carried out. The results of chemical crosslinking and pull down assay revealed that KChIP1, KChIP2.1, and KChIP2.2 could form homo- as well as hetero-oligomer, and this oligomerization exhibited a Ca(2+)-dependent manner. Moreover, homomeric and heteromeric assembly of KChIPs did not perturb their interaction with Kv4.2 K(+) channel, indicating that the region associated with oligomerization of KChIPs was distinct from that for binding with Kv4.2. Together with previous findings that the net effects of KChIP proteins on the molecular properties and trafficking of Kv channel were different, these observations open a fascinating possibility that the electrophysiological properties of Kv channel may be differently regulated by homomeric and heteromeric assembly of KChIPs.     

Evidence showing an intermolecular interaction between KChIP proteins and Taiwan cobra cardiotoxins

Direct protein-protein interaction between Taiwan cobra cardiotoxin3 (CTX3) and potassium channel-interacting proteins (KChIPs) was investigated in the present study. It was found that KChIPs bound with CTX3, in which KChIP and CTX3 formed a 1:1 complex as evidenced by the results of chemical cross-linking. Pull-down assay revealed that the intact EF-hands 3 and 4 of KChIP1 were critical for CTX3-binding. Likewise, removal of EF-hands 3 and 4 distorted the ability of KChIP1 to bind with Kv4.2 N-terminal fragment (KvN) as well as fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS). In contrast to the interaction between KChIP1 and KvN, the binding of CTX3 to KChIP1 showed a Ca(2+)-independent manner. Fluorescence measurement revealed that CTX3 affected the binding of ANS to Ca(2+)-bound KChIP1, but not Ca(2+)-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 CTX for this biomedical application could be considered.     

Enhanced Trafficking of Tetrameric Kv4.3 Channels by KChIP1 Clamping

The cytoplamsic auxiliary KChIPs modulate surface expression and gating properties of Kv4 channels. Recent co-crystal structure of Kv4.3 N-terminus and KChIP1 reveals a clamping action of the complex in which a single KChIP1 molecule laterally binds two neighboring Kv4.3 N-termini at different locations, thus forming two contact interfaces involved in the protein–protein interaction. In the second interface, it functions to stabilize the tetrameric assembly, but the role it plays in channel trafficking remains elusive. In this study, we examined the effects of KChIP1 on Kv4 protein trafficking in COS-7 cells expressing EGFP-tagged Kv4.3 channels using confocal microscopy. Mutations either in KChIP1 (KChIP1 L39E-Y57A-K61A) or Kv4.3 (Kv4.3 E70A-F73E) that disrupt the protein–protein interaction within the second interface can reduce surface expression of Kv4 channel proteins. Kv4.3 C110A, the Zn²⁺ binding site mutation in T1 domain, that disrupts the tetrameric assembly of the channels can be rescued by WT KChIP1, but not the KChIP1 triple mutant. These results were further confirmed by whole cell current recordings in oocytes. Our findings show that key residues of second interface involved in stabilizing tetrameric assembly can regulate the channel trafficking, indicating an intrinsic link between tetrameric assembly and channel trafficking. The results also suggest that formation of octameric Kv4 and KChIP complex by KChIPs clamping takes place before their trafficking to final destination on the cell surface. Special issue article in honor of Dr. Ji-Sheng Han.     

Functional role of EF-hands 3 and 4 in membrane-binding of KChIP1

The aim of the present study is to explore whether membrane targeting of K⁺ channel-interacting protein 1 (KChIP1) is associated with its EF-hand motifs and varies with specific phospholipids. Truncated KChIP1, in which the EFhands 3 and 4 were deleted, retained the α-helix structure, indicating that the N-terminal half of KChIP1 could fold appropriately. Compared with wild-type KChIP1, truncated KChIP1 exhibited lower lipid-binding capability. Compared with wild-type KChIP1, increasing membrane permeability by the use of digitonin caused a marked loss of truncated KChIP1, suggesting that intact EF-hands 3 and 4 were crucial for the anchorage of KChIP1 on membrane. KChIP1 showed a higher binding capability with phosphatidylserine (PS) than truncated KChIP1. Unlike that of truncated KChIP1, the binding of wild-type KChIP1 with membrane was enhanced by increasing the PS content. Moreover, the binding of KChIP1 with phospholipid vesicles induced a change in the structure of KChIP1 in the presence of PS. Taken together, our data suggest that EF-hands 3 and 4 of KChIP1 are functionally involved in a specific association with PS on the membrane.     

Expression and high glucose-mediated regulation of K channel interacting protein 3 (KChIP3) and KV4 channels in retinal Müller glial cells

Normal vision depends on the correct function of retinal neurons and glia and it is impaired in the course of diabetic retinopathy. Müller cells, the main glial cells of the retina, suffer morphological and functional alterations during diabetes participating in the pathological retinal dysfunction. Recently, we showed that Müller cells express the pleiotropic protein potassium channel interacting protein 3 (KChIP3), an integral component of the voltage-gated K⁺ channels K_V4. Here, we sought to analyze the role of KChIP3 in the molecular mechanisms underlying hyperglycemia-induced phenotypic changes in the glial elements of the retina. The expression and function of KChIp3 was analyzed in vitro in rat Müller primary cultures grown under control (5.6 mM) or high glucose (25 mM) (diabetic-like) conditions. We show the up-regulation of KChIP3 expression in Müller cell cultures under high glucose conditions and demonstrate a previously unknown interaction between the K_V4 channel and KChIP3 in Müller cells. We show evidence for the expression of a 4-AP-sensitive transient outward voltage-gated K⁺ current and an alteration in the inactivation of the macroscopic outward K⁺ currents expressed in high glucose-cultured Müller cells. Our data support the notion that induction of KChIP3 and functional changes of K_V4 channels in Müller cells could exert a physiological role in the onset of diabetic retinopathy.     

DREAM/Calsenilin/KChIP3:神经系统的多功能蛋白

新近发现的转录因子DREAM(downstreamregulatoryelementantagonistmodulator)可结合到基因(包括PPD、Hrk、cfos等)的DRE(downstreamregulatoryelement)位点,抑制基因转录。它是第一个已知的可以直接与DNA结合发挥转录抑制作用的Ca2 结合蛋白,为Ca2 调节基因表达除蛋白激酶/磷酸酶这条主要通路外提供了另一条通路。由于DREAM与另两个研究小组发现的蛋白calsenilin和KChIP3实为同一种物质,所以DREAM具有PS(presenilin)作用蛋白、Kv4通道调节蛋白和转录因子的多重功能特性。本文将就DREAM的分布、功能及其调控、DREAM与疼痛的关系作简要综述。     

The Stoichiometry and Biophysical Properties of the Kv4 Potassium Channel Complex with K+ Channel-interacting Protein (KChIP) Subunits Are Variable,Depending on the Relative Expression Level

Kv4 is a voltage-gated K⁺ channel, which underlies somatodendritic subthreshold A-type current (I_SA) and cardiac transient outward K⁺ (I_to) current. Various ion channel properties of Kv4 are known to be modulated by its auxiliary subunits, such as K⁺ channel-interacting protein (KChIP) or dipeptidyl peptidase-like protein. KChIP is a cytoplasmic protein and increases the current amplitude, decelerates the inactivation, and accelerates the recovery from inactivation of Kv4. Crystal structure analysis demonstrated that Kv4 and KChIP form an octameric complex with four Kv4 subunits and four KChIP subunits. However, it remains unknown whether the Kv4·KChIP complex can have a different stoichiometry other than 4:4. In this study, we expressed Kv4.2 and KChIP4 with various ratios in Xenopus oocytes and observed that the biophysical properties of Kv4.2 gradually changed with the increase in co-expressed KChIP4. The tandem repeat constructs of Kv4.2 and KChIP4 revealed that the 4:4 (Kv4.2/KChIP4) channel shows faster recovery than the 4:2 channel, suggesting that the biophysical properties of Kv4.2 change, depending on the number of bound KChIP4s. Subunit counting by single-molecule imaging revealed that the bound number of KChIP4 in each Kv4.2·KChIP4 complex was dependent on the expression level of KChIP4. Taken together, we conclude that the stoichiometry of Kv4·KChIP complex is variable, and the biophysical properties of Kv4 change depending on the number of bound KChIP subunits.     

Co-assembly of Kv4 α Subunits with K+ Channel-interacting Protein 2 Stabilizes Protein Expression and Promotes Surface Retention of Channel Complexes

Members of the K⁺ channel-interacting protein (KChIP) family bind the distal N termini of members of the Shal subfamily of voltage-gated K⁺ channel (Kv4) pore-forming (α) subunits to generate rapidly activating, rapidly inactivating neuronal A-type (I_A) and cardiac transient outward (I_to) currents. In heterologous cells, KChIP co-expression increases cell surface expression of Kv4 α subunits and Kv4 current densities, findings interpreted to suggest that Kv4·KChIP complex formation enhances forward trafficking of channels (from the endoplasmic reticulum or the Golgi complex) to the surface membrane. The results of experiments here, however, demonstrate that KChIP2 increases cell surface Kv4.2 protein expression (∼40-fold) by an order of magnitude more than the increase in total protein (∼2-fold) or in current densities (∼3-fold), suggesting that mechanisms at the cell surface regulate the functional expression of Kv4.2 channels. Additional experiments demonstrated that KChIP2 decreases the turnover rate of cell surface Kv4.2 protein by inhibiting endocytosis and/or promoting recycling. Unexpectedly, the experiments here also revealed that Kv4.2·KChIP2 complex formation stabilizes not only (total and cell surface) Kv4.2 but also KChIP2 protein expression. This reciprocal protein stabilization and Kv4·KChIP2 complex formation are lost with deletion of the distal (10 amino acids) Kv4.2 N terminus. Taken together, these observations demonstrate that KChIP2 differentially regulates total and cell surface Kv4.2 protein expression and Kv4 current densities.     

Modulation of the Voltage-gated Potassium Channel (Kv4.3) and the Auxiliary Protein (KChIP3) Interactions by the Current Activator NS5806

KChIP3 (potassium channel interacting protein 3) is a calcium-binding protein that binds at the N terminus of the Kv4 voltage-gated potassium channel through interactions at two contact sites and has been shown to regulate potassium current gating kinetics as well as channel trafficking in cardiac and neuronal cells. Using fluorescence spectroscopy, isothermal calorimetry, and docking simulations we show that the novel potassium current activator, NS5806, binds at a hydrophobic site on the C terminus of KChIP3 in a calcium-dependent manner, with an equilibrium dissociation constant of 2–5 μm in the calcium-bound form. We further determined that the association between KChIP3 and the hydrophobic N terminus of Kv4.3 is calcium-dependent, with an equilibrium dissociation constant in the apo-state of 70 ± 3 μm and 2.7 ± 0.1 μm in the calcium-bound form. NS5806 increases the affinity between KChIP3 and the N terminus of Kv4.3 (K_d = 1.9 ± 0.1 μm) in the presence and absence of calcium. Mutation of Tyr-174 or Phe-218 on KChIP3 abolished the enhancement of Kv4.3 site 1 binding in the apo-state, highlighting the role of these residues in drug and K4.3 binding. Kinetic studies show that NS5806 decreases the rate of dissociation between KChIP3 and the N terminus of K_V4.3. Overall, these studies support the idea that NS5806 directly interacts with KChIP3 and modulates the interactions between this calcium-binding protein and the T1 domain of the Kv4.3 channels through reorientation of helix 10 on KChIP3.