Kv Channel-interacting Proteins Research Articles

KV Channel-Interacting Proteins in the Neurological and Cardiovascular Systems: An Updated Review.

KV channel-interacting proteins (KChIP1-4) belong to a family of Ca2+-binding EF-hand proteins that are able to bind to the N-terminus of the KV4 channel α-subunits. KChIPs are predominantly expressed in the brain and heart, where they contribute to the maintenance of the excitability of neurons and cardiomyocytes by modulating the fast inactivating-KV4 currents. As the auxiliary subunit, KChIPs are critically involved in regulating the surface protein expression and gating properties of KV4 channels. Mechanistically, KChIP1, KChIP2, and KChIP3 promote the translocation of KV4 channels to the cell membrane, accelerate voltage-dependent activation, and slow the recovery rate of inactivation, which increases KV4 currents. By contrast, KChIP4 suppresses KV4 trafficking and eliminates the fast inactivation of KV4 currents. In the heart, IKs, ICa,L, and INa can also be regulated by KChIPs. ICa,L and INa are positively regulated by KChIP2, whereas IKs is negatively regulated by KChIP2. Interestingly, KChIP3 is also known as downstream regulatory element antagonist modulator (DREAM) because it can bind directly to the downstream regulatory element (DRE) on the promoters of target genes that are implicated in the regulation of pain, memory, endocrine, immune, and inflammatory reactions. In addition, all the KChIPs can act as transcription factors to repress the expression of genes involved in circadian regulation. Altered expression of KChIPs has been implicated in the pathogenesis of several neurological and cardiovascular diseases. For example, KChIP2 is decreased in failing hearts, while loss of KChIP2 leads to increased susceptibility to arrhythmias. KChIP3 is increased in Alzheimer's disease and amyotrophic lateral sclerosis, but decreased in epilepsy and Huntington's disease. In the present review, we summarize the progress of recent studies regarding the structural properties, physiological functions, and pathological roles of KChIPs in both health and disease. We also summarize the small-molecule compounds that regulate the function of KChIPs. This review will provide an overview and update of the regulatory mechanism of the KChIP family and the progress of targeted drug research as a reference for researchers in related fields.

Identification of Kv4.2 protein complex and modifications by tandem affinity purification-mass spectrometry in primary neurons.

Proteins usually form complexes to fulfill variable physiological functions. In neurons, communication relies on synapses where receptors, channels, and anchoring proteins form complexes to precisely control signal transduction, synaptic integration, and action potential firing. Although there are many published protocols to isolate protein complexes in cell lines, isolation in neurons has not been well established. Here we introduce a method that combines lentiviral protein expression with tandem affinity purification followed by mass-spectrometry (TAP-MS) to identify protein complexes in neurons. This protocol can also be used to identify post-translational modifications (PTMs) of synaptic proteins. We used the A-type voltage-gated K+ channel subunit Kv4.2 as the target protein. Kv4.2 is highly expressed in the hippocampus where it contributes to learning and memory through its regulation of neuronal excitability and synaptic plasticity. We tagged Kv4.2 with the calmodulin-binding-peptide (CBP) and streptavidin-binding-peptide (SBP) at its C-terminus and expressed it in neurons via lentivirus. Kv4.2 was purified by two-step TAP and samples were analyzed by MS. MS identified two prominently known Kv4.2 interacting proteins [dipeptidyl peptidase like (DPPs) and Kv channel-interacting proteins (KChIPs)] in addition to novel synaptic proteins including glutamate receptors, a calcium channel, and anchoring proteins. Co-immunoprecipitation and colocalization experiments validated the association of Kv4.2 with glutamate receptors. In addition to protein complex identification, we used TAP-MS to identify Kv4.2 phosphorylation sites. Several known and unknown phosphorylation sites were identified. These findings provide a novel path to identify protein-protein interactions and PTMs in neurons and shed light on mechanisms of neuronal signaling potentially involved in the pathology of neurological diseases.

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 β-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.

Modulation of Kv4.2/KChIP3 interaction by the ceroid lipofuscinosis neuronal 3 protein CLN3

Voltage-gated potassium (Kv) channels of the Kv4 subfamily associate with Kv channel-interacting proteins (KChIPs), which leads to enhanced surface expression and shapes the inactivation gating of these channels. KChIP3 has been reported to also interact with the late endosomal/lysosomal membrane glycoprotein CLN3 (ceroid lipofuscinosis neuronal 3), which is modified because of gene mutation in juvenile neuronal ceroid lipofuscinosis (JNCL). The present study was undertaken to find out whether and how CLN3, by its interaction with KChIP3, may indirectly modulate Kv4.2 channel expression and function. To this end, we expressed KChIP3 and CLN3, either individually or simultaneously, together with Kv4.2 in HEK 293 cells. We performed co-immunoprecipitation experiments and found a lower amount of KChIP3 bound to Kv4.2 in the presence of CLN3. In whole-cell patch-clamp experiments, we examined the effects of CLN3 co-expression on the KChIP3-mediated modulation of Kv4.2 channels. Simultaneous co-expression of CLN3 and KChIP3 with Kv4.2 resulted in a suppression of the typical KChIP3-mediated modulation; i.e. we observed less increase in current density, less slowing of macroscopic current decay, less acceleration of recovery from inactivation, and a less positively shifted voltage dependence of steady-state inactivation. The suppression of the KChIP3-mediated modulation of Kv4.2 channels was weaker for the JNCL-related missense mutant CLN3R334C and for a JNCL-related C-terminal deletion mutant (CLN3ΔC). Our data support the notion that CLN3 is involved in Kv4.2/KChIP3 somatodendritic A-type channel formation, trafficking, and function, a feature that may be lost in JNCL.

SUMOylating Two Distinct Sites on the A-type Potassium Channel, Kv4.2, Increases Surface Expression and Decreases Current Amplitude.

Post-translational conjugation of Small Ubiquitin-like Modifier (SUMO) peptides to lysine (K) residues on target proteins alters their interactions. SUMOylation of a target protein can either promote its interaction with other proteins that possess SUMO binding domains, or it can prevent target protein interactions that normally occur in the absence of SUMOylation. One subclass of voltage-gated potassium channels that mediates an A-type current, IA, exists as a ternary complex comprising Kv4 pore-forming subunits, Kv channel interacting proteins (KChIP) and transmembrane dipeptidyl peptidase like proteins (DPPL). SUMOylation could potentially regulate intra- and/or intermolecular interactions within the complex. This study began to test this hypothesis and showed that Kv4.2 channels were SUMOylated in the rat brain and in human embryonic kidney (HEK) cells expressing a GFP-tagged mouse Kv4.2 channel (Kv4.2g). Prediction software identified two putative SUMOylation sites in the Kv4.2 C-terminus at K437 and K579. These sites were conserved across mouse, rat, and human Kv4.2 channels and across mouse Kv4 isoforms. Increasing Kv4.2g SUMOylation at each site by ~30% produced a significant ~22%–50% decrease in IA Gmax, and a ~70%–95% increase in channel surface expression. Site-directed mutagenesis of Kv4.2g showed that K437 SUMOylation regulated channel surface expression, while K579 SUMOylation controlled IA Gmax. The K579R mutation mimicked and occluded the SUMOylation-mediated decrease in IA Gmax, suggesting that SUMOylation at K579 blocked an intra- or inter-protein interaction involving K579. The K437R mutation did not obviously alter channel surface expression or biophysical properties, but it did block the SUMOylation-mediated increase in channel surface expression. Interestingly, enhancing K437 SUMOylation in the K579R mutant roughly doubled channel surface expression, but produced no change in IA Gmax, suggesting that the newly inserted channels were electrically silent. This is the first report that Kv4.2 channels are SUMOylated and that SUMOylation can independently regulate Kv4.2 surface expression and IA Gmax in opposing directions. The next step will be to determine if/how SUMOylation affects Kv4 interactions within the ternary complex.

OR23-3 Differential Effects of Chronic Exposure to Bisphenol-A on Ion Channel Activity and Expression in Mouse Pancreatic Beta-Cells

Many dairy products found in plastics, cosmetics and food containers disrupt human health due to their actions as endocrine-disrupting chemicals (EDCs). Studies in different cellular and animal models have reported that low doses of the EDC bisphenol A (BPA), modify pancreatic β-cell function, induce insulin resistance and other metabolic alterations. Here, we aimed to investigate whether administration of low doses of BPA could regulate ion channels expression and function in mice pancreatic islets. For this purpose, we used microarrays to analyze the global gene expression profile, focusing on the modulation of ion channels-related genes. For in vivo experiments, mice were subcutaneously injected with BPA (100 μg/kg/day during 4 days). Islets were isolated 12 h after the last injection and prepared for RNA extraction and hybridization on Affymetrix GeneChip Mouse Genome 430 2.0 Array. Array scanning and data analysis were performed using Affymetrix Expression Console and the Affymetrix Transcriptome Analysis Console (TAC) Softwares. Expression level analysis was performed using the normalization method based on the processing algorithm called robust multi-array average (RMA). Whole islets or dispersed islets from C57BL/6 or estrogen receptor beta knockout (βERKO) mice were treated in vitro with BPA or vehicle during 48 h. Whole-cell patch-clamp recordings were used to measure sodium and potassium currents. Microarray analysis of islets isolated from mice treated with BPA showed that some ion channels, such as sodium channel voltage-gated type IX, alpha (Scn9a), potassium large conductance calcium-activated channel alpha member 1 (Kcnma1), and Kv channel-interacting protein 1 (Kcnip1), were modulated. These results were confirmed by quantitative RT-PCR. Electrophysiological measurements showed a decrease in both sodium currents and total potassium currents. Additionally, the currents through the voltage-gated potassium channel subunits Kv2.1 and 2.2 as well as the KCa1.1 (encoded by Kcnma1 gene) channels were also reduced. Interestingly, beta-cells from βERKO mice did not present such reductions, suggesting that these effects occur mainly via ERβ. Our results show that, acting as a xenoestrogen, BPA modulates sodium and potassium currents as well as gene expression via ERβ. Funding: Ministerio de Economia y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER), EU Grants SAF2014-58335-P, BFU2017-86579-R, BFU2016-77125-R and Generalitat Valenciana PROMETEO II/2015/016. LM holds a Juan de la Cierva fellowship from the Ministry of Economy, Industry and Competitiveness (IJCI-2015-24482). CIBERDEM is an initiative of the Instituto de Salud Carlos III. J-AG was supported by the Robert A. Welch Foundation (E-0004).

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 β-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.

Interaction of nectin-2α with the auxiliary protein of the voltage-gated A-type K+ channel Kv4.2 dipeptidyl aminopeptidase-like protein at the boundary between the adjacent somata of clustered cholinergic neurons in the medial habenula

The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.

Somatodendritic surface expression of epitope-tagged and KChIP binding-deficient Kv4.2 channels in hippocampal neurons.

Kv4.2 channels mediate a subthreshold-activating somatodendritic A-type current (ISA) in hippocampal neurons. We examined the role of accessory Kv channel interacting protein (KChIP) binding in somatodendritic surface expression and activity-dependent decrease in the availability of Kv4.2 channels. For this purpose we transfected cultured hippocampal neurons with cDNA coding for Kv4.2 wild-type (wt) or KChIP binding-deficient Kv4.2 mutants. All channels were equipped with an externally accessible hemagglutinin (HA)-tag and an EGFP-tag, which was attached to the C-terminal end. Combined analyses of EGFP self-fluorescence, surface HA immunostaining and patch-clamp recordings demonstrated similar dendritic trafficking and functional surface expression for Kv4.2[wt]HA,EGFP and the KChIP binding-deficient Kv4.2[A14K]HA,EGFP. Coexpression of exogenous KChIP2 augmented the surface expression of Kv4.2[wt]HA,EGFP but not Kv4.2[A14K]HA,EGFP. Notably, activity-dependent decrease in availability was more pronounced in Kv4.2[wt]HA,EGFP + KChIP2 coexpressing than in Kv4.2[A14K]HA,EGFP + KChIP2 coexpressing neurons. Our results do not support the notion that accessory KChIP binding is a prerequisite for dendritic trafficking and functional surface expression of Kv4.2 channels, however, accessory KChIP binding may play a potential role in Kv4.2 modulation during intrinsic plasticity processes.

Notch signaling modulates the electrical behavior of cardiomyocytes.

Notch receptor signaling is active during cardiac development and silenced in myocytes after birth. Conversely, outward K+ Kv currents progressively appear in postnatal myocytes leading to shortening of the action potential (AP) and acquisition of the mature electrical phenotype. In the present study, we tested the possibility that Notch signaling modulates the electrical behavior of cardiomyocytes by interfering with Kv currents. For this purpose, the effects of Notch receptor activity on electrophysiological properties of myocytes were evaluated using transgenic mice with inducible expression of the Notch1 intracellular domain (NICD), the functional fragment of the activated Notch receptor, and in neonatal myocytes after inhibition of the Notch transduction pathway. By patch clamp, NICD-overexpressing cells presented prolonged AP duration and reduced upstroke amplitude, properties that were coupled with reduced rapidly activating Kv and fast Na+ currents, compared with cells obtained from wild-type mice. In cultured neonatal myocytes, inhibition of the proteolitic release of NICD with a γ-secretase antagonist increased transcript levels of the Kv channel-interacting proteins 2 (KChIP2) and enhanced the density of Kv currents. Collectively, these results indicate that Notch signaling represents an important regulator of the electrophysiological behavior of developing and adult myocytes by repressing, at least in part, repolarizing Kv currents. NEW & NOTEWORTHY We investigated the effects of Notch receptor signaling on the electrical properties of cardiomyocytes. Our results indicate that the Notch transduction pathway interferes with outward K+ Kv currents, critical determinants of the electrical repolarization of myocytes.

Modulation of human Kv4.3/KChIP2 channel inactivation kinetics by cytoplasmic Ca2.

The transient outward current (I to) in the human heart is mediated by Kv4.3 channels complexed with Kv channel interacting protein (KChIP) 2, a cytoplasmic Ca2+-binding EF-hand protein known to modulate Kv4.3 inactivation gating upon heterologous co-expression. We studied Kv4.3 channels co-expressed with wild-type (wt) or EF-hand-mutated (ΔEF) KChIP2 in human embryonic kidney (HEK) 293 cells. Co-expression took place in the absence or presence of BAPTA-AM, and macroscopic currents were recorded in the whole-cell patch-clamp configuration with different free Ca2+ concentrations in the patch-pipette. Our data indicate that Ca2+ is not necessary for Kv4.3/KChIP2 complex formation. The Kv4.3/KChIP2-mediated current decay was faster and the recovery of Kv4.3/KChIP2 channels from inactivation slower with 50μM Ca2+ than with BAPTA (nominal Ca2+-free) in the patch-pipette. The apparent Ca2+-mediated slowing of recovery kinetics was still observed when EF-hand 4 of KChIP2 was mutated (ΔEF4) but not when EF-hand 2 (ΔEF2) was mutated, and turned into a Ca2+-mediated acceleration of recovery kinetics when EF-hand 3 (ΔEF3) was mutated. In the presence of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93 cytoplasmic Ca2+ (50μM) induced an acceleration of Kv4.3/KChIP2 recovery kinetics, which was still observed when EF-hand 2 was mutated (ΔEF2) but not when EF-hand 3 (ΔEF3) or EF-hand 4 (ΔEF4) was mutated. Our results support the notion that binding of Ca2+ to KChIP2 EF-hands can acutely modulate Kv4.3/KChIP2 channel inactivation gating, but the Ca2+-dependent gating modulation depends on CaMKII action. Our findings speak for an acute modulation of I to kinetics and frequency-dependent I to availability in cardiomyocytes under conditions with elevated Ca2+ levels and CaMKII activity.

Effect of eicosapentaenoic acid and pitavastatin on electrophysiology and anticoagulant gene expression in mice with rapid atrial pacing.

Atrial remodeling is considered to be any persistent change in atrial structure or function, and is responsible for the development and perpetuation of atrial fibrillation (AF). Oxidative stress and intracellular pH regulation may also be linked to AF; however it remains unclear whether eicosapentaenoic acid (EPA) or statins have beneficial therapeutic effects. The aim of the present study was to investigate the effects of EPA and pitavastatin on the electrophysiology of and gene expressions in mice with rapidly-paced atria. Mice were treated with EPA (10 mg/g/day) or pitavastatin (30 ng/g/day) for 6 weeks, following which AF was simulated by 8-h atrial pacing at 1,800 bpm. The atrial electrophysiological properties and the expression of cardiac genes, potassium voltage-gated channel subfamily A member 5 (Kcna5), Kcn subfamily D member 2 (Kcnd2), Kv channel-interacting protein 2 (KChIP2), solute carrier family 9 member A1, thrombomodulin (TM) and tissue factor pathway inhibitor (TFPI) were examined using reverse transcription-quantitative polymerase chain reaction. In control mice, significant atrial electrical remodeling was observed (P<0.05); however, treatment with either EPA or pitavastatin ameliorated these electrophysiological changes (P>0.05). mRNA levels of Kcnd2, KChIP2 and Kcna5 were significantly upregulated in control mice (P<0.05), whereas treatment with EPA or pitavastatin attenuated this upregulation (P>0.05). Administration of pitavastatin significantly reduced the downregulation of both TFPI and TM (P<0.05). EPA treatment attenuated the TFPI downregulation compared with control mice (P>0.05), however no significant effect on TM expression was observed. In addition, both EPA (P>0.05) and pitavastatin (P<0.05) suppressed the overexpression of endothelial nitric oxide synthase. This was also exhibited in Ras-related C3 botulinum toxin substrate 1 genes (P<0.01 for both treatments). In conclusion, the results of the present study suggested that EPA and pitavastatin are able to prevent atrial electrical remodeling, thrombotic states and oxidative stress in rapidly-paced murine atria.

Vascular Endothelial Growth Factor-B Induces a Distinct Electrophysiological Phenotype in Mouse Heart.

Vascular endothelial growth factor B (VEGF-B) is a potent mediator of vascular, metabolic, growth, and stress responses in the heart, but the effects on cardiac muscle and cardiomyocyte function are not known. The purpose of this study was to assess the effects of VEGF-B on the energy metabolism, contractile, and electrophysiological properties of mouse cardiac muscle and cardiac muscle cells. In vivo and ex vivo analysis of cardiac-specific VEGF-B TG mice indicated that the contractile function of the TG hearts was normal. Neither the oxidative metabolism of isolated TG cardiomyocytes nor their energy substrate preference showed any difference to WT cardiomyocytes. Similarly, myocyte Ca2+ signaling showed only minor changes compared to WT myocytes. However, VEGF-B overexpression induced a distinct electrophysiological phenotype characterized by ECG changes such as an increase in QRSp time and decreases in S and R amplitudes. At the level of isolated TG cardiomyocytes, these changes were accompanied with decreased action potential upstroke velocity and increased duration (APD60–70). These changes were partly caused by downregulation of sodium current (INa) due to reduced expression of Nav1.5. Furthermore, TG myocytes had alterations in voltage-gated K+ currents, namely decreased density of transient outward current (Ito) and total K+ current (Ipeak). At the level of transcription, these were accompanied by downregulation of Kv channel-interacting protein 2 (Kcnip2), a known modulatory subunit for Kv4.2/3 channel. Cardiac VEGF-B overexpression induces a distinct electrophysiological phenotype including remodeling of cardiomyocyte ion currents, which in turn induce changes in action potential waveform and ECG.

KChIP2 genotype dependence of transient outward current (Ito) properties in cardiomyocytes isolated from male and female mice.

The transient outward current (Ito) in cardiomyocytes is largely mediated by Kv4 channels associated with Kv Channel Interacting Protein 2 (KChIP2). A knockout model has documented the critical role of KChIP2 in Ito expression. The present study was conducted to characterize in both sexes the dependence of Ito properties, including current magnitude, inactivation kinetics, recovery from inactivation and voltage dependence of inactivation, on the number of functional KChIP2 alleles. For this purpose we performed whole-cell patch-clamp experiments on isolated left ventricular cardiomyocytes from male and female mice which had different KChIP2 genotypes; i.e., wild-type (KChIP2+/+), heterozygous knockout (KChIP2+/-) or complete knockout of KChIP2 (KChIP2-/-). We found in both sexes a KChIP2 gene dosage effect (i.e., a proportionality between number of alleles and phenotype) on Ito magnitude, however, concerning other Ito properties, KChIP2+/- resembled KChIP2+/+. Only in the total absence of KChIP2 (KChIP2-/-) we observed a slowing of Ito kinetics, a slowing of recovery from inactivation and a negative shift of a portion of the voltage dependence of inactivation. In a minor fraction of KChIP2-/- myocytes Ito was completely lost. The distinct KChIP2 genotype dependences of Ito magnitude and inactivation kinetics, respectively, seen in cardiomyocytes were reproduced with two-electrode voltage-clamp experiments on Xenopus oocytes expressing Kv4.2 and different amounts of KChIP2. Our results corroborate the critical role of KChIP2 in controlling Ito properties. They demonstrate that the Kv4.2/KChIP2 interaction in cardiomyocytes is highly dynamic, with a clear KChIP2 gene dosage effect on Kv4 channel surface expression but not on inactivation gating.

Abstract 386: Notch1 Signaling Negatively Modulates Repolarizing Kv Currents in Cardiomyocytes

Notch1 is a critical signaling pathway during embryonic and early postnatal development and activity of this transduction system gradually declines after birth. Conversely, outward K + Kv currents are progressively increased in myocytes during postnatal life leading to shortening of the action potential (AP) duration and acquisition of the mature electrical phenotype. Thus, we tested the possibility that Notch1 signaling modulates the electrophysiological properties of developing cells by interfering with Kv current densities. For this purpose, molecular and physiological studies were conducted in vitro using mouse neonatal myocytes (NMs). These assays were complemented with tests in adult cells obtained from a conditional mouse model of Notch1 intracellular domain (NICD) overexpression. NMs were treated with a γ-secretase inhibitor (DAPT) to prevent the cleavage of Notch1 receptor and subsequent translocation of the active NICD to the nucleus. By RT-PCR, Notch1 targets Hes1 and Hey1 were reduced with DAPT-treatment, confirming the effective perturbation of Notch1 signaling. Importantly, inhibition of Notch signaling led to upregulation of the three splice variants of the Kv channel-interacting proteins 2 (KChIP2) gene, which controls the appearance of outward Kv currents in myocytes. By patch-clamp, Kv currents were identified in only 15% of NMs (n=20) cultured for 1-2 days in in control condition, whereas this fraction increased to 50% (n=18) in cells treated with DAPT. Thus, Notch inhibition promotes the expression of KChIP2 and appearance of Kv currents. To clarify these in vitro findings, the consequences of ectopic activation of Notch1 signaling on the electrical behavior of adult myocytes were established. By patch-clamp, NICD-overexpressing myocytes presented a &gt;2-fold longer duration of the early repolarization phase of the AP, with respect to cells from wild type hearts. This alteration was coupled with a &gt;50% reduction of Kv currents I to and I Kslow1 in NICD-overexpressing cells, with respect to wild-type myocytes. Thus, Notch signaling represses KChIP2 and Kv currents in cardiomyocytes representing an important modulator of the electrical phenotype of the developing heart.

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.

Effect of tyrphostin AG879 on Kv 4.2 and Kv 4.3 potassium channels.

A-type potassium channels (IA) are important proteins for modulating neuronal membrane excitability. The expression and activity of Kv 4.2 channels are critical for neurological functions and pharmacological inhibitors of Kv 4.2 channels may have therapeutic potential for Fragile X syndrome. While screening various compounds, we identified tyrphostin AG879, a tyrosine kinase inhibitor, as a Kv 4.2 inhibitor from. In the present study we characterized the effect of AG879 on cloned Kv 4.2/Kv channel-interacting protein 2 (KChIP2) channels. To screen the library of pharmacologically active compounds, the thallium flux assay was performed on HEK-293 cells transiently-transfected with Kv 4.2 cDNA using the Maxcyte transfection system. The effects of AG879 were further examined on CHO-K1 cells expressing Kv 4.2/KChIP2 channels using a whole-cell patch-clamp technique. Tyrphostin AG879 selectively and dose-dependently inhibited Kv 4.2 and Kv 4.3 channels. In Kv 4.2/KChIP2 channels, AG879 induced prominent acceleration of the inactivation rate, use-dependent block and slowed the recovery from inactivation. AG879 induced a hyperpolarizing shift in the voltage-dependence of the steady-state inactivation of Kv 4.2 channels without apparent effect on the V1/2 of the voltage-dependent activation. The blocking effect of AG879 was enhanced as channel inactivation increased. Furthermore, AG879 significantly inhibited the A-type potassium currents in the cultured hippocampus neurons. AG879 was identified as a selective and potent inhibitor the Kv 4.2 channel. AG879 inhibited Kv 4.2 channels by preferentially interacting with the open state and further accelerating their inactivation.

Dynamic Subunit Stoichiometry of Kv4.3-KChIP4A Channel Complexes Visualized by Single-Molecule Subunit Counting

Auxiliary Kv channel-interacting proteins 1-4 (KChIPs1-4) co-assemble with pore-forming Kv4 α-subunits to underlie 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 dynamic channel complexes that can exhibit distinct biophysical properties for modulation of neural function. To test this hypothesis, we utilized single molecule subunit counting to investigate whether different isoforms of auxiliary KChIPs such as KChIP4a and KChIP4bl can compete for binding of Kv4.3 to co-assemble hetero-multimeric channel complexes. Single-molecule imaging subunit counting revealed that the number of KChIP4 proteins in Kv4.3-KChIP channel complexes can vary depending on KChIP4 expression level. Increasing amount of KChIP4bl gradually reduces the bleaching steps of GFP for KChIP4a proteins and vice-versa. To further demonstrate Kv4 gating affected by different KChIP4 subunit stoichiometry, we generated two tandem constructs to mimic the situations of KChIP4a half-occupied channel complexes (KChIP4a-2xKv4.3) and KChIP4a saturated channel complexes (KChIP4a-Kv4.3) expressed in Xenopus oocytes. Gating kinetics of KChIP4a-2xKv4.3 co-expressed with KChIP4bl (to mimic channel complex like Kv4.3:KChIP4a:KChIP4bl with the ratio of 4:2:2) shows that both KChIP4a and KChIP4bl can simultaneously modulate the function of channel complexes upon co-assembly. The significance of dynamic channel complexes was further investigated in hippocampal neurons from kainic acid seizure model in rats by detecting a shift of expression profile from KChIP4bl to KChIP4a. Our preliminary findings demonstrate that auxiliary KChIPs can hetero-assemble with Kv4 in a competitive manner to form hetero-multimeric Kv4-KChIP4 complexes that are biophysically distinct and dynamically regulated under pathological conditions.

Loss of K+ Currents in Heart Failure Is Accentuated in KChIP2 Deficient Mice

KV 4 together with KV Channel-Interacting Protein 2 (KChIP2) mediate the fast recovering transient outward potassium current (I(to,f)) in the heart. KChIP2 is downregulated in human heart failure (HF), potentially underlying the loss of I(to,f). We investigated remodeling associated with HF hypothesizing that KChIP2 plays a central role in the modulation of outward K(+) currents in HF. HF was induced by aortic banding in wild-type (WT) and KChIP2 deficient (KChIP2(-/-)) mice, evaluated by echocardiography. Action potentials were measured by floating microelectrodes in intact hearts. Ventricular cardiomyocytes were isolated and whole-cell currents were recorded by patch clamp. Left ventricular action potentials in KChIP2(-/-) mice were prolonged in a rate dependent manner, consistent with patch-clamp data showing loss of a fast recovering outward K(+) current and upregulation of the slow recovering I(to,s) and I(Kur). HF decreased all outward K(+) currents in WT mice and did not change the relative contribution of I(to,f) in WT mice. Compared to WT HF, KChIP2(-/-) HF had a larger reduction of K(+) -current density. However, the relative APD prolongation caused by HF was shorter for KChIP2(-/-) compared with WT, and the APs of the 2 HF mouse types were indistinguishable. I(to,f) is just one of many K(+) currents being downregulated in murine HF. The downregulation of repolarizing currents in HF is accentuated in KChIP2(-/-) mice. However, the prolongation of APs associated with HF is less in KChIP2(-/-) compared to WT, suggesting other compensatory mechanism(s) in the KChIP2(-/-) mouse.

Modulatory mechanisms and multiple functions of somatodendritic A-type K (+) channel auxiliary subunits.

Auxiliary subunits are non-conducting, modulatory components of the multi-protein ion channel complexes that underlie normal neuronal signaling. They interact with the pore-forming α-subunits to modulate surface distribution, ion conductance, and channel gating properties. For the somatodendritic subthreshold A-type potassium (ISA) channel based on Kv4 α-subunits, two types of auxiliary subunits have been extensively studied: Kv channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPLPs). KChIPs are cytoplasmic calcium-binding proteins that interact with intracellular portions of the Kv4 subunits, whereas DPLPs are type II transmembrane proteins that associate with the Kv4 channel core. Both KChIPs and DPLPs genes contain multiple start sites that are used by various neuronal populations to drive the differential expression of functionally distinct N-terminal variants. In turn, these N-terminal variants generate tremendous functional diversity across the nervous system. Here, we focus our review on (1) the molecular mechanism underlying the unique properties of different N-terminal variants, (2) the shaping of native ISA properties by the concerted actions of KChIPs and DPLP variants, and (3) the surprising ways that KChIPs and DPLPs coordinate the activity of multiple channels to fine-tune neuronal excitability. Unlocking the unique contributions of different auxiliary subunit N-terminal variants may provide an important opportunity to develop novel targeted therapeutics to treat numerous neurological disorders.