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.

KChIPs have been implicated in the pathogenesis of several diseases, including cardiac arrhythmias, cardiac hypertrophy, neurodegenerative diseases, and epilepsy. As research deepens and the role of KChIPs in physiological and pathological conditions become better understood through the use of some transgenic or gene knockout animal models, the evidence for the association of KChIPs with these diseases is increasing. Small molecules acting on KChIPs have been shown to enhance or inhibit the activity of KChIPs [12], which is instructive for the development of drugs targeting KChIPs to treat related diseases. However, a growing number of recent studies have shown the diversity of functions of KChIPs. For example, KChIP3 knockout rats and mice appear to have different pain hypersensitivity responses to drug stimulation. The small molecule NS5806 has different effects in different species, even in different parts of the same species. The emergence of these questions indicates the complexity of the function of KChIPs, and there are still many questions in this field that researchers urgently need to answer. In this review, we summarized the function of KChIPs and their multiple roles in disease progression. Meanwhile, we updated the small-molecule drugs targeting KChIPs to provide new guidance for future basic and translational research in this field.  KChIP4.4 (KChIP4a) [25] and KChIP3x (KChIP3b) [30], which possess the functional K V 4 channel inhibitory domain (KID), an N-terminal membrane-spanning segment, are able to exert the inhibitory effect on K V 4 channels. The KID contains an ER retention motif consisting of six hydrophobic and aliphatic residues 12-17, which interferes with K V 4 surface expression. Residues 19-21 (VKL motif), adjacent to the ER retention motif, enhance K V 4 inactivation and keep it in the closed state, thereby inhibiting channel current [35,37,42].

Regulation of KChIPs Expression 2.2.1. Transcriptional Level
Some physiological functions or pathological conditions are underpinned by changes in the expression levels of KChIPs. Unfortunately, mechanistic studies of the expression regulation of KChIPs are limited and have mainly focused on KChIP2. A number of signaling pathways have been identified that affect the transcription of KChIP2, including NF-κB [43], CaMKII [44], NFAT [45], MAPK [46][47][48], and Notch [49]. Our previous work showed that NF-κB can directly bind to the promoter region of the Kcnip2 gene and repress its transcription [43]. Several factors that downregulate KChIP2 expression, including ligands of TGF-β receptors [50], α-adrenergic receptors [51], β-adrenergic receptors [52], and C-reactive protein [53], are partially dependent on the activation of the NF-κB pathway. Additionally, Ca 2+ reduces the mRNA expression of KChIP2 through the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) [44] and the calcineurin/NFAT signaling pathway [45]. Under physiological conditions, the inhibition of MEK1 increases, whereas the activation of MEK1 decreases KChIP2 mRNA level. In phenylephrine-induced hypertrophic cardiomyocytes, inhibition of JNK1 rescues the downregulation of KChIP2. These findings suggest that mitogen-activated protein kinase (MAPK) pathways are also involved in the regulation of KChIP2 expression [30]. Notch signaling inhibits the expression of KChIP2 expression and thereby contributes to the electrophysiological differences between neonatal and adult cardiomyocytes [49]. Several transcription factors have also been reported to bind directly to the promoter region of KCNIP2 to regulate its expression. For example, cyclic AMP response element binding protein (CREB) [54], Krüppel-like factor-4 [55], and Krüppel-like factor-5 [56] are able to promote KCNIP2 transcription ( Figure 3). signaling pathways have been identified that affect the transcription of KChIP2, including NF-κB [43], CaMKII [44], NFAT [45], MAPK [46][47][48], and Notch [49]. Our previous work showed that NF-κB can directly bind to the promoter region of the Kcnip2 gene and repress its transcription [43]. Several factors that downregulate KChIP2 expression, including ligands of TGF-β receptors [50], α-adrenergic receptors [51], β-adrenergic receptors [52], and C-reactive protein [53], are partially dependent on the activation of the NF-κB pathway. Additionally, Ca 2+ reduces the mRNA expression of KChIP2 through the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) [44] and the calcineurin/NFAT signaling pathway [45]. Under physiological conditions, the inhibition of MEK1 increases, whereas the activation of MEK1 decreases KChIP2 mRNA level. In phenylephrine-induced hypertrophic cardiomyocytes, inhibition of JNK1 rescues the downregulation of KChIP2. These findings suggest that mitogen-activated protein kinase (MAPK) pathways are also involved in the regulation of KChIP2 expression [30]. Notch signaling inhibits the expression of KChIP2 expression and thereby contributes to the electrophysiological differences between neonatal and adult cardiomyocytes [49]. Several transcription factors have also been reported to bind directly to the promoter region of KCNIP2 to regulate its expression. For example, cyclic AMP response element binding protein (CREB) [54], Krüppel-like factor-4 [55], and Krüppel-like factor-5 [56] are able to promote KCNIP2 transcription ( Figure 3).

Protein Level
It is worth noting that as an integral component of the K V 4 channel complex, the protein level of KChIPs is tightly coupled to K V 4 and dipeptidyl-peptidase-related proteins (DPPs). On the one hand, deletion of K V 4.2 significantly reduces KChIP1, KChIP2, and KChIP3 expression in the mouse brain. In particular, deletion of K V 4.2 results in reduced expression of KChIP2 and KChIP3 in the hippocampus, KChIP2 in the striatum, and KChIP1 and KChIP3 in the cerebellum [57]. Furthermore, KChIP2, KChIP3, and KChIP4 protein expression levels in cortical pyramidal neurons were extremely low in mice with a targeted deletion of either K V 4.2 or K V 4.3 [15]. Nerbonne et al. showed that loss of K V 4.2 in cortical pyramidal neurons resulted in targeted degradation of KChIP3 protein [58]. On the other hand, DPPs also affect the expression of KChIPs. Downregulation of DPP6 reduces K V 4.2 and KChIPs in CA1 hippocampal neurons [59]. Similarly, knockdown of DPP10 in the dorsal root ganglion (DRG) neurons resulted in downregulation of KChIP1 and KChIP2 [60].
In addition, members of the KChIP subfamily can have an effect on the expression of other KChIPs. For example, when KChIP3 was deficient in the cortex, the expression of other KChIPs were increased to compensate for KChIP3 deficiency [15,61]. Interestingly, there is evidence that KChIP3 can be a negative regulator of its own expression [61].

Biological Function of KChIPs
3.1. KChIPs Are Auxiliary Subunits of K V 4 Channels 3.1.1. The Interaction of KChIPs with K V 4 Channels K V 4 channels are members of the K V channel superfamily. In mammals, the K V 4family consists of four members: K V 4.1, K V 4.2, and two splice variants of K V 4.3. All K V 4 channels share a functional core that is assembled as a tetramer of pore-forming α-subunits around a central pore [12]. The K V 4 α-subunit contains an N-terminal cytoplasmic domain with an N-terminal hydrophobic segment, the K V channel assembly domain (T1 domain) under the tetrameric channel pore domains, a transmembrane domain with six transmembrane helices S1-S6, and the C-terminal cytoplasmic domain [62]. The K V 4 channels are highly expressed in the brain, heart, and smooth muscle cells. The neuronal K V 4 channels underlie the transient A-type current (I A ), sustaining the homeostatic excitability of neurons [32]. In cardiomyocytes, K V 4 channels control the early repolarization phase of the action potential by mediating the transient outward current (I to ) [63]. In gastrointestinal smooth muscle cells, K V 4 currents are involved in shaping the slow-wave activity and mechanical responses [4].
However, K V 4 channels can not carry out normal physiological processes on their own. Two auxiliary subunits, KChIPs and DPPs, are essential for the physiological function of K V 4 channels. Mechanistically, KChIP1-3 can bind to the cytoplasmic domains of K V 4 α-subunits, thereby increasing total K V 4 current, slowing channel inactivation, and accelerating recovery from inactivation. An et al. also found that KChIP4a, which contains the KID, can eliminate the fast inactivation of the K V 4 current. KChIPs are anchored laterally to the T1 domains of K V 4 and clamp two adjacent K V 4 N-terminals in a 4:4 ratio [64,65] through hydrophobic interactions and hydrogen bonds. The second site is mainly formed by the interaction between the KChIP1 H2 helix and the KChIP-specific docking loop of the neighboring T1 domain through the hydrophobic interactions and salt bridges, stabilizing the K V 4.3 tetramerization. In the second site, the conserved Phe73 of K V 4 fits tightly into a hydrophobic cavity that is formed by the residues Leu39, Leu42, Leu53, Tyr57, and Phe108 in KChIP1. Meanwhile, residues Lys50, Arg51, and Lys61 of KChIP1 form salt bridges with Glu77, Asp78, and Glu70 of K V 4.3, respectively [65]. The first site is responsible for K V 4 inactivation and the second site for stabilizing the tetramer of K V 4. Based on crystallographic studies by Wang et al., Cattee et al. defined the third site of K V 4.3-KChIP1 interaction using all-atom molecular dynamics simulations. Residues R51, R58, and E63 on the KChIP1 H2 helix, which is also involved in the formation of the second site, interact with residues D39 and R60 on the K V 4.3 T1N linker to form the third site, providing further stability to the K V 4.3 tetrameric intracellular domain [65,66]. In addition, KChIP1 is able to capture the C-terminal cytoplasmic S6 helix on K V 4.2 through the hydrophobic interactions [62].

KChIPs
Modulate the Gating Properties of K V 4 Channels K V 4 are the rapidly inactivating (A-type) K V potassium channels that generate currents at subthreshold membrane potentials. They are characterized by fast activation, fast inactivation, and fast recovery from inactivation. The inactivation of K V 4 channels is classified into two types: open-state inactivation and closed-state inactivation. Closed-state inactivation is the main type, indicating that K V 4 channels can be inactivated directly from the closed state [67]. Binding of KChIPs to the N-terminus of K V 4 modulates the gating properties of K V 4 channels. Specifically, KChIP1-3 augment K V 4 currents through the following electrophysiological effects: shifting the activation midpoint of voltage activation to more negative potentials, slower inactivation, and acceleration of recovery from inactivation [9]. When co-expressed with KChIPs, the activation time of K V 4 was slightly prolonged compared to K V 4 alone, while the midpoint for K V 4 of voltage activation significantly shifted to more hyperpolarized potentials [9]. In contrast, the modulation of K V 4 gating by KChIPs is mainly manifested in the inactivation kinetics. Heterologous co-expression of K V 4 and KChIPs significantly prolongs the inactivation time of K V 4 channels. To be specific, KChIPs eliminate open-state inactivation and accelerate closedstate inactivation of K V 4 channels [9,27,62]. However, it is still unclear about the molecular mechanism by which KChIPs control K V 4 inactivation. The EF-hands were reportedly involved in the regulation of K V 4.3 inactivation by sensing intracellular Ca 2+ levels [68]. Recently, breakthroughs have been made in the structural basis of KChIPs that regulate the inactivation kinetics of K V 4 channels. Kise et al. reported that KChIP1 is able to capture and sequester both the N-terminal hydrophobic segment and the C-terminus of K V 4.2 channels. KChIP1, on the one hand, binds the C-terminal intracellular S6 helix to stabilize the S6 conformation. It also binds the N-terminal hydrophobic segment and two T1 domains from the neighboring subunit of K V 4.2. Together, these KChIP1-mediated structural features prevent open-state inactivation and accelerate closed-state inactivation of K V 4.2 [62]. By truncating the N-terminal or C-terminal helix of K V 4.2, respectively, Ye et al. demonstrated that the interactions of KChIP2 with the K V 4.2 N-terminal helix play a more prominent role in modulating channel inactivation [69]. Moreover, KChIPs accelerate the rate of recovery of K V 4 channels from inactivation in a Ca 2+ -independent manner [68].
It is interesting to note that a specific KChIP isoform, KChIP4a, has been reported to delay K V 4.3 channel activation, abolish rapid inactivation, and impede channel closure after opening. Therefore, co-expression of KChIP4a with K V 4 α-subunits converts the A-type K V 4 current to a slowly inactivating delayed rectifier-type potassium current [25]. The similar suppressive effect on K V 4 currents was also found for KChIP3x (KChIP3b) in subsequent research [30]. . KChIPs allow KV4 trafficking to the cell surface. KV4 channels cannot be trafficked to the cell membrane on their own because they are aggregated, misfolded, and retained in the ER for degradation. Co-expression of KChIP1-3 releases KV4 channels from ER retention and redistributes them to the cell surface.

Role of KChIPs in Regulating Other Ion Channels
KV1.5 is another vital potassium channel characterized by rapid activation and rapid inactivation that is highly expressed in both the brain and heart. In the human heart, KV1.5 is expressed abundantly in atrial myocytes and mediates the ultra-rapid delayed rectifier current (IKr). In the adult mouse heart, KV1.5, which encodes the slow delayed rectifier K + current (IKs), is involved in the repolarization of ventricular myocytes [85]. Several studies have demonstrated the contribution of KChIPs in the modulation of KV1.5 channels. In Since KChIPs are essential for the control of electrophysiological processes in cells, inhibiting or enhancing their effect on K V 4 channels is a promising avenue for pharmacological research in the treatment of K V 4-mediated channelopathies. Several small molecules that bind to KChIPs have been shown to modulate K V 4 currents ( Table 2). For example, the binding of arachidonic acid to the hydrophobic C-terminus of KChIP1 accelerates K V 4 inactivation and decreases current amplitude [26]. CL-888, a diaryl-urea compound, can bind to KChIP1 [73] and KChIP3 [74] to counteract their regulatory effects on K V 4, reducing peak current amplitude and accelerating inactivation kinetics. IQM-PC330 and IQM-PC332, the derivatives of CL-888 modified by Lopez-Hurtado et al., have a more refined blocking effect on K V 4 currents. IQM-PC330 and IQM-PC332 inhibit K V 4.3 channels not only by reducing peak current amplitude and accelerating their inactivation but also by delaying their recovery from inactivation. Mechanistically, they act as KChIP3 ligands to reverse the regulatory function of KChIP3 on K V 4.3 channel gating properties. It is interesting to note that the hydrophobic combination of IQM-PC332 and KChIP3 accelerates the activation kinetics of K V 4.3 current at low concentrations (0.01 to 0.1 M) and reverses its effect on channel gating at higher concentrations. However, inactivation recovery kinetics were significantly reduced at all concentrations [75]. They also identified IQM-266 as a novel KChIP3 ligand with similar inhibitory properties to IQM-PC330 and IQM-PC332 [76]. Recently, IQM-266 was reported to bind KChIP2 and increase K V 4.3/KChIP2 currents [77].
The sulfonylurea compound NS5806, a ligand of KChIP3, has been demonstrated to activate K V 4.3 channels in neurons. NS5806 delays the inactivation of I A and slightly reduces the maximum peak current [78]. This effect is based on the Ca 2+ -dependent binding of NS5806 to the hydrophobic site at the C-terminus of KChIP3, which facilitated the binding affinity between KChIP3 and K V 4.3 as well as reducing their dissociation rate [34]. In cardiomyocytes, however, the control of K V 4 currents by NS5806 is controversial. In canine cardiomyocytes, NS5806 functions as an I to activator to increase the amplitude of the K V 4.3/KChIP2 peak current and significantly slow the current decay [79], while in mouse ventricular myocytes NS5806 has the opposite effect, as shown by a decrease in the amplitude of native I to and significant acceleration of current inactivation [80]. Furthermore, in rabbit ventricular myocytes, NS5806 dramatically raised I to amplitude, while in atrial myocytes, the I to amplitude was repressed [81]. These conflicting results suggest that more than one site may be involved in the interaction between NS5806 and K V 4 channel complexes, and that NS5806 may act as both an agonist and an antagonist of I to on the same channel complexes. The exact mechanism by which NS5806 elicits different responses in different species and cell types remains unclear. Indeed, studies comparing the effects of reported small molecule drugs targeting KChIPs in different species are still lacking. It is therefore necessary to test and compare these small molecules in more animal models. More interestingly, repaglinide, a commonly used antidiabetic drug, can bind directly to KChIP3 and exert an inhibitory effect on K V 4 channels [74]. In fact, accumulated research has shown that repaglinide and glibenclamide can competitively bind to the SUR subunit of cardiovascular and neurological K ATP (ATP-sensitive potassium) channels to inhibit I KATP [82,83]. However, the magnitude and kinetics of K V 4 currents were not affected by glibenclamide [74]. Mechanistically, repaglinide can bind to the Ca 2+ -dependent EF-hand protein of the neuronal calcium sensor family to antagonize its biological function [84]. It is regrettable that the effects of repaglinide against other KChIPs in other organs have not been reported. Given that repaglinide is already a mature drug that can improve type 2 diabetes and cardiovascular events, further investigation of the effect of repaglinide on K V 4 currents in different tissue will be a potential way to improve KChIPs-related neurological and cardiovascular diseases. Altogether, the discovery of these KChIP ligands has improved our knowledge of the interaction between K V 4 and KChIPs and has paved the way for future pharmaceutical development for the treatment of neurodegenerative and cardiovascular diseases involving K V 4/KChIPs.

Role of KChIPs in Regulating Other Ion Channels
is another vital potassium channel characterized by rapid activation and rapid inactivation that is highly expressed in both the brain and heart. In the human heart, K V 1.5 is expressed abundantly in atrial myocytes and mediates the ultra-rapid delayed rectifier current (I Kr ). In the adult mouse heart, K V 1.5, which encodes the slow delayed rectifier K + current (I Ks ), is involved in the repolarization of ventricular myocytes [85]. Several studies have demonstrated the contribution of KChIPs in the modulation of K V 1.5 channels. In contrast to their augmenting effect on K V 4 currents, KChIPs negatively regulate K V 1.5-encoded K + currents. In transiently transfected HEK293 cells, KChIP1 and KChIP2 attenuate K V 1.5 currents by inhibiting the trafficking of K V 1.5 channels to the cell surface [86]. Moreover, in the ventricles of Kcnip2 −/− mice, K V 1.5 mRNA levels were significantly increased and I Ks were upregulated [87]. However, the structural basis of KChIP1 and KChIP2 negative regulation of cardiomyocyte K V 1.5 channels in vivo remains unclear and needs further investigation.

Ca V 1.2
The high-voltage-activated L-type Ca 2+ channel, Ca V 1.2, located on the t-tubule sarcolemma, is the major calcium channel type in cardiomyocytes. Ca V 1.2 channels are macromolecular complexes that are composed of α 1 , α 2 δ, and β subunits [88]. The cardiac L-type Ca 2+ current (I Ca,L ) mediated by Ca V 1.2 is essential for cardiomyocyte depolarization and contraction. KChIPs have a dual effect on the regulation of the cardiac L-type Ca 2+ current (I Ca,L ). On one hand, KChIP2 regulates the I Ca,L through direct interaction with the intracellular N-terminus of the Ca V 1.2 α 1C subunit. Without raising Ca V 1.2 protein expression or trafficking to the plasma membrane, KChIP2 increases I Ca,L current density by impeding the N-terminal inhibitory module [89]. On the other hand, KChIP2 and KChIP3 bind and repress the transcription of the Cacnb2 [90] and Cacna1c [17] genes, respectively, which encode the corresponding β 2 -subunit and α 1C -subunit of Ca V 1.2 channels.
3.2.3. Na V 1.5 Na V 1.5, which is encoded by SCN5A, is highly expressed in the heart. By mediating the rapid influx of Na + , Na V 1.5 dominates the rapid depolarization phase of action poten-tial. Previous studies suggest that K V 4.3 and Na V 1.5 work coordinately and can regulate each other [91]. Deschênes et al. found that co-expression of KChIP2 with Na V 1.5 increased Na V 1.5 current density but had no effect on Na + current gating properties, whereas silencing of Kcnip2 resulted in a significant decrease in Scn5a mRNA level and complete inhibition of voltage-dependent Na + currents [92]. However, this evidence is based on heterologous expression systems. The electrophysiological significance of the cross-regulation of KChIP2, Na V 1.5, and K V 4.3 in cardiomyocytes requires further study.

KChIPs Are Ca 2+ -Dependent Transcriptional Factors
KChIP3 is the first KChIP identified to have transcriptional activity, as it can bind to the downstream regulatory element (DRE) downstream of the TATA box in the human PDYN gene promoter [8]. Subsequent studies have demonstrated that all the KChIPs have Ca 2+ -dependent DRE binding affinities that block gene transcription in the form of homotetramer or heterotetramer. The binding of KchIP3 to DRE sequences is regulated by divalent cations [8]. Tetramer formation is required for KchIP3 in binding to DRE. Mg 2+ and Ca 2+ are the most important factors affecting the ability of KchIP3 to bind to DNA. The combination of Mg 2+ and KchIP3 stabilizes the tertiary structure of the protein and promotes DNA binding [93]. In contrast, as intracellular Ca 2+ levels increase, the Ca 2+ -bound KchIP3 switches to a denser dimer structure [94], preventing KchIP3-DRE interaction. Furthermore, direct interaction of nucleoprotein C-terminal binding protein [95] and αCREM [96] with KChIP3 enhances and inhibits their transcriptional repressive functions, respectively. In the nucleus, in addition to direct binding to DRE in target genes, KChIP3 also forms transcriptional regulatory complexes with nuclear proteins such as cAMP response element-binding protein [97], thyroid transcription factor 1 [98], and nuclear receptors [99]. Genes regulated by KChIP3 include but are not limited to the following: Pdyn, Fos [ Figure 5).
Through the transcriptional regulation of the above-mentioned genes, KChIP3 has been shown to be multifunctional and is critically involved in the regulation of physiological and pathological functions of neurons, endocrine cells, immune cells, endothelial cells, and hematopoietic cells. For example, in the pineal gland, KChIP1-4 are involved in the regulation of rhythmically expressed genes engaged in circadian rhythms. By binding to the DRE sites of arylalkylamine N-acetyltransferase (Aanat), inducible cAMP early repressor (Crem), and Fos-related antigen-2 (Fra-2), KChIP1-4 are able to repress the basal and induced transcription of these circadian-rhythm-related genes [106]. In the pancreas, KChIP3 has been detected in islet αand β-cells. KChIP3 represses the transcription of the Pdyn gene in a Ca 2+ -dependent manner and thereby affects glucagon release [114]. In the thyroid, KChIP3 inhibits the expression of thyroglobulin by binding directly to the DRE of the thyroglobulin gene and blocking thyroid-specific transcription factors such as TTF-1, TTF-2, and Pax8 [98,105]. KChIP3, as well as KChIP2, are expressed in T-and B cells to regulate immunological responses. In T lymphocytes, KChIP2 and KChIP3 act as transcriptional repressors to inhibit the expression of IFN-γ, IL-2, and IL-4 [103]. By inhibiting the transcription of the proliferation-related gene Klf9 and the protein-translationrelated gene Eif4g3 in B cells, KChIP2 and KChIP3 govern B-cell proliferation and IgM and IgG protein synthesis [16]. In lung endothelial cells, neutrophils, and macrophages, KChIP3 can promote the NF-κB-pathway-mediated inflammatory response by suppressing the expression of the TNFAIP3 gene, which encodes the anti-inflammatory deubiquitinase A20 [104,115]. to the nucleus to repress the transcription of associated genes by interacting with the DRE sequences. This interaction can be enhanced by Mg 2+ and C-terminal binding protein (CtBP) and inhibited by Ca 2+ and cyclic AMP-responsive element modulator α (αCREM). KChIP3 can also repress target gene transcription by interacting with the transcription factor CREB to interfere with CREB phosphorylation and CREB-binding protein (CBP) recruitment. Palmitoylation and phosphorylation of KChIP3 increase its location in the cytoplasm, where it interacts with presenilin (PS) to regulate the enzymatic activity of the γ-secretase. In the cytoplasm, KChIP3 regulates intracellular K + , Ca 2+ , and Na + currents by interacting with KV4, N-methyl-D-aspartate receptor (NMDAR), transient receptor potential vanilloid 1 (TRPV1), and the ryanodine receptor.
Through the transcriptional regulation of the above-mentioned genes, KChIP3 has been shown to be multifunctional and is critically involved in the regulation of physiological and pathological functions of neurons, endocrine cells, immune cells, endothelial cells, and hematopoietic cells. For example, in the pineal gland, KChIP1-4 are involved in the regulation of rhythmically expressed genes engaged in circadian rhythms. By binding to the DRE sites of arylalkylamine N-acetyltransferase (Aanat), inducible cAMP early repressor (Crem), and Fos-related antigen-2 (Fra-2), KChIP1-4 are able to repress the basal and induced transcription of these circadian-rhythm-related genes [106]. In the pancreas, KChIP3 has been detected in islet α-and β-cells. KChIP3 represses the transcription of the Pdyn gene in a Ca 2+ -dependent manner and thereby affects glucagon release [114]. In the thyroid, KChIP3 inhibits the expression of thyroglobulin by binding directly to the DRE of the thyroglobulin gene and blocking thyroid-specific transcription factors such as TTF-1, TTF-2, and Pax8 [98,105]. KChIP3, as well as KChIP2, are expressed in T-and B cells to regulate immunological responses. In T lymphocytes, KChIP2 and KChIP3 act as transcriptional repressors to inhibit the expression of IFN-γ, IL-2, and IL-4 [103]. By inhibiting KChIP3 has physiological functions in both the cytoplasm and the nucleus. Sumoylated KChIP3 translocates to the nucleus to repress the transcription of associated genes by interacting with the DRE sequences. This interaction can be enhanced by Mg 2+ and C-terminal binding protein (CtBP) and inhibited by Ca 2+ and cyclic AMP-responsive element modulator α (αCREM). KChIP3 can also repress target gene transcription by interacting with the transcription factor CREB to interfere with CREB phosphorylation and CREB-binding protein (CBP) recruitment. Palmitoylation and phosphorylation of KChIP3 increase its location in the cytoplasm, where it interacts with presenilin (PS) to regulate the enzymatic activity of the γ-secretase. In the cytoplasm, KChIP3 regulates intracellular K + , Ca 2+ , and Na + currents by interacting with K V 4, N-methyl-D-aspartate receptor (NMDAR), transient receptor potential vanilloid 1 (TRPV1), and the ryanodine receptor.

Epilepsy
The International League Against Epilepsy (ILAE) classification of epilepsy types includes focal epilepsy, generalized epilepsy, combined generalized and focal epilepsy, and unknown epilepsy [116]. All of these seizure types share the pathophysiological characteristic of increased neuronal excitability and synchronicity. Among them, temporal lobe epilepsy is the most common focal epilepsy that can further be subdivided into mesial temporal lobe epilepsy and lateral or neocortical temporal lobe epilepsy [117]. Adults with intractable epilepsy are most commonly affected by epilepsy of the mesial temporal lobe, which is the chronic and pharmacoresistant form of epilepsy. In this type of epilepsy, seizures originate from the hippocampus, entorhinal cortex, amygdala, and parahippocampal gyrus [118]. Abundantly expressed in the hippocampus, KChIPs control the frequency of slow repetitive spike firing and attenuate action potential backpropagation by modulating the properties of K V 4 channels. Thus, KChIPs play a key role in maintaining neuronal excitability [119]. To date, several studies have implicated the downregulation of KChIPs in temporal lobe epilepsy. For example, the hippocampus of patients with medically refractory temporal lobe epilepsy had significantly reduced KChIP3 immunoreactivity compared to normal brains [120]. Consistently, downregulation of KChIP expression in specific hippocampal subfields (CA1 and CA3, but not CA2) was observed in rodent models of temporal lobe epilepsy that progressed to status epilepticus, i.e., very prolonged seizures. For example, in the hippocampus of the pilocarpine-induced rat epilepsy models, loss of KChIP1 immunoreactivity in interneurons and reduction of KChIP2 in the stratum radiatum of the CA1 region were observed [121]. In the kainic-acid-induced mouse epilepsy model, KChIP3 expression was reduced in the cortical area and CA3 region of the hippocampus in status epilepticus [120]. The use of KChIP-deficient transgenic mice provided further evidence for the role of KChIPs in the pathogenesis of epilepsy. In Kc-nip2 −/− mice, the excitability of hippocampal neurons was enhanced and the susceptibility to epilepsy induced by kindling was increased. In the hippocampal pyramidal neurons from Kcnip2 −/− mice, I A showed a reduced amplitude and shift in V1 /2 for steady-state inactivation to hyperpolarized potentials [122]. Nevertheless, the role of KChIPs in the pathogenesis of epilepsy remains to be further elucidated using neuron-specific Kcnip overexpression or knockout animal models.

Pain
A key factor in the development and maintenance of neuropathic pain is neuronal excitability. The physiological pain circuit can be briefly summarized as follows: physicochemical signals from noxious stimuli transduces through ion channels and purinergic channels to evoke action potentials. These action potentials are amplified by Na + channels to produce pain. The electrical signals are carried by unmyelinated C-fibres and thinly myelinated Aδ-fibres to the DRG in the body and the trigeminal ganglion (TG) in the face, where their cell bodies are located and project to the dorsal horn of the spinal cord and the medulla oblongata, respectively. After integration and processing, the pain input is transmitted via several ascending tracts to various projection sites in the brain. To process the sensory and discriminative aspects of pain, the lateral spinothalamic tract projects to the lateral thalamus. Medial projections of the spinothalamic and parabrachial tracts to the medial thalamus and limbic structures mediate the emotional and aversive components of pain. In pathological conditions such as inflammation, neuropathy, and diabetes, physiological pain is converted into pathological pain, which manifests as increased sensitivity to painful stimuli (hyperalgesia) [123].
As proposed by Costigan et al.: "No DREAM, no pain" [124], indicating that KChIP3 is critically involved in the regulation of pain. KChIP3 is expressed in the neurons of the ventral and dorsal horns of the spinal cord and the TG of mice [22,100]. Using Kcnip3 −/− mice, Cheng et al. proposed that loss of KChIP3 attenuates pain responses. Kcnip3 −/− mice showed significantly reduced pain behaviors in models of visceral pain induced by MgSO 4 and acetic acid, chemical pain induced by formalin, inflammatory pain induced by capsaicin and carrageenan, and neuropathic pain induced by cuff implantation around the sciatic nerve [100]. This result was further substantiated by Rivera et al. using daDREAM (dominant active DREAM)-transgenic mice. daDREAM is a Ca 2+ -insensitive double mutant (EF-hand, leucine-charged residue-rich domains) KChIP3 that actively represses KChIP3 target genes and prevents the Ca 2+ -dependent derepression function of KChIP3. In contrast to Kcnip3 −/− mice, which showed a basal state of analgesia, daDREAM transgenic mice displayed a state of basal hyperalgesia. However, the daDREAM transgenic mice showed impaired response to inflammatory pain induced by Complete Freund's adjuvant (CFA) [102]. In the above studies, the modulation of the basal threshold of pain by KChIP3 was attributed to the inhibition of the expression of PDYN, which can be cleaved into dynorphin in the spinal cord and acts as an endogenous ligand of κ-opioid receptors to exert analgesic effects [125]. Meanwhile, the regulation of spinal sensitization by KChIP3 was considered to be dependent on BDNF which is a well-established regulator of synaptic plasticity and plays a modulatory role in spinal nociceptive processing. BDNF in the physiological dose range facilitates nociception, whereas high doses produce analgesia or hypoalgesia [126]. Both Pdyn and Bdnf promoter regions contain the DRE sequence [8,61], the transcription of which could be inhibited by KChIP3. Notably, in daDREAM transgenic mice, orofacial injection of formalin (acute trigeminal nerve stimulation) induced hyperalgesia with a concomitant reduction in BDNF [127]. This finding was contrary to that reported by Rivera above, which may be because BDNF plays different and specific roles in the control of nociception in the trigeminal ganglion and in the spinal cord. Furthermore, in trigeminal neurons, KChIP3 specifically downregulates Mgll (encodes monoglyceride lipase) and Ctsl (encodes cathepsin L), which are associated with nociception [127].
Most of the conclusions drawn from the mouse model have been confirmed in the rat model. For example, upregulation of KChIP3 in the nuclear compartment was observed in a formalin-injection-induced acute pain model in rats [128]. Minocycline administration attenuated tactile allodynia and chemical hyperalgesia in diabetic rats, accompanied by downregulation of KChIP3 protein in the spinal cord [129]. However, the "no DREAM, no pain" hypothesis does not seem to apply to Kcnip3 −/− rats. Guo et al. recently found that global knockout of KChIP3 using CRISPR/Cas9 technology increased pain sensitivity in rats to acute and chronic inflammatory pain induced by formalin and CFA [130,131], which is in contrast to what was found in studies of Kcnip3 −/− mice by Cheng et al. [100]. This is a big wake-up call that KChIP3, in addition to repressing the expression of genes involved in pain processing, may have other functions in pain transmission and processing in rats. But the specificity of KChIP3 in nociception and pain processing at different levels could not be demonstrated in transgenic animals with global KChIP3 knockout. Indeed, KChIP3 is highly expressed in rat DRG neurons [132], whereas previously reported KChIP3 protein in mouse DRG could not be detected by Western blot [100]. Some reports suggest that the mechanism of KChIP3 in pain modulation in rats involves its interaction with several receptors and ion channels involved in pain-sensing and transmission in DRG neurons. These include the N-methyl-D-aspartate receptor (NMDAR) [133], transient receptor potential vanilloid 1 (TRPV1) channel [131], and K V 4 [132]. Activation of NMDAR in the central nervous system is required for central sensitization. KChIP3 significantly inhibited the surface expression of NMDARs and NMDAR-mediated currents by directly interacting with NMDAR through its N-terminal residues 21-40 [131]. TRPV1, which is also known as the thermosensitive protein, can sense noxious heat and chemical stimuli simultaneously. In primary sensory neurons, TRPV1 acts as an integrator of the painful afferent sensation and as a key initiator of the efferent neurogenic inflammation [134]. In the rat inflammatory pain model induced by CFA, TRPV1 in nociceptive sensory neurons undergoes functional sensitization, resulting in Ca 2+ influx. Meanwhile, upregulation of KChIP3 protein expression resulted in enhanced binding of KChIP3 to the TRPV1, inhibiting TRPV1 cell surface localization and thereby exerting an analgesic effect [131]. Furthermore, by regulating K V 4 in DRG neurons that meditate somatodendritic I SA , KChIP3 is involved in the transmission of pain signals [132]. Notably, Ca V 3 channels and K V 4 channels, the important contributors to pain control, were found to form a signaling complex in rat cerebellar, hippocampal, and neocortical regions [135]. Recently, a novel KChIP3 ligand, IQM-PC332, was reported to reduce mechanical hypersensitivity in rats subjected to chronic constriction injury of the sciatic nerve. Mechanistically, IQM-PC332 binds to KChIP3 and reduces ionic currents mediated by TRPV1 channels, K V 4.3 channels, low-voltage-activated T-type Ca 2+ channels, and high-voltage-activated Ca 2+ channels in DRG neurons [136]. This is a potential chemical tool and the elucidation of the structural basis of the analgesic effect of IQM-PC332 will be one of the possible steps towards a better understanding of how KChIP3 controls pain. In conclusion, KChIP3 may act at multiple levels to modulate pain. It will be necessary to use tissue-specific KChIP3 knockout models or site-specific inhibition of KChIP3 to further elucidate its complex mechanism in pain modulation ( Figure 6).

Memory Dysfunction
Learning is the process by which new information about the world is acquired, and memory is the process by which knowledge is stored. The cellular basis of learning and memory is thought to be the process by which synapses undergo bidirectional changes in synaptic strength, known as synaptic plasticity. Among the different types of synaptic plasticity, two opposite forms of synaptic plasticity, long-term potentiation (LTP) and long-term depression (LTD), have been the most studied [137]. LTP is the strengthening of synapses following repeated synaptic activity. Inhibition of LTP results in impaired learning or failure to retain memories, while LTD is the synaptic framework of weakening synaptic strengths, contributing to learning by engaging in a functional interplay with LTP [138]. Several brain regions are involved in memory, including the hippocampus, neocortex, amygdala, basal ganglia, and cerebellum. Of these, the hippocampus is essential for the spatial representation of the environment and the ability to recall specific events, or 'episodic memory' [139]. KChIP3 is highly expressed in the hippocampus and is also involved in the regulation of learning and memory. Alexander et al. reported that KChIP3 knockout mice exhibited enhanced memory in a contextual fear-conditioning paradigm. They also found that translocation of KChIP3 from the membrane to the nucleus was increased in mouse hippocampal neurons following the fear conditioning training paradigm [33]. In the cell nucleus, KChIP3 functions as a transcriptional repressor to regulate the expression of genes involved in memory formation, such as Pdyn, Fos, Bdnf, and Npas4 [33,61]. In addition, Fontán et al. reported that loss of KChIP3 not only enhances LTP and improves learning and memory in young mice, but also improves cognition and slows age-related brain degeneration in old age [140]. Mechanistically, KChIP3 interacts with CREB, a key transcription factor involved in memory, and negatively regulates CREB-dependent transcription. This raises the threshold for CREB activation in learning and memory. Therefore, in the Kcnip3 −/− mice, the threshold for CREB phosphorylation and CREB-CBP interaction in learning and memory is lowered, and the timing of CREB phosphorylation and CBP recruitment to CREB is shortened, thereby promoting CREB-dependent transcription during learning. Consistent results were also confirmed in daDREAM-transgenic mice. Transgenic mice expressing the daDREAM showed significant impairments in learning and memory [61].
The mechanism by which KChIP3 is involved in memory regulation also involves its protein-protein interactions. NMDAR is the major ionotropic glutamate receptor in the central nervous system and plays an important role in both synaptic transmission and plasticity [141]. NMDARs are required for LTP and LTD of synaptic transmission [142]. When Ca 2+ binds to KChIP3, the interaction between KChIP3 and PSD95 is released, allowing NMDA to function as a normal receptor. The EF-hands mutant KChIP3, which lacks Ca 2+ binding ability, inhibits NMDAR function in mouse hippocampal CA1, impairs LTD, and thereby reduces consolidation of hippocampus-dependent contextual fear-conditioned reflexive memory [143]. In addition, K V 4.2 channels play an important role in both synaptic plasticity and cognition, which in part contributes to the regulatory effect of KChIP3 on learning and memory [144]. channels, low-voltage-activated T-type Ca 2+ channels, and high-voltage-activated Ca 2+ channels in DRG neurons [136]. This is a potential chemical tool and the elucidation of the structural basis of the analgesic effect of IQM-PC332 will be one of the possible steps towards a better understanding of how KChIP3 controls pain. In conclusion, KChIP3 may act at multiple levels to modulate pain. It will be necessary to use tissue-specific KChIP3 knockout models or site-specific inhibition of KChIP3 to further elucidate its complex mechanism in pain modulation ( Figure 6).

Alzheimer's Disease
Amyloid beta-protein (Aβ) deposition is the main feature of AD, which has a toxic effect on neurons [145]. PS is the catalytic core of the high-molecular-weight enzyme complex γ-secretase, which can mediate γ-cleavage of β-amyloid precursor protein to produce Aβ [146]. KChIP3 was initially discovered as being capable of binding to the C-terminal fragment of presenilin 1 (PS1) and PS2 via the C-terminal 103 amino acids encoded by ALG3 [7]. The binding of KChIP3 to PS is dependent on the presence of Ca 2+ [147]. Existing studies have shown that mutations of PS1 and PS2 lead to increased Aβ formation and apoptosis, thus causing early familial Alzheimer's disease (AD) [148]. In the brains of patients with AD, the expression of KChIP3 was increased in neurons and reactive astrocytes, which is consistent with the regions with pathological changes of AD [149]. This suggests that KChIP3 may be involved in the pathogenesis of AD. Zaidi et al. subsequently showed that KChIP3 stabilizes the structure of the PS in the cerebellum and hippocampus [150]. Jo et al. reported KChIP3 as a pro-apoptotic protein that facilitates cell death induced by Aβ production [151]. Overexpression of KChIP3 increased the enzymatic activity of the presenilin-γ-secretase complex in HEK293 cells [152]. Aβ increased KChIP3 expression in cultured neurons while blocking its expression protected neuronal cells from Aβ toxicity [149]. Repaglinide has been shown to inhibit the KChIP3-PS2 interaction, suggesting a novel avenue for future AD treatment [153].
KChIP3 also regulates the intracellular Ca 2+ signaling, the dysregulation of which has been implicated in the development of AD [154]. In AD models, increased Ca 2+ release from ryanodine-sensitive Ca 2+ stores have been observed in neurons [155]. Lilliehook et al. showed that overexpression of KChIP3 can enhance apoptosis by increasing ER Ca 2+ release in glioma cells [156]. Fedrizzi et al. reported that transient co-expression of KChIP3 with PS facilitated Ca 2+ release from the ER in neurons [157]. Mechanistically, KChIP3 binds to the DRE sequence of the isoform 3 of the Na + /Ca 2+ exchanger (Ncx3) gene to repress its transcription, thereby regulating intracellular Ca 2+ homeostasis [109]. In mouse hippocampal and cortical neurons, KChIP3 regulates intracellular calcium-induced Ca 2+ release through direct protein-protein interaction with the ryanodine receptor [158]. Furthermore, KChIP3 appears to be involved in age-related brain degeneration [140], which is a well-known clinical symptom of AD. Although the current molecular understanding of the relationship between KChIP3 and AD pathogenesis is limited and insufficient, these findings clearly support KChIP3 as an appropriate target for further research into AD. Therefore, unravelling the diversity and complexity of KChIP3's functions would pave the way for developing more effective treatments for AD.

Other Neurodegenerative Disorders
In addition to AD, KChIP3 has been implicated in the pathogenesis of other neurodegenerative disorders. Recent evidence suggests that KChIP3 is closely linked to Huntington's disease (HD) [159] and amyotrophic lateral sclerosis (ASL) [160]. Firstly, the levels of KChIP3 protein are significantly reduced in the hippocampus samples of HD patients. To be specific, KChIP3 interacts with activating transcription factor 6 (ATF6) to suppress the pro-survival unfolded protein response, a common feature of neurodegenerative diseases. In the mouse model of HD, inhibition of the KChIP3-ATF6 interaction delays the onset of cognitive deficits. In addition, treatment with repaglinide has been shown to delay the onset and progression of motor and cognitive decline and extend lifespan by blocking the interaction of KChIP3 and ATF6 [74,159], suggesting that KChIP3 may be a novel target for the treatment of related diseases. Second, upregulation of KChIP3 has been observed in motor neurons and astrocytes in the spinal cord and frontal cortex of ALS patients [160]. The main feature of ALS is the relentless loss of motor neurons and increased reactive astrogliosis [161]. Cebolla et al. showed that KChIP3 is able to promote astrocyte differentiation of cortical precursors via cAMP-dependent calcium signaling. The neonatal cortex of Kcnip3 −/− mice has a reduced number of astrocytes and an increased number of neurons [108]. Nevertheless, studies linking KChIP3 to these diseases have been limited. More evidence is needed to elucidate the mechanisms by which KChIP3 is involved in these neurodegenerative diseases.

Arrhythmias
Cardiac arrhythmias are classified mechanistically into two categories: focal activity due to enhanced or abnormal pulse generation and reentry due to conduction disturbances [162]. Early afterdepolarizations preceding full repolarization and delayed afterdepolarizations occurring after full repolarization are the most common causes of focal arrhythmias. Prolongation of action potentials caused by I Na,L (late Na + current), the I Ca,L , or I NCX , or decreases in the repolarizing potassium currents (I Kr , I Ks , I K1 ) can lead to early afterdepolarizations. As mentioned above, KChIPs have been shown to regulate a wide variety of ion channels in cardiomyocytes, including those that control the depolarization and repolarization of cardiac action potentials. Thus, KChIPs are critically involved in the pathogenesis of arrhythmias, as demonstrated by some of the available evidence, either directly or indirectly. For example, KChIP2 was upregulated in aging porcine atria [163] and was significantly reduced in TLR4-activated inflammatory responses [164] and chronic NM-DAR activation [165]. Atrial fibrillation could be induced in zebrafish hearts overexpressing KCNIP1 [13]. Consistently, Kcnip2 −/− mice have an increased susceptibility to arrhythmias, manifested by a prolonged elevation in the ST segment on the electrocardiogram [166]. In fact, the ST segment coincides in time with the action potential plateau, and increasing outward current (mainly I to and I K-ATP ) or decreasing inward current (mainly I Na and I Ca ) can promote the occurrence of ST segment elevation [167]. Therefore, Kcnip2 −/− mice may have compensatory remodeling against the loss of I to . Mechanistically, KChIP2 on the one hand directly regulates the subcellular localization and gating properties of several ion channels as an auxiliary subunit in the cardiomyocyte. On the other hand, KChIP2 also controls the expression of ion channel subunits at the transcriptional level. In ventricular myocytes from Kcnip2 −/− mice, I to,f and I Na are abolished, I Ca,L is downregulated, and I Ks and I to,s (the slow transient outward K + current) are upregulated [87,92,168]. Recently, KChIP2 was reported to act as a transcriptional repressor by binding directly to the promoters of miR-34b/c, a miRNA that directly affects SCN5A (Na V 1.5), SCN1B (Na V β1), and KCND3 (K V 4.3) gene expression. Inhibition of miR-34b/c can block the induction of arrhythmia [169]. Whether there are other potential ion channel proteins, or whether the genes encoding these ion channels are regulated by KChIP2, is a topic that deserves further investigation. Due to the multiple roles of KChIP2 in regulating ion channels in the heart, a thorough understanding of the detailed molecular mechanisms by which abnormal KChIP2 levels increase arrhythmias susceptibility is key to the development of novel therapies for the prevention and treatment of cardiac arrhythmias.

Cardiac Remodeling
Cardiac remodeling is defined as persistent changes in cardiac structure and function in response to physiological or pathological stimuli. Pathophysiological events that cause a decrease in contractility and/or an increase in wall stress, such as ischemia/reperfusion, myocardial infarction, pressure and volume overload, hypertension, and neuroendocrine stimulation, often result in adverse cardiac remodeling [170]. KChIP2 expression is altered in many cardiovascular events, including ischemic cardiomyopathy [171], myocarditis [172], mitral valve disease [173], inflammatory cytokine-induced myocardial injury [50,174], myocardial infarction [175], and type 2 diabetes mellitus [176]. Previous studies have shown that KChIP2 expression is significantly downregulated in hypertrophic myocardium, which is associated with reduced I to [166,177]. Some of these mechanisms have been validated in vitro models of hypertrophy. For example, overactivation of the JNK pathway was found to cause downregulation of KChIP2 in neonatal ventricular myocytes treated with phenylephrine, a robust inducer of hypertrophy [46]. In addition, our previous study demonstrated that the KChIP2 expression was decreased in phenylephrine-induced hypertrophic cardiomyocytes. Mechanistically, phenylephrine-activated NF-κB binds to the promoter region of the Kcnip2 gene and directly represses its transcription. Overexpression of muscle-specific mitsugumin 53 upregulates KChIP2 through inhibition of NF-κB and thereby reversed phenylephrine-induced cardiomyocyte hypertrophy [43]. In addition, Jin et al. found that adenoviral overexpression of KChIP2 in vivo significantly attenuated the development of left ventricular hypertrophy in aortic-banded rats. This protective effect of KChIP2 was achieved by inhibiting MAPK signaling activity and reducing calcineurin/NFAT expression [178].
Cardiac memory is a specific form of cardiac remodeling that is manifested by the persistence of inverted T waves after the restoration of sinus rhythm. The T wave "remembers" the QRS complex from the paced or arrhythmia phase following a short alteration in the sequence of ventricular depolarization caused by pacing or arrhythmia [179]. An important electrophysiological mechanism responsible for cardiac memory is the reduction in I to density and its significantly prolonged recovery from inactivation [180]. The decreased expression of KChIP2 during this phase is essential for the occurrence of cardiac memory. According to Ozgen et al., left ventricular pacing induces the degradation of the transcription factor CREB by initiating the production of myocardial angiotensin II and the synthesis of reactive oxygen species, which results in the downregulation of KChIP2 expression [54].

Heart Failure
There is a growing number of evidence that KChIP2 also plays an important role in the pathogenesis of heart failure. The mRNA and protein expression of KChIP2 was significantly downregulated in the failing heart [181]. Meanwhile, autoantibodies against KChIP2 have been detected in patients with dilated cardiomyopathy. In vitro incubation of anti-KChIP2 antibody facilitates necrotic cell death in rat cardiomyocytes, suggesting that KChIP2 autoantibodies may be involved in the pathogenesis of dilated cardiomyopathy [182]. Candesartan, the angiotensin II receptor blocker, attenuates KChIP2 downregulation in dilated cardiomyopathy and is involved in preventing severe electrical remodeling in inherited dilated cardiomyopathy [183]. Nassal et al. found that reduction of KChIP2 in guinea pig cardiomyocytes significantly increased I Ca,L and prolonged action potentials by increasing Ca V 1.2 protein expression [184]. They also found that KChIP2, like the neuronal KChIP isoforms, can regulate ryanodine receptor activity by interacting with PS. Loss of KChIP2 resulted in reduced ryanodine receptor activity due to a decrease in its binding affinity to PS, which disrupted calcium-induced calcium release events. This further leads to impaired contractility of cardiomyocytes and promotes the onset of heart failure [185]. Interestingly, Speerschneider et al. showed that although KChIP2 is downregulated in heart failure, the reduction of I to,f does not promote the development of heart failure. On the contrary, reduction of I to exerts antiarrhythmic effects in mouse heart [186]. Likewise, Grubb et al. showed that while the downregulation of repolarization currents in heart failure was exacerbated in Kcnip2 −/− mice, there was less prolongation of action potentials associated with heart failure due to compensation by upregulation of I Ks and I to,s [187]. In summary, the role of KChIP2 in heart failure remains controversial. Further research is needed to determine whether targeting KChIP2 has therapeutic implications in heart failure.

Concluding Remarks
KChIPs are collections of multifunctional proteins involved in a wide range of physiological processes and pathological diseases. Thanks to the contributions of many outstanding researchers in this field over the last 25 years, the understanding of their structure, regulation, biological function in health and diseases, and small-molecule drugs targeting KChIPs has been achieved. KChIPs are auxiliary subunits of K V 4 channels and are critical for the trafficking and gating properties of K V 4 channels. Meanwhile, KChIPs are also transcriptional regulators that repress the transcription of target genes in various systems throughout the body to maintain normal physiological functions, including the nervous, cardiovascular, respiratory, immune, and endocrine systems. To date, an increasing number of reports have partially linked dysregulation of KChIPs expression to disease. These include AD, HD, epilepsy, and memory dysfunction in the nervous system, arrhythmia, remodeling, and heart failure in the cardiovascular system. Some small-molecule drugs that regulate them have been shown to alleviate symptoms or provide therapeutic benefits in these diseases, most notably repaglinide, which has the most significant therapeutic effect in HD. In addition, many small molecule ligands targeting KChIPs have emerged in recent years, and basic research has shown good effects in activating or inhibiting KChIPs. However, our understanding of KChIPs, a class of proteins with similar functions but different characteristics, is still only the tip of the iceberg. There are still many questions that researchers need to answer. For example, the structural basis for the regulation of K V 4 channels by KChIPs as auxiliary subunits remains elusive. The regulatory function of KChIP2 on K V 1.5 and Na V 1.5 found in the heterologous expression system has not yet been verified in vivo. Altered expression of KChIPs has been found in many clinical disease samples, but the elucidation of their causal relationship to these diseases and the mechanisms involved is far from complete. In addition, some conflicting results that have been reported in this field further confirm the functional complexity of KChIPs. For example, the role of KChIP3 in pain appears to differ between rats and mice. Small-molecule compounds such as NS5806, a ligand for KChIP2, has different effects in different species or even different parts of the same species, which will significantly hamper the development of targeted therapies. Therefore, despite the development of several small molecules targeting KChIPs, further efforts are needed to demonstrate their translational value in treating disease.