Structural basis of gating modulation of Kv4 channel complexes.

Modulation of voltage-gated potassium (Kv) channels by auxiliary subunits is central to the physiological function of channels in the brain and heart1,2. Native Kv4 tetrameric channels form macromolecular ternary complexes with two auxiliary ß-subunits-intracellular Kv channel-interacting proteins (KChIPs) and transmembrane dipeptidyl peptidase-related proteins (DPPs)-to evoke rapidly activating and inactivating A-type currents, which prevent the backpropagation of action potentials1-5. However, the modulatory mechanisms of Kv4 channel complexes remain largely unknown. Here we report cryo-electron microscopy structures of the Kv4.2-DPP6S-KChIP1 dodecamer complex, the Kv4.2-KChIP1 and Kv4.2-DPP6S octamer complexes, and Kv4.2 alone. The structure of the Kv4.2-KChIP1 complex reveals that the intracellular N terminus of Kv4.2 interacts with its C terminus that extends from the S6 gating helix of the neighbouring Kv4.2 subunit. KChIP1 captures both the N and the C terminus of Kv4.2. In consequence, KChIP1 would prevent N-type inactivation and stabilize the S6 conformation to modulate gating of the S6 helices within the tetramer. By contrast, unlike the reported auxiliary subunits of voltage-gated channel complexes, DPP6S interacts with the S1 and S2 helices of the Kv4.2 voltage-sensing domain, which suggests that DPP6S stabilizes the conformation of the S1-S2 helices. DPP6S may therefore accelerate the voltage-dependent movement of the S4 helices. KChIP1 and DPP6S do not directly interact with each other in the Kv4.2-KChIP1-DPP6S ternary complex. Thus, our data suggest that two distinct modes of modulation contribute in an additive manner to evoke A-type currents from the native Kv4 macromolecular complex.

A polybasic motif in alternatively spliced KChIP2 isoforms prevents Ca2+ regulation of Kv4 channels.

The Kv4 family of A-type voltage-gated K+ channels regulates the excitability in hippocampal pyramidal neuron dendrites and are key determinants of dendritic integration, spike timing-dependent plasticity, long-term potentiation, and learning. Kv4.2 channel expression is down-regulated following hippocampal seizures and in epilepsy, suggesting A-type currents as therapeutic targets. In addition to pore-forming Kv4 subunits, modulatory auxiliary subunits called K+ channel-interacting proteins (KChIPs) modulate Kv4 expression and activity and are required to recapitulate native hippocampal A-type currents in heterologous expression systems. KChIP mRNAs contain multiple start sites and alternative exons that generate considerable N-terminal variation and functional diversity in shaping Kv4 currents. As members of the EF-hand domain-containing neuronal Ca2+ sensor protein family, KChIP auxiliary proteins may convey Ca2+ sensitivity upon Kv4 channels; however, to what degree intracellular Ca2+ regulates KChIP-Kv4.2 complexes is unclear. To answer this question, we expressed KChIP2 with Kv4.2 in HEK293T cells, and, with whole-cell patch-clamp electrophysiology, measured an ∼1.5-fold increase in Kv4.2 current density in the presence of elevated intracellular Ca2+ Intriguingly, the Ca2+ regulation of Kv4 current was specific to KChIP2b and KChIP2c splice isoforms that lack a putative polybasic domain that is present in longer KChIP2a1 and KChIP2a isoforms. Site-directed acidification of the basic residues within the polybasic motif of KChIP2a1 rescued Ca2+-mediated regulation of Kv4 current density. These results support divergent Ca2+ regulation of Kv4 channels mediated by alternative splicing of KChIP2 isoforms. They suggest that distinct KChIP-Kv4 interactions may differentially control excitability and function of hippocampal dendrites.

In silico investigation of the interaction between the voltage-gated potassium channel Kv4.3 and its auxiliary protein KChIP1.

The voltage-gated potassium channel Kv4.3 plays a vital role in shaping the timing, frequency, and backpropagation of electrical signals in the brain and heart by generating fast transient currents at subthreshold membrane potentials in repetitive firing neurons. To achieve its physiological function, Kv4.3 is assisted by auxiliary ß-subunits that become integral parts of the native A-type potassium channels, among which there are the Kv channel-interacting proteins (KChIPs). KChIPs are a family of cytosolic proteins that, when coexpressed with Kv4, lead to higher current density, modulation of channel inactivation and faster recovery from inactivation, while the loss of KChIP function may lead to severe pathological states. Recently, the structural basis of the KChIP1-Kv4.3 interaction was reported by using two similar X-ray crystallographic structures, which supported a crucial role for KChIP1 in enhancing the stability of the Kv4.3 tetrameric assembly, thus helping the trafficking of the channel to the plasma membrane. Here, we investigate through fully atomistic simulations the structure and stability of the human Kv4.3 tetramerization (T1) domain in complex with KChIP1 upon specific mutations located in the first and second interfaces of the complex, as compared to the wild-type (WT). Our results nicely complement the available structural and biophysical information collected so far on these complex variants. In particular, the degree of structural deviations and energetic instability, from small to substantial, observed in these variants with respect to the WT model seems to parallel well the level of channel dysfunction known from electrophysiology data. Our simulations provide an octameric structure of the WT KChIP1-Kv4.3 assembly very similar to the known crystal structures, and, at the same time, highlight the importance of a previously overlooked site of interaction between KChIP1 and the Kv4.3 T1 domain.

Calsenilin, a Presenilin Interactor, Regulates RhoA Signaling and Neurite Outgrowth.

Calsenilin modulates A-type potassium channels, regulates presenilin-mediated γ-secretase activity, and represses prodynorphin and c-fos genes expression. RhoA is involved in various cellular functions including proliferation, differentiation, migration, transcription, and regulation of the actin cytoskeleton. Although recent studies demonstrate that calsenilin can directly interact with RhoA and that RhoA inactivation is essential for neuritogenesis, it is uncertain whether there is a link between calsenilin and RhoA-regulated neuritogenesis. Here, we investigated the role of calsenilin in RhoA-regulated neuritogenesis using in vitro and in vivo systems. We found that calsenilin induced RhoA inactivation, which accompanied RhoA phosphorylation and the reduced phosphorylation levels of LIM kinase (LIMK) and cofilin. Interestingly, PC12 cells overexpressing either full-length (FL) or the caspase 3-derived C-terminal fragment (CTF) of calsenilin significantly inactivated RhoA through its interaction with RhoA and p190 Rho GTPase-activating protein (p190RhoGAP). In addition, cells expressing FL and the CTF of calsenilin had increased neurite outgrowth compared to cells expressing the N-terminal fragment (NTF) of calsenilin or vector alone. Moreover, Tat-C3 and Y27632 treatment significantly increased the percentage of neurite-bearing cells, neurite length, and the number of neurites in cells. Finally, calsenilin deficiency in the brains of calsenilin-knockout mice significantly interfered with RhoA inactivation. These findings suggest that calsenilin contributes to neuritogenesis through RhoA inactivation.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca2+-binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca2+ but detaches from DRE under Ca2+ stimulation, allowing gene expression. The Ca2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca2+. Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca2+-binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca2+ signal to CREB-mediated gene transcription.

Characterization of the Photophysical, Thermodynamic, and Structural Properties of the Terbium(III)-DREAM Complex.

DREAM (also known as K(+) channel interacting protein 3 and calsenilin) is a calcium binding protein and an active modulator of KV4 channels in neuronal cells as well as a novel Ca(2+)-regulated transcriptional modulator. DREAM has also been associated with the regulation of Alzheimer's disease through the prevention of presenilin-2 fragmentation. Many interactions of DREAM with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by binding of metals to DREAM is essential for elucidating the mechanism of signal transduction and biological activity of this protein. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analogue, Tb(3+), are enhanced upon binding to the EF-hands of DREAM due to a mechanism of energy transfer between Trp and Tb(3+). We also observe that unlike Tb(3+)-bound calmodulin, the luminescence lifetime of terbium bound to DREAM decays as a complex multiexponential (τaverage ∼ 1.8 ms) that is sensitive to perturbation of the protein structure and drug (NS5806) binding. Using isothermal calorimetry, we have determined that Tb(3+) binds to at least three sites with high affinity (Kd = 1.8 µM in the presence of Ca(2+)) and displaces bound Ca(2+) through an entropically driven mechanism (ΔH ∼ 12 kcal mol(-1), and TΔS ∼ 22 kcal mol(-1)). Furthermore, the hydrophobic probe 1,8-ANS shows that Tb(3+), like Ca(2+), triggers the exposure of a hydrophobic surface on DREAM, which modulates ligand binding. Analogous to Ca(2+) binding, Tb(3+) binding also induces the dimerization of DREAM. Secondary structural analyses using far-UV circular dichroism and trapped ion mobility spectrometry-mass spectrometry reveal that replacement of Ca(2+) with Tb(3+) preserves the folding state with minimal changes to the overall structure of DREAM. These findings pave the way for further investigation of the metal binding properties of DREAM using lanthanides as well as the study of DREAM-protein complexes by lanthanide resonance energy transfer or nuclear magnetic resonance.

Different KChIPs compete for heteromultimeric assembly with pore-forming Kv4 subunits.

Auxiliary Kv channel-interacting proteins 1-4 (KChIPs1-4) coassemble with pore-forming Kv4 α-subunits to form channel complexes underlying somatodendritic subthreshold A-type current that regulates neuronal excitability. It has been hypothesized that different KChIPs can competitively bind to Kv4 α-subunit to form variable channel complexes that can exhibit distinct biophysical properties for modulation of neural function. In this study, we use single-molecule subunit counting by total internal reflection fluorescence microscopy in combinations with electrophysiology and biochemistry to investigate whether different isoforms of auxiliary KChIPs, KChIP4a, and KChIP4bl, can compete for binding of Kv4.3 to coassemble heteromultimeric channel complexes for modulation of channel function. To count the number of photobleaching steps solely from cell membrane, we take advantage of a membrane tethered k-ras-CAAX peptide that anchors cytosolic KChIP4 proteins to the surface for reduction of background noise. Single-molecule subunit counting reveals that the number of KChIP4 isoforms in Kv4.3-KChIP4 complexes can vary depending on the KChIP4 expression level. Increasing the amount of KChIP4bl gradually reduces bleaching steps of KChIP4a isoform proteins, and vice versa. Further analysis of channel gating kinetics from different Kv4-KChIP4 subunit compositions confirms that both KChIP4a and KChIP4bl can modulate the channel complex function upon coassembly. Taken together, our findings show that auxiliary KChIPs can heteroassemble with Kv4 in a competitive manner to form heteromultimeric Kv4-KChIP4 channel complexes that are biophysically distinct and regulated under physiological or pathological conditions.

Amphiphilic Residues 29-44 of DREAM N-Termini Mediate Calmodulin:DREAM Complex Formation.

DREAM (downstream regulatory element antagonist modulator) is a neuronal calcium sensor that has been shown to modulate gene expression as well as to be involved in numerous neuronal processes. In this report, we show that association of calcium-bound calmodulin (CaM) with DREAM is mediated by a short amphipathic amino acid sequence located between residues 29 and 44 on DREAM. The association of CaM with a peptide analogous to DREAM(29-44) or to full-length DREAM protein is calcium-dependent with a dissociation constant of 136 nM or 3.4 µM, respectively. Thermodynamic and kinetic studies show that the observed decrease in affinity for the native protein is due to electrostatic interactions between the basic N-terminus and an electronegative surface on DREAM. These results are further supported by circular dichroism, binding studies, and molecular dynamics simulations. Additionally, fluorescence anisotropy decay measurements show a rotational correlation time of 10.8 ns for a complex of CaM with a DREAM(29-44) peptide, supporting a wraparound semispherical model with 1:1 stoichiometry. Furthermore, the interaction between an IEDANS-labeled CaM construct with DREAM is best modeled as a heterotetramer that adopts an elongated conformation with a correlation time of 45 ns in the presence of Ca(2+). We also demonstrate that association of CaM with DREAM eliminates the nonspecific interaction of DREAM with the DRE double-stranded DNA sequence of the human prodynorphin gene. This work provides molecular insight into the CaM:DREAM complex and its potential role in modulation of gene expression.

Application of ANS fluorescent probes to identify hydrophobic sites on the surface of DREAM.

DREAM (calsenilin or KChIP-3) is a calcium sensor involved in regulation of diverse physiological processes by interactions with multiple intracellular partners including DNA, Kv4 channels, and presenilin, however the detailed mechanism of the recognition of the intracellular partners remains unclear. To identify the surface hydrophobic surfaces on apo and Ca(2+)DREAM as a possible interaction sites for target proteins and/or specific regulators of DREAM function the binding interactions of 1,8-ANS and 2,6-ANS with DREAM were characterized by fluorescence and docking studies. Emission intensity of ANS-DREAM complexes increases upon Ca(2+) association which is consistent with an overall decrease in surface polarity. The dissociation constants for ANS binding to apoDREAM and Ca(2+)DREAM were determined to be 195±20µM and 62±4µM, respectively. Fluorescence lifetime measurements indicate that two ANS molecules bind in two independent binding sites on DREAM monomer. One site is near the exiting helix of EF-4 and the second site is located in the hydrophobic crevice between EF-3 and EF-4. 1,8-ANS displacement studies using arachidonic acid demonstrate that the hydrophobic crevice between EF-3 and EF-4 serves as a binding site for fatty acids that modulate functional properties of Kv4 channel:KChIP complexes. Thus, the C-terminal hydrophobic crevice may be involved in DREAM interactions with small hydrophobic ligands as well as other intracellular proteins.

Evolution of CpG island promoter function underlies changes in KChIP2 potassium channel subunit gene expression in mammalian heart.

Scaling of cardiac electrophysiology with body mass requires large changes in the ventricular action potential duration and heart rate in mammals. These changes in cellular electrophysiological function are produced by systematic and coordinated changes in the expression of multiple ion channel and transporter genes. Expression of one important potassium current, the transient outward current (I(to)), changes significantly during mammalian evolution. Changes in I(to) expression are determined, in part, by variation in the expression of an obligatory auxiliary subunit encoded by the KChIP2 gene. The KChIP2 gene is expressed in both cardiac myocytes and neurons and transcription in both cell types is initiated from the same CpG island promoter. Species-dependent variation of KChIP2 expression in heart is mediated by the evolution of the cis-regulatory function of this gene. Surprisingly, the major locus of evolutionary change for KChIP2 gene expression in heart lies within the CpG island core promoter. The results demonstrate that CpG island promoters are not simply permissive for gene expression but can also contribute to tissue-selective expression and, as such, can function as an important locus for the evolution of cis-regulatory function. More generally, evolution of the cis-regulatory function of voltage-gated ion channel genes appears to be an effective and efficient way to modify channel expression levels to optimize electrophysiological function.

Interactions of KChIP4a and its mutants with Ca2+ or Kv4.3 N-terminus by affinity capillary electrophoresis.

The specific binding of auxiliary Kv channel-interacting proteins (KChIPs) to the N terminus of Kv4 pore-forming α-subunits results in modulation of gating properties, surface expression, and subunit assembly of Kv4 channels. However, the interactions between KChIPs and Kv4 remain elusive. Thus, affinity capillary electrophoresis (ACE) was employed to quantitatively evaluate the interactions between KChIPs and Kv4.3 N terminus (KvN) and between KChIP4a/related mutants and Ca(2+) for the first time. The mobility ratio, derivatives calculated from the mobility shift method, was used to deduce the binding constants (Kb). As a result, the binding constants for KChIP4a/KvN and KChIP1/KvN complexes were (8.32±1.66)×10(6) L mol(-1) and (5.26±0.71)×10(6) L mol(-1), respectively. In addition, in the presence of calcium (10 µmol L(-1)), the binding constant of KChIP4a/KvN increased to (6.72±1.66)×10(7) L mol(-1). In addition, the binding constant of KChIP4a with Ca(2+) was (7.1±1.5)×10(7) L mol(-1). Besides, studies on the effect of truncated mutants revealed that the third EF hand of KChIP4a was related to high-affinity binding with Ca(2+), and the integrity of the molecular structure of KChIP4a was important for Ca(2+) binding. This method profits from small samples, rapid analysis, and simple operation without being time-consuming.

Protein aggregation due to nsSNP resulting in P56S VABP protein is associated with amyotrophic lateral sclerosis.

Mutations in the gene encoding vesicle-associated membrane protein (VAPB) cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. The VAPB gene is mapped to chromosome number 20 and can be found at cytogenetic location 20q13.33 of the chromosome. VAPB is seen to play a significant role in the unfolded protein response (UPR), which is a process that suppresses the accumulation of unfolded proteins in the endoplasmic reticulum. Earlier studies have reported two points; which we have analyzed in our study. Firstly, the mutation P56S in the VAPB is seen to increase the stability of the protein and secondly, the mutation P56S in VAPB is seen to interrupt the functioning of the gene and loses its ability to be involved in the activation of the IRE1/XBP1 pathway which leads to ALS. With correlation on the previous research studies on the stability of this protein, we carried out Molecular dynamics (MD) simulation. We analyzed the SNP results of 17 nsSNPs obtained from dbSNP using SIFT, polyphen, I-Mutant, SNP&GO, PhDSNP and Mutpred to predict the role of nsSNPs in VAPB. MD simulation is carried out and plots for RMSD, RMSF, Rg, SASA, H-bond and PCA are obtained to check and prove the stability of the wild type and the mutant protein structure. The protein is checked for its aggregation and the results obtained show changes in the protein structure that might result in the loss of function.

The potassium channel interacting protein 3 (DREAM/KChIP3) heterodimerizes with and regulates calmodulin function.

Downstream regulatory element antagonistic modulator (DREAM/KChIP3), a neuronal EF-hand protein, modulates pain, potassium channel activity, and binds presenilin 1. Using affinity capture of neuronal proteins by immobilized DREAM/KChIP3 in the presence and absence of calcium (Ca(2+)) followed by mass spectroscopic identification of interacting proteins, we demonstrate that in the presence of Ca(2+), DREAM/KChIP3 interacts with the EF-hand protein, calmodulin (CaM). The interaction of DREAM/KChIP3 with CaM does not occur in the absence of Ca(2+). In the absence of Ca(2+), DREAM/KChIP3 binds the EF-hand protein, calcineurin subunit-B. Ca(2+)-bound DREAM/KChIP3 binds CaM with a dissociation constant of ∼3 µM as assessed by changes in DREAM/KChIP3 intrinsic protein fluorescence in the presence of CaM. Two-dimensional (1)H,(15)N heteronuclear single quantum coherence spectra reveal changes in chemical shifts and line broadening upon the addition of CaM to (15)N DREAM/KChIP3. The amino-terminal portion of DREAM/KChIP3 is required for its binding to CaM because a construct of DREAM/KChIP3 lacking the first 94 amino-terminal residues fails to bind CaM as assessed by fluorescence spectroscopy. The addition of Ca(2+)-bound DREAM/KChIP3 increases the activation of calcineurin (CN) by calcium CaM. A DREAM/KChIP3 mutant incapable of binding Ca(2+) also stimulates calmodulin-dependent CN activity. The shortened form of DREAM/KChIP3 lacking the NH(2)-terminal amino acids fails to activate CN in the presence of calcium CaM. Our data demonstrate the interaction of DREAM/KChIP3 with the important EF-hand protein, CaM, and show that the interaction alters CN activity.

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.

Sumoylation regulates nuclear localization of repressor DREAM.

DREAM is a Ca(2+)-binding protein with specific functions in different cell compartments. In the nucleus, DREAM acts as a transcriptional repressor, although the mechanism that controls its nuclear localization is unknown. Yeast two-hybrid assay revealed the interaction between DREAM and the SUMO-conjugating enzyme Ubc9 and bioinformatic analysis identified four sumoylation-susceptible sites in the DREAM sequence. Single K-to-R mutations at positions K26 and K90 prevented in vitro sumoylation of recombinant DREAM. DREAM sumoylation mutants retained the ability to bind to the DRE sequence but showed reduced nuclear localization and failed to regulate DRE-dependent transcription. In PC12 cells, sumoylated DREAM is present exclusively in the nucleus and neuronal differentiation induced nuclear accumulation of sumoylated DREAM. In fully differentiated trigeminal neurons, DREAM and SUMO-1 colocalized in nuclear domains associated with transcription. Our results show that sumoylation regulates the nuclear localization of DREAM in differentiated neurons. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

The Murine PSE/TATA-dependent transcriptome: evidence of functional homologies with its human counterpart.

A series of recent studies demonstrated an unexpectedly high frequency of intronic RNA polymerase (pol) III transcription units spread throughout the human genome. The investigation of a subset of these transcripts revealed their tissue/cell-specific transcription together with the involvement in relevant physiopathological pathways. Despite this evidence, these transcripts did not seem to have murine orthologs, based on their nucleotide sequence, resulting in a limitation of the experimental approaches aimed to study their function. In this work, we have extended our investigation to the murine genome identifying 121 pairs of mouse/human transcripts displaying syntenic subchromosomal localization. The analysis in silico of this set of putative noncoding (nc)RNAs suggest their association with alternative splicing as suggested by recent experimental evidence. The investigation of one of these pairs taken as experimental model in mouse hippocampal neurons provided evidence of a human/mouse functional homology that does not depend on underlying sequence conservation. In this light, the collection of transcriptional units here reported can be considered as a novel source for the identification and the study of novel regulatory elements involved in relevant biological processes.

Three-dimensional structure of the KChIP1-Kv4.3 T1 complex reveals a cross-shaped octamer.

Brain I(A) and cardiac I(to) currents arise from complexes containing Kv4 voltage-gated potassium channels and cytoplasmic calcium-sensor proteins (KChIPs). Here, we present X-ray crystallographic and small-angle X-ray scattering data that show that the KChIP1-Kv4.3 N-terminal cytoplasmic domain complex is a cross-shaped octamer bearing two principal interaction sites. Site 1 comprises interactions between a unique Kv4 channel N-terminal hydrophobic segment and a hydrophobic pocket formed by displacement of the KChIP H10 helix. Site 2 comprises interactions between a T1 assembly domain loop and the KChIP H2 helix. Functional and biochemical studies indicate that site 1 influences channel trafficking, whereas site 2 affects channel gating, and that calcium binding is intimately linked to KChIP folding and complex formation. Together, the data resolve how Kv4 channels and KChIPs interact and provide a framework for understanding how KChIPs modulate Kv4 function.

Structural basis for modulation of Kv4 K+ channels by auxiliary KChIP subunits.

KChIPs coassemble with pore-forming Kv4 alpha subunits to form a native complex in the brain and heart and regulate the expression and gating properties of Kv4 K(+) channels, but the mechanisms underlying these processes are unknown. Here we report a co-crystal structure of the complex of human Kv4.3 N-terminus and KChIP1 at a 3.2-A resolution. The structure reveals a unique clamping action of the complex, in which a single KChIP1 molecule, as a monomer, laterally clamps two neighboring Kv4.3 N-termini in a 4:4 manner, forming an octamer. The proximal N-terminal peptide of Kv4.3 is sequestered by its binding to an elongated groove on the surface of KChIP1, which is indispensable for the modulation of Kv4.3 by KChIP1, and the same KChIP1 molecule binds to an adjacent T1 domain to stabilize the tetrameric Kv4.3 channels. Taken together with biochemical and functional data, our findings provide a structural basis for the modulation of Kv4 by KChIPs.

CIB2 and CIB3 are auxiliary subunits of the mechanotransduction channel of hair cells.

CIB2 is a Ca2+- and Mg2+-binding protein essential for mechanoelectrical transduction (MET) by cochlear hair cells, but not by vestibular hair cells that co-express CIB2 and CIB3. Here, we show that in cochlear hair cells, CIB3 can functionally substitute for CIB2. Using X-ray crystallography, we demonstrate that CIB2 and CIB3 are structurally similar to KChIP proteins, auxiliary subunits of voltage-gated Kv4 channels. CIB2 and CIB3 bind to TMC1/2 through a domain in TMC1/2 flanked by transmembrane domains 2 and 3. The co-crystal structure of the CIB-binding domain in TMC1 with CIB3 reveals that interactions are mediated through a conserved CIB hydrophobic groove, similar to KChIP1 binding of Kv4. Functional studies in mice show that CIB2 regulates TMC1/2 localization and function in hair cells, processes that are affected by deafness-causing CIB2 mutations. We conclude that CIB2 and CIB3 are MET channel auxiliary subunits with striking similarity to Kv4 channel auxiliary subunits.

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.