E3 ubiquitin ligase rififylin has yin and yang effects on rabbit cardiac transient outward potassium currents (Ito) and corresponding channel proteins.

Genome-wide association studies have reported a correlation between a SNP of the RING finger E3 ubiquitin protein ligase rififylin (RFFL) and QT interval variability in humans (Newton-Cheh et al., 2009). Previously, we have shown that RFFL downregulates expression and function of the human-like ether-a-go-go-related gene potassium channel and corresponding rapidly activating delayed rectifier potassium current (IKr) in adult rabbit ventricular cardiomyocytes. Here, we report that RFFL also affects the transient outward current (Ito), but in a peculiar way. RFFL overexpression in adult rabbit ventricular cardiomyocytes significantly decreases the contribution of its fast component (Ito,f) from 35% to 21% and increases the contribution of its slow component (Ito,s) from 65% to 79%. Since Ito,f in rabbits is mainly conducted by Kv4.3, we investigated the effect of RFFL on Kv4.3 expressed in HEK293A cells. We found that RFFL overexpression reduced Kv4.3 expression and corresponding Ito,f in a RING domain-dependent manner in the presence or absence of its accessory subunit Kv channel-interacting protein 2. On the other hand, RFFL overexpression in Kv1.4-expressing HEK cells leads to an increase in both Kv1.4 expression level and Ito,s, similarly in a RING domain-dependent manner. Our physiologically detailed rabbit ventricular myocyte computational model shows that these yin and yang effects of RFFL overexpression on Ito,f, and Ito,s affect phase 1 of the action potential waveform and slightly decrease its duration in addition to suppressing IKr. Thus, RFFL modifies cardiac repolarization reserve via ubiquitination of multiple proteins that differently affect various potassium channels and cardiac action potential duration.

Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2.

The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.

Kv4.2 phosphorylation by PKA drives Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization.

Sympathetic regulation of the Kv4.2 transient outward potassium current (Ito) is critical for the acute electrical and contractile response of the myocardium under physiological and pathological conditions. Previous studies have suggested that KChIP2, the key auxiliary subunit of Kv4 channels, is required for the sympathetic regulation of Kv4.2 current densities. Of interest, Kv4.2 and KChIP2, and key components mediating acute sympathetic signaling transduction are present in lipid rafts, which are profoundly involved in regulation of Ito densities in rat ventricular myocytes. However, little is known about the mechanisms of Kv4.2-raft association and its connection with acute sympathetic regulation. With the aid of high-resolution fluorescent microscope, we demonstrated that KChIP2 assisted Kv4.2 localization in lipid rafts in HEK293 cells. Moreover, PKA-mediated Kv4.2 phosphorylation, the downstream signaling event of acute sympathetic stimulation, induced dissociation between Kv4.2 and KChIP2, resulting in Kv4.2 shifting out of lipid rafts in KChIP2-expressed HEK293. The mutation that mimics Kv4.2 phosphorylation by PKA (K4.2-S552D) similarly disrupted Kv4.2 interaction with KChIP2 and also decreased the surface stability of Kv4.2. The attenuated Kv4.2-KChIP2 interaction was also observed in native neonatal rat ventricular myocytes (NRVMs) upon acute adrenergic stimulation with phenylephrine (PE). Furthermore, PE stimulation decreased Kv4.2 location at lipid rafts and induced internalization of Kv4.2 as well as the effect of lipid rafts disruption. In conclusion, KChIP2 contributes to targeting Kv4.2 to lipid rafts. Acute adrenergic stimulation induces Kv4.2-KChIP2 dissociation, leading to Kv4.2 out of lipid rafts and internalization, reinforcing the critical role of Kv4.2-lipid raft association in the essential physiological response of Ito to acute sympathetic regulation.

Regulation of Kv4.3 and hERG potassium channels by KChIP2 isoforms and DPP6 and response to the dual K+ channel activator NS3623.

Transient outward potassium current (Ito) contributes to early repolarization of many mammalian cardiac action potentials, including human, whilst the rapid delayed rectifier K+ current (IKr) contributes to later repolarization. Fast Ito channels can be produced from the Shal family KCNDE gene product Kv4.3s, although accessory subunits including KChIP2.x and DPP6 are also needed to produce a near physiological Ito. In this study, the effect of KChIP2.1 & KChIP2.2 (also known as KChIP2b and KChIP2c respectively), alone or in conjunction with the accessory subunit DPP6, on both Kv4.3 and hERG were evaluated. A dual Ito and IKr activator, NS3623, has been recently proposed to be beneficial in heart failure and the action of NS3623 on the two channels was also investigated. Whole-cell patch-clamp experiments were performed at 33 ±â€¯1 °C on HEK293 cells expressing Kv4.3 or hERG in the absence or presence of these accessory subunits. Kv4.3 current magnitude was augmented by co-expression with either KChIP2.2 or KChIP2.1 and KChIP2/DPP6 with KChIP2.1 producing a greater effect than KChIP2.2. Adding DPP6 removed the difference in Kv4.3 augmentation between KChIP2.1 and KChIP2.2. The inactivation rate and recovery from inactivation were also altered by KChIP2 isoform co-expression. In contrast, hERG (Kv11.1) current was not altered by co-expression with KChIP2.1, KChIP2.2 or DPP6. NS3623 increased Kv4.3 amplitude to a similar extent with and without accessory subunit co-expression, however KChIP2 isoforms modulated the compound's effect on inactivation time course. The agonist effect of NS3623 on hERG channels was not affected by KChIP2.1, KChIP2.2 or DPP6 co-expression.

K+ Channel Modulatory Subunits KChIP and DPP Participate in Kv4-Mediated Mechanical Pain Control.

The K+ channel pore-forming subunit Kv4.3 is expressed in a subset of nonpeptidergic nociceptors within the dorsal root ganglion (DRG), and knockdown of Kv4.3 selectively induces mechanical hypersensitivity, a major symptom of neuropathic pain. K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 are coexpressed in Kv4.3+ DRG neurons, but whether they participate in Kv4.3-mediated pain control is unknown. Here, we show the existence of a Kv4.3/KChIP1/KChIP2/DPP10 complex (abbreviated as the Kv4 complex) in the endoplasmic reticulum and cell surface of DRG neurons. After intrathecal injection of a gene-specific antisense oligodeoxynucleotide to knock down the expression of each component in the Kv4 complex, mechanical hypersensitivity develops in the hindlimbs of rats in parallel with a reduction in all components in the lumbar DRGs. Electrophysiological data further indicate that the excitability of nonpeptidergic nociceptors is enhanced. The expression of all Kv4 complex components in DRG neurons is downregulated following spinal nerve ligation (SNL). To rescue Kv4 complex downregulation, cDNA constructs encoding Kv4.3, KChIP1, and DPP10 were transfected into the injured DRGs (defined as DRGs with injured spinal nerves) of living SNL rats. SNL-evoked mechanical hypersensitivity was attenuated, accompanied by a partial recovery of Kv4.3, KChIP1, and DPP10 surface levels in the injured DRGs. By showing an interdependent regulation among components in the Kv4 complex, this study demonstrates that K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 participate in Kv4.3-mediated mechanical pain control. Thus, these modulatory subunits could be potential drug targets for neuropathic pain.SIGNIFICANCE STATEMENT Neuropathic pain, a type of moderate to severe chronic pain resulting from nerve injury or disorder, affects 6.9%-10% of the global population. However, less than half of patients report satisfactory pain relief from current treatments. K+ channels, which act to reduce nociceptor activity, have been suggested to be novel drug targets for neuropathic pain. This study is the first to show that K+ channel modulatory subunits KChIP1, KChIP2, and DPP10 are potential drug targets for neuropathic pain because they form a channel complex with the K+ channel pore-forming subunit Kv4.3 in a subset of nociceptors to selectively inhibit mechanical hypersensitivity, a major symptom of neuropathic pain.

KChIP2 is a core transcriptional regulator of cardiac excitability.

Arrhythmogenesis from aberrant electrical remodeling is a primary cause of death among patients with heart disease. Amongst a multitude of remodeling events, reduced expression of the ion channel subunit KChIP2 is consistently observed in numerous cardiac pathologies. However, it remains unknown if KChIP2 loss is merely a symptom or involved in disease development. Using rat and human derived cardiomyocytes, we identify a previously unobserved transcriptional capacity for cardiac KChIP2 critical in maintaining electrical stability. Through interaction with genetic elements, KChIP2 transcriptionally repressed the miRNAs miR-34b and miR-34c, which subsequently targeted key depolarizing (INa) and repolarizing (Ito) currents altered in cardiac disease. Genetically maintaining KChIP2 expression or inhibiting miR-34 under pathologic conditions restored channel function and moreover, prevented the incidence of reentrant arrhythmias. This identifies the KChIP2/miR-34 axis as a central regulator in developing electrical dysfunction and reveals miR-34 as a therapeutic target for treating arrhythmogenesis in heart disease.

Circadian rhythm in QT interval is preserved in mice deficient of potassium channel interacting protein 2.

Potassium Channel Interacting Protein 2 (KChIP2) is suggested to be responsible for the circadian rhythm in repolarization duration, ventricular arrhythmias, and sudden cardiac death. We investigated the hypothesis that there is no circadian rhythm in QT interval in the absence of KChIP2. Implanted telemetric devices recorded electrocardiogram continuously for 5 days in conscious wild-type mice (WT, n = 9) and KChIP2-/- mice (n = 9) in light:dark periods and in complete darkness. QT intervals were determined from all RR intervals and corrected for heart rate (QT100 = QT/(RR/100)1/2). Moreover, QT intervals were determined from complexes within the RR range of mean-RR ± 1% in the individual mouse (QTmean-RR). We find that RR intervals are 125 ± 5 ms in WT and 123 ± 4 ms in KChIP2-/- (p = 0.81), and QT intervals are 52 ± 1 and 52 ± 1 ms, respectively(p = 0.89). No ventricular arrhythmias or sudden cardiac deaths were observed. We find similar diurnal (light:dark) and circadian (darkness) rhythms of RR intervals in WT and KChIP2-/- mice. Circadian rhythms in QT100 intervals are present in both groups, but at physiological small amplitudes: 1.6 ± 0.2 and 1.0 ± 0.3 ms in WT and KChIP2-/-, respectively (p = 0.15). A diurnal rhythm in QT100 intervals was only found in WT mice. QTmean-RR intervals display clear diurnal and circadian rhythms in both WT and KChIP2-/-. The amplitude of the circadian rhythm in QTmean-RR is 4.0 ± 0.3 and 3.1 ± 0.5 ms in WT and KChIP2-/-, respectively (p = 0.16). In conclusion, KChIP2 expression does not appear to underlie the circadian rhythm in repolarization duration.

Ventricular repolarization time, location of pacing stimulus and current pulse amplitude conspire to determine arrhythmogenicity in mice.

AIM: In this study, we investigate the impact of altered action potential durations (APD) on ventricular repolarization time and proarrhythmia in mice with and without genetic deletion of the K+ -channel-interacting protein 2 (KChIP2-/- and WT respectively). Moreover, we examine the interrelationship between the dispersion of repolarization time and current pulse amplitude in provoking ventricular arrhythmia. METHODS: Intracardiac pacing in anesthetized mice determined refractory periods and proarrhythmia susceptibility. Regional activation time (AT), APD and repolarization time (=AT + APD) were measured in isolated hearts using floating microelectrodes. RESULTS: Proarrhythmia in WT and KChIP2-/- was not sensitive to changes in refractory periods. Action potentials were longer in KChIP2-/- hearts compared to WT hearts. Isolated WT hearts had large apico-basal dispersion of repolarization time, whereas hearts from KChIP2-/- mice had large left-to-right ventricular dispersion of repolarization time. Pacing from the right ventricle in KChIP2-/- mice in vivo revealed significant lower current pulse amplitudes needed to induce arrhythmias in these mice. CONCLUSION: Large heterogeneity of repolarization time is proarrhythmic when pacing is delivered from the location of earlier repolarization time. Ventricular repolarization time, location of the pacing stimulus and the amplitude of the stimulating current pulse are critical parameters underlying arrhythmia vulnerability.

Coexpression of auxiliary subunits KChIP and DPPL in potassium channel Kv4-positive nociceptors and pain-modulating spinal interneurons.

Subthreshold A-type K(+) currents (ISA s) have been recorded from the somata of nociceptors and spinal lamina II excitatory interneurons, which sense and modulate pain, respectively. Kv4 channels are responsible for the somatodendritic ISA s. Accumulative evidence suggests that neuronal Kv4 channels are ternary complexes including pore-forming Kv4 subunits and two types of auxiliary subunits: K(+) channel-interacting proteins (KChIPs) and dipeptidyl peptidase-like proteins (DPPLs). Previous reports have shown Kv4.3 in a subset of nonpeptidergic nociceptors and Kv4.2/Kv4.3 in certain spinal lamina II excitatory interneurons. However, whether and which KChIP and DPPL are coexpressed with Kv4 in these ISA -expressing pain-related neurons is unknown. In this study we mapped the protein distribution of KChIP1, KChIP2, KChIP3, DPP6, and DPP10 in adult rat dorsal root ganglion (DRG) and spinal cord by immunohistochemistry. In the DRG, we found colocalization of KChIP1, KChIP2, and DPP10 in the somatic surface and cytoplasm of Kv4.3(+) nociceptors. KChIP3 appears in most Aß and Aδ sensory neurons as well as a small population of peptidergic nociceptors, whereas DPP6 is absent in sensory neurons. In the spinal cord, KChIP1 is coexpressed with Kv4.3 in the cell bodies of a subset of lamina II excitatory interneurons, while KChIP1, KChIP2, and DPP6 are colocalized with Kv4.2 and Kv4.3 in their dendrites. Within the dorsal horn, besides KChIP3 in the inner lamina II and lamina III, we detected DPP10 in most projection neurons, which transmit pain signal to brain. The results suggest the existence of Kv4/KChIP/DPPL ternary complexes in ISA -expressing nociceptors and pain-modulating spinal interneurons.

Effects of C-reactive protein on K(+) channel interaction protein 2 in cardiomyocytes.

Several studies have found that C-reactive protein (CRP) was associated with QTc interval prolongation and ventricular arrhythmia. However, little is known about the mechanisms involved. K(+) channel interaction protein 2 (KChIP2) is a necessary subunit for the formation of transient outward potassium current (Ito.f) which plays a critical role in early repolarization and QTc interval of heart. In this study, we aimed to evaluate the effects of CRP on KChIP2 and Ito.f in cardiomyocytes and to explore the potential mechanism. The neonatal mice ventricular cardiomyocytes were cultured and treated with CRP at different concentrations. The expression of KChIP2 was detected by real time quantitative PCR and Western blot. In addition, Ito.f current density was evaluated by whole cell patch clamp techniques. Our results showed that CRP significantly decreased the mRNA and protein expression of KChIP2 in time and doses dependent manners (P < 0.05), and also reduced the current density of Ito.f (P < 0.05). In addition, CRP increased the expression of NF-κB and decreased IκBα expression without significant influence on the expression of ERK1/2 and JNK. Meanwhile, the NF-κB inhibitor PDTC significantly attenuated the effects of CRP on KChIP2 and Ito.f current density. In conclusion, CRP could significantly down-regulate KChIP2 expression and reduce current density of Ito.f partly through NF-κB pathway, suggesting that CRP may directly or indirectly influence QTc interval and arrhythmia via influencing KChIP2 expression and Ito.f current density of cardiomyocytes.

Loss of K+ currents in heart failure is accentuated in KChIP2 deficient mice.

INTRODUCTION: 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. METHODS AND RESULTS: 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. CONCLUSION: 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.

New insights into the roles of Xin repeat-containing proteins in cardiac development, function, and disease.

Since the discovery of Xin repeat-containing proteins in 1996, the importance of Xin proteins in muscle development, function, regeneration, and disease has been continuously implicated. Most Xin proteins are localized to myotendinous junctions of the skeletal muscle and also to intercalated discs (ICDs) of the heart. The Xin gene is only found in vertebrates, which are characterized by a true chambered heart. This suggests that the evolutionary origin of the Xin gene may have played a key role in vertebrate origins. Diverse vertebrates including mammals possess two paralogous genes, Xinα (or Xirp1) and Xinß (or Xirp2), and this review focuses on the role of their encoded proteins in cardiac muscles. Complete loss of mouse Xinß (mXinß) results in the failure of forming ICD, severe growth retardation, and early postnatal lethality. Deletion of mouse Xinα (mXinα) leads to late-onset cardiomyopathy with conduction defects. Molecular studies have identified three classes of mXinα-interacting proteins: catenins, actin regulators/modulators, and ion-channel subunits. Thus, mXinα acts as a scaffolding protein modulating the N-cadherin-mediated adhesion and ion-channel surface expression. Xin expression is significantly upregulated in early stages of stressed hearts, whereas Xin expression is downregulated in failing hearts from various human cardiomyopathies. Thus, mutations in these Xin loci may lead to diverse cardiomyopathies and heart failure.

Antibodies against potassium channel interacting protein 2 induce necrosis in isolated rat cardiomyocytes.

Auto-antibodies against cardiac proteins have been described in patients with dilated cardiomyopathy. Antibodies against the C-terminal part of KChIP2 (anti-KChIP2 [C-12]) enhance cell death of rat cardiomyocytes. The underlying mechanisms are not fully understood. Therefore, we wanted to explore the mechanisms responsible for anti-KChIP2-mediated cell death. Rat cardiomyocytes were treated with anti-KChIP2 (C-12). KChIP2 RNA and protein expressions, nuclear NF-κB, mitochondrial membrane potential Δψm, caspase-3 and -9 activities, necrotic and apoptotic cells, total Ca(2+) and K(+) concentrations, and the effects on L-type Ca(2+) channels were quantified. Anti-KChIP2 (C-12) induced nuclear translocation of NF-κB. Anti-KChIP2 (C-12)-treatment for 2 h significantly reduced KChIP2 mRNA and protein expression. Anti-KChIP2 (C-12) induced nuclear translocation of NF-κB after 1 h. After 6 h, Δψm and caspase-3 and -9 activities were not significantly changed. After 24 h, anti-KChIP2 (C-12)-treated cells were 75 ± 3% necrotic, 2 ± 1% apoptotic, and 13 ± 2% viable. Eighty-six ± 1% of experimental buffer-treated cells were viable. Anti-KChIP2 (C-12) induced significant increases in total Ca(2+) (plus 11 ± 2%) and K(+) (plus 18 ± 2%) concentrations after 5 min. Anti-KChIP2 (C-12) resulted in an increased Ca(2+) influx through L-type Ca(2+) channels. In conclusion, our results suggest that anti-KChIP2 (C-12) enhances cell death of rat cardiomyocytes probably due to necrosis.

The auxiliary subunit KChIP2 is an essential regulator of homeostatic excitability.

BACKGROUND: The necessity for, or redundancy of, distinctive KChIP proteins is not known. RESULTS: Deletion of KChIP2 leads to increased susceptibility to epilepsy and to a reduction in IA and increased excitability in pyramidal hippocampal neurons. CONCLUSION: KChIP2 is essential for homeostasis in hippocampal neurons. SIGNIFICANCE: Mutations in K(A) channel auxiliary subunits may be loci for epilepsy. The somatodendritic IA (A-type) K(+) current underlies neuronal excitability, and loss of IA has been associated with the development of epilepsy. Whether any one of the four auxiliary potassium channel interacting proteins (KChIPs), KChIP1-KChIP4, in specific neuronal populations is critical for IA is not known. Here we show that KChIP2, which is abundantly expressed in hippocampal pyramidal cells, is essential for IA regulation in hippocampal neurons and that deletion of Kchip2 affects susceptibility to limbic seizures. The specific effects of Kchip2 deletion on IA recorded from isolated hippocampal pyramidal neurons were a reduction in amplitude and shift in the V½ for steady-state inactivation to hyperpolarized potentials when compared with WT neurons. Consistent with the relative loss of IA, hippocampal neurons from Kchip2(-/-) mice showed increased excitability. WT cultured neurons fired only occasional single action potentials, but the average spontaneous firing rate (spikes/s) was almost 10-fold greater in Kchip2(-/-) neurons. In slice preparations, spontaneous firing was detected in CA1 pyramidal neurons from Kchip2(-/-) mice but not from WT. Additionally, when seizures were induced by kindling, the number of stimulations required to evoke an initial class 4 or 5 seizure was decreased, and the average duration of electrographic seizures was longer in Kchip2(-/-) mice compared with WT controls. Together, these data demonstrate that the KChIP2 is essential for physiologic IA modulation and homeostatic stability and that there is a lack of functional redundancy among the different KChIPs in hippocampal neurons.