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.     

Functional stoichiometry underlying KChIP regulation of Kv4.2 functional expression

K channel‐interacting proteins (KChIPs) enhance functional expression of Kv4 channels by binding to an N‐terminal regulatory region located in the first 40 amino acids of Kv4.2 that we call the functional expression regulating N‐terminal (FERN) domain. Mutating two residues in the FERN domain to alanines, W8A and F11A, disrupts KChIP binding and regulation of Kv4.2 without eliminating the FERN domain's control of basal expression level or regulation by DPP6. When Kv4.2(W8A,F11A) is co‐expressed with wild type Kv4.2 and KChIP3 subunits, a dominant negative effect is seen where the current expression is reduced to levels normally seen without KChIP addition. The dominant negative effect correlates with heteromultimeric channels remaining on intracellular membranes despite KChIP binding to non‐mutant Kv4.2 subunits. In contrast, the deletion mutant Kv4.2(Δ1‐40), eliminating both KChIP binding and the FERN domain, has no dominant negative effect even though the maximal conductance level is 5x lower than seen with KChIP3. The 5x increased expression seen with KChIP integration into the channel is fully apparent even when a reduced number of KChIP subunits are incorporated as long as all FERN domains are bound. Our results support the hypothesis that KChIPs enhances Kv4.2 functional expression by a 1 : 1 suppression of the N‐terminal FERN domain and by producing additional positive regulatory effects on functional channel expression.     

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.     

Altered K(+) channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4

Expression of voltage-gated K(+) channels encoding the K(+) independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K(+) current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in alpha-MHC mRNA and a 259% increase in beta-MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K(+) channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.     

Molecular correlates of altered expression of potassium currents in failing rabbit myocardium

Action potential (AP) prolongation is a hallmark of failing myocardium. Functional downregulation of K currents is a prominent feature of cells isolated from failing ventricles. The detailed changes in K current expression differ depending on the species, the region of the heart, and the mechanism of induction of heart failure. We used complementary approaches to study K current downregulation in pacing tachycardia-induced heart failure in the rabbit. The AP duration (APD) at 90% repolarization was significantly longer in cells isolated from failing hearts compared with controls (539 +/- 162 failing vs. 394 +/- 114 control, P < 0.05). The major K currents in the rabbit heart, inward rectifier potassium current (I(K1)), transient outward (I(to)), and delayed rectifier current (I(K)) were functionally downregulated in cells isolated from failing ventricles. The mRNA levels of Kv4.2, Kv1.4, KChIP2, and Kir2.1 were significantly downregulated, whereas the Kv4.3, Erg, KvLQT1, and minK were unaltered in the failing ventricles compared with the control left ventricles. Significant downregulation in the long splice variant of Kv4.3, but not in the total Kv4.3, Kv4.2, and KChIP2 immunoreactive protein, was observed in cells isolated from the failing ventricle with no change in Kv1.4, KvLQT1, and in Kir2.1 immunoreactive protein levels. Multiple cellular and molecular mechanisms underlie the downregulation of K currents in the failing rabbit ventricle.     

Functional Rescue of Kv4.3 Channel Tetramerization Mutants by KChIP4a

KChIP4a shows a high homology with other members of the family of Kv channel-interacting proteins (KChIPs) in the conserved C-terminal core region, but exhibits a unique modulation of Kv4 channel gating and surface expression. Unlike KChIP1, the KChIP4 splice variant KChIP4a has been shown to inhibit surface expression and function as a suppressor of channel inactivation of Kv4. In this study, we sought to determine whether the multitasking KChIP4a modulates Kv4 function in a clamping fashion similar to that shown by KChIP1. Injection of Kv4.3 T1 zinc mutants into Xenopus oocytes resulted in the nonfunctional expression of Kv4.3 channels. Coexpression of Kv4.3 zinc mutants with WT KChIP4a gave rise to the functional expression of Kv4.3 current. Oocyte surface labeling results confirm the correlation between functional rescue and enhanced surface expression of zinc mutant proteins. Chimeric mutations that replace the Kv4.3 N-terminus with N-terminal KChIP4a or N-terminal deletion of KChIP4a further demonstrate that the functional rescue of Kv4.3 channel tetramerization mutants depends on the KChIP4a core region, but not its N-terminus. Structure-guided mutation of two critical residues of core KChIP4a attenuated functional rescue and tetrameric assembly. Moreover, size exclusion chromatography combined with fast protein liquid chromatography showed that KChIP4a can drive zinc mutant monomers to assemble as tetramers. Taken together, our results show that KChIP4a can rescue the function of tetramerization-defective Kv4 monomers. Therefore, we propose that core KChIP4a functions to promote tetrameric assembly and enhance surface expression of Kv4 channels by a clamping action, whereas its N-terminus inhibits surface expression of Kv4 by a mechanism that remains elusive.     

Differential expression of Kv4 pore-forming and KChIP auxiliary subunits in rat uterus during pregnancy

Regulation of voltage-gated K(+) (K(v)) channel expression may be involved in controlling contractility of uterine smooth muscle cells during pregnancy. Functional expression of these channels is not only controlled by the levels of pore-forming subunits, but requires their association with auxiliary subunits. Specifically, rapidly inactivating K(v) current is prominent in myometrial cells and may be carried by complexes consisting of Kv4 pore-forming and KChIP auxiliary subunits. To determine the molecular identity of the channel complexes and their changes during pregnancy, we examined the expression and localization of these subunits in rat uterus. RT-PCR analysis revealed that rat uterus expressed all three Kv4 pore-forming subunits and KChIP2 and -4 auxiliary subunits. The expression of mRNAs for these subunits was dynamically and region selectively regulated during pregnancy. In the corpus, Kv4.2 mRNA level increased before parturition, whereas the expression of Kv4.1 and Kv4.3 mRNAs decreased during pregnancy. A marked increase in KChIP2 mRNA level was also seen at late gestation. In the cervix, the expression of all three pore-forming and two auxiliary subunit mRNAs increased at late gestation. Immunoprecipitation followed by immunoblot analysis indicated that Kv4.2-KChIP2 complexes were significant in uterus at late pregnancy. Kv4.2- and KChIP2-immunoreactive proteins were present in both circular and longitudinal myometrial cells. Finally, Kv4.2 and KChIP2 mRNA levels were similarly elevated in pregnant and nonpregnant corpora of one side-conceived rats. These results suggest that diffusible factors coordinate the pregnancy-associated changes in molecular compositions of myometrial Kv4-KChIP channel complexes.     

Multi-Walled Carbon Nanotubes Impair Kv4.2/4.3 Channel Activities,Delay Membrane Repolarization and Induce Bradyarrhythmias in the Rat

Purpose

The potential hazardous effects of multi-walled carbon nanotubes (MWCNTs) on cardiac electrophysiology are seldom evaluated. This study aimed to investigate the impacts of MWCNTs on the Kv4/I _to channel, action potential and heart rhythm and the underlying mechanisms.

Methods

HEK293 cells were engineered to express Kv4.2 or Kv4.3 with or without KChIP2 expression. A series of approaches were introduced to analyze the effects of MWCNTs on Kv4/I _to channel kinetics, current densities, expression and trafficking. Transmission electron microscopy was performed to observe the internalization of MWCNTs in HEK293 cells and rat cardiomyocytes. Current clamp was employed to record the action potentials of isolated rat cardiomyocytes. Surface ECG and epicardial monophasic action potentials were recorded to monitor heart rhythm in rats in vivo. Vagal nerve discharge monitoring and H&E staining were also performed.

Results

Induction of MWCNTs into the cytosole through pipette solution soon accelerated the decay of I _Kv4 in HEK293 cells expressing Kv4.2/4.3 and KChIP2, and promoted the recovery from inactivation when Kv4.2 or Kv4.3 was expressed alone. Longer exposure (6 h) to MWCNTs decreased the I _Kv4.2 density, Kv4.2/Kv4.3 (but not KChIP2) expression and trafficking towards the plasma membrane in HEK293 cells. In acutely isolated rat ventricular myocytes, pipette MWCNTs also quickly accelerated the decay of I _Kv4 and prolonged the action potential duration (APD). Intravenous infusion of MWCNTs (2 mg/rat) induced atrioventricular (AV) block and even cardiac asystole. No tachyarrhythmia was observed after MWCNTs administration. MWCNTs did not cause coronary clot but induced myocardial inflammation and increased vagus discharge.

Conclusions

MWCNTs suppress Kv4/I _to channel activities likely at the intracellular side of plasma membrane, delay membrane repolarization and induce bradyarrhythmia. The delayed repolarization, increased vagus output and focal myocardial inflammation may partially underlie the occurrence of bradyarrhythmias induced by MWCNTs. The study warns that MWCNTs are hazardous to cardiac electrophysiology.     

Traffic of Kv4 K+ channels mediated by KChIP1 is via a novel post-ER vesicular pathway

The traffic of Kv4 K+ channels is regulated by the potassium channel interacting proteins (KChIPs). Kv4.2 expressed alone was not retained within the ER, but reached the Golgi complex. Coexpression of KChIP1 resulted in traffic of the channel to the plasma membrane, and traffic was abolished when mutations were introduced into the EF-hands with channel captured on vesicular structures that colocalized with KChIP1(2-4)-EYFP. The EF-hand mutant had no effect on general exocytic traffic. Traffic of Kv4.2 was coat protein complex I (COPI)-dependent, but KChIP1-containing vesicles were not COPII-coated, and expression of a GTP-loaded Sar1 mutant to block COPII function more effectively inhibited traffic of vesicular stomatitis virus glycoprotein (VSVG) than did KChIP1/Kv4.2 through the secretory pathway. Therefore, KChIP1seems to be targeted to post-ER transport vesicles, different from COPII-coated vesicles and those involved in traffic of VSVG. When expressed in hippocampal neurons, KChIP1 co-distributed with dendritic Golgi outposts; therefore, the KChIP1 pathway could play an important role in local vesicular traffic in neurons.     

C-terminal domain of Kv4.2 and associated KChIP2 interactions regulate functional expression and gating of Kv4.2

The Kv4.2 transient voltage-dependent potassium current contributes to the morphology of the cardiac action potential as well as to neuronal excitability and firing frequency. Here we report profound effects of the Kv4.2 C terminus on the surface expression and activation gating properties of Kv4.2 that are modulated by the direct interaction between KChIP2, an auxiliary regulatory subunit, and the C terminus of Kv4.2. We show that increasingly large truncations of the C terminus of rat Kv4.2 (wild type) cause a progressive decrease of Kv4.2 current along with a shift in voltage-dependent activation that is closely correlated with negative charge deletion. Co-expression of more limited Kv4.2 C-terminal truncation mutants (T588 and T528) with KChIP2 results in a doubling of Kv4.2 protein expression and up to an 8-fold increase in Kv4.2 current amplitude. Pulsechase experiments show that co-expression with KChIP2 slows Kv4.2 wild type degradation 8-fold. Co-expression of KChIP2 with an intermediate-length C-terminal truncation mutant (T474) shifts Kv4.2 activation voltage dependence and enhances expression of Kv4.2 current. The largest truncation mutants (T417 and DeltaC) show an intracellular localization with no measurable currents and no response to KChIP2 co-expression. Co-immunoprecipitation and competitive glutathione S-transferase-binding assays indicate a direct interaction between KChIP2 and the Kv4.2 C terminus with a relative binding affinity comparable with that of the N terminus. Overall, these results suggest that the C-terminal domain of Kv4.2 plays a critical role in voltage-dependent activation and functional expression that is mediated by direct interaction between the Kv4.2 C terminus and KChIP2.     

Interaction of syntaxin 1A with the N-terminus of Kv4.2 modulates channel surface expression and gating

Kv4.2 channels are responsible in the heart for the Ca2+-independent transient outward currents and are important in regulating myocardial excitability and Ca2+ homeostasis. We have identified previously the expression of syntaxin 1A (STX1A) on the cardiac ventricular myocyte plasma membranes, and its modulation of cardiac ATP-sensitive K+ channels. We speculated that STX1A interacts with other cardiac ion channels, thus we examined the interaction of STX1A with Kv4.2 channels. Co-immunoprecipitation and GST pulldown assays demonstrated a direct interaction of STX1A with the Kv4.2 N-terminus. We next investigated the functional alterations of Kv4.2 gating by STX1A in Xenopus oocytes. Coexpression of Kv4.2 with STX1A (1) resulted in a reduction of Kv4.2 current amplitude; (2) caused a depolarizing shift of the steady-state inactivation curve; (3) enhanced the rate of current decay; and (4) accelerated the rate of recovery from inactivation. Additional coexpression of botulinum neurotoxin C, which cleaves STX1A, reversed the effects of STX1A on Kv4.2. STX1A inhibited partially the gating changes by KChIP2, suggesting a competitive interaction of these proteins for an overlapping binding region on the N-terminus of Kv4.2. Indeed, the N-terminal truncation mutants of Kv4.2 (Kv4.2Delta2-40 and Kv4.2Delta7-11), which form part of the KChIP2 binding site, displayed reduced sensitivity to STX1A modulation. Our study suggests that STX1A directly modulates Kv4.2 current amplitude and gating through its interaction with an overlapping region of the KChIP binding motif domain on the Kv4.2 N-terminus.     

Auxiliary KChIP4a Suppresses A-type K+ Current through Endoplasmic Reticulum (ER) Retention and Promoting Closed-state Inactivation of Kv4 Channels

In the brain and heart, auxiliary Kv channel-interacting proteins (KChIPs) co-assemble with pore-forming Kv4 α-subunits to form a native K⁺ channel complex and regulate the expression and gating properties of Kv4 currents. Among the KChIP1–4 members, KChIP4a exhibits a unique N terminus that is known to suppress Kv4 function, but the underlying mechanism of Kv4 inhibition remains unknown. Using a combination of confocal imaging, surface biotinylation, and electrophysiological recordings, we identified a novel endoplasmic reticulum (ER) retention motif, consisting of six hydrophobic and aliphatic residues, 12–17 (LIVIVL), within the KChIP4a N-terminal KID, that functions to reduce surface expression of Kv4-KChIP complexes. This ER retention capacity is transferable and depends on its flanking location. In addition, adjacent to the ER retention motif, the residues 19–21 (VKL motif) directly promote closed-state inactivation of Kv4.3, thus leading to an inhibition of channel current. Taken together, our findings demonstrate that KChIP4a suppresses A-type Kv4 current via ER retention and enhancement of Kv4 closed-state inactivation.     

N-type Inactivation Features of Kv4.2 Channel Gating

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Δ40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K⁺. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Δ40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Δ40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.     

KChIP3 rescues the functional expression of Shal channel tetramerization mutants

KChIP proteins regulate Shal, Kv4.x, channel expression by binding to a conserved sequence at the N terminus of the subunit. The binding of KChIP facilitates a redistribution of Kv4 protein to the cell surface, producing a large increase in current along with significant changes in channel gating kinetics. Recently we have shown that mutants of Kv4.2 lacking the ability to bind an intersubunit Zn(2+) between their T1 domains fail to form functional channels because they are unable to assemble to tetramers and remain trapped in the endoplasmic reticulum. Here we find that KChIPs are capable of rescuing the function of Zn(2+) site mutants by driving the mutant subunits to assemble to tetramers. Thus, in addition to known trafficking effects, KChIPs play a direct role in subunit assembly by binding to monomeric subunits within the endoplasmic reticulum and promoting tetrameric channel assembly. Zn(2+)-less Kv4.2 channels expressed with KChIP3 demonstrate several distinct kinetic changes in channel gating, including a reduced time to peak and faster entry into the inactivated state as well as extending the time to recover from inactivation by 3-4 fold.     

Structural insights into the functional interaction of KChIP1 with Shal-type K(+) channels

Four Kv channel-interacting proteins (KChIP1 through KChIP4) interact directly with the N-terminal domain of three Shal-type voltage-gated potassium channels (Kv4.1, Kv4.2, and Kv4.3) to modulate cell surface expression and function of Kv4 channels. Here we report a 2.0 Angstrom crystal structure of the core domain of KChIP1 (KChIP1*) in complex with the N-terminal fragment of Kv4.2 (Kv4.2N30). The complex reveals a clam-shaped dimeric assembly. Four EF-hands from each KChIP1 form each shell of the clam. The N-terminal end of Kv4.2 forming an alpha helix (alpha1) and the C-terminal alpha helix (H10) of KChIP1 are enclosed nearly coaxially by these shells. As a result, the H10 of KChIP1 and alpha1 of Kv4.2 mediate interactions between these two molecules, structurally reminiscent of the interactions between calmodulin and its target peptides. Site-specific mutagenesis combined with functional characterization shows that those interactions mediated by alpha1 and H10 are essential to the modulation of Kv4.2 by KChIPs.     

Multiple Kv channel-interacting proteins contain an N-terminal transmembrane domain that regulates Kv4 channel trafficking and gating

Kv channel-interacting proteins (KChIPs) are auxiliary subunits of the heteromultimeric channel complexes that underlie neuronal I(SA), the subthreshold transient K(+) current that dynamically regulates membrane excitability, action potential firing properties, and long term potentiation. KChIPs form cytoplasmic associations with the principal pore-forming Kv4 subunits and typically mediate enhanced surface expression and accelerated recovery from depolarization-induced inactivation. An exception is KChIP4a, which dramatically suppresses Kv4 inactivation while promoting neither surface expression nor recovery. These unusual properties are attributed to the effects of a K channel inactivation suppressor domain (KISD) encoded within the variable N terminus of KChIP4a. Here, we have functionally and biochemically characterized two brain KChIP isoforms, KChIP2x and KChIP3x (also known as KChIP3b) and show that they also contain a functional KISD. Like KChIP4a and in contrast with non-KISD-containing KChIPs, both KChIP2x and KChIP3x strongly suppress inactivation and slow activation and inhibit the typical increases in surface expression of Kv4.2 channels. We then examined the properties of the KISD to determine potential mechanisms for its action. Subcellular fractionation shows that KChIP4a, KChIP2x, and KChIP3x are highly associated with the membrane fraction. Fluorescent confocal imaging of enhanced green fluorescent proteins (eGFP) N-terminally fused with KISD in HEK293T cells indicates that KISDs of KChIP4a, KChIP2x, and KChIP3x all autonomously target eGFP to intracellular membranes. Cell surface biotinylation experiments on KChIP4a indicate that the N terminus is exposed extracellularly, consistent with a transmembrane KISD. In summary, KChIP4a, KChIP2x, and KChIP3x comprise a novel class of KChIP isoforms characterized by an unusual transmembrane domain at their N termini that modulates Kv4 channel gating and trafficking.     

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.