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Post-traumatic stress disorder (PTSD) is an anxiety disorder caused by traumatic or frightening events, with intensified anxiety, fear memories, and cognitive impairment caused by a dysfunctional hippocampus. Owing to its complex phenotype, currently prescribed treatments for PTSD are limited. This study investigated the psychopharmacological effects of novel COMBINATION herbal medicines on the hippocampus of a PTSD murine model induced by combining single prolonged stress (SPS) and foot shock (FS). We designed a novel herbal formula extract (HFE) from Chaenomeles sinensis , Glycyrrhiza uralensis , and Atractylodes macrocephala . SPS+FS mice were administered HFE (500 and 1000 mg/kg) once daily for 14 days. The effects of HFE of HFE on the hippocampus were analyzed using behavioral tests, immunostaining, Golgi staining, and Western blotting. HFE alleviated anxiety-like behavior and fear response, improved short-term memory, and restored hippocampal dysfunction, including hippocampal neurogenesis alteration and aberrant migration and hyperactivation of dentate granule cells in SPS+FS mice. HFE increased phosphorylation of the Kv4.2 potassium channel, extracellular signal-regulated kinase, and cAMP response element-binding protein, which were reduced in the hippocampus of SPS+FS mice. Therefore, our study suggests HFE as a potential therapeutic drug for PTSD by improving behavioral impairment and hippocampal dysfunction and regulating Kv4.2 potassium channel-related pathways in the hippocampus.

Long noncoding RNAs (lncRNAs) are known to be the important regulators in cancer progression. However, the role of lncRNA FAM66C (FAM66C) is yet to be investigated in intrahepatic cholangiocarcinoma (ICC). This study aimed to investigate the effects and related mechanisms of FAM66C in ICC. Human ICC tissues and cell lines were collected. The expression levels of FAM66C, hsa-miR-23b-3p (miR-23b-3p), and KCND2 were detected by qRT-RCR. The transfection experiments were employed to measure the effect of FAM66C on cell viabilities, migration, and invasion in ICC cells by CCK-8, transwell assays. Glycolysis was investigated by glucose consumption, lactate production and ATP levels. The dual-luciferase reporter and RNA pull down assays were conducted as a means of confirming the interactions between FAM66C, miR-23b-3p, and KCND2. Furthermore, the levels of the EMT-associated proteins (KCND2, GLUT1, PKM2, and LDHA) in ICC cells were detected by western blot. FAM66C was increased in ICC tissues and cells, increased cell viability, glycolysis, migration and invasion, and decreased apoptosis were shown in FAM66C overexpressing cells. Mechanistic analyses revealed that FAM66C regulated the downstream target gene KCND2 by sponging miR-23b-3p. FAM66C effect on ICC was further validated in murine xenograft assays. FAM66C knockdown cells gave rise to tumors that were smaller in size, consistent with the role of FAM66C as a promoter of in vivo tumor growth. These data revealed that FAM66C was able to drive ICC tumor progression and glycolytic activity via the miR-23b-3p/KCND2 axis, indicating FAM66C may be a viable target for treating ICC., (© 2021 Wiley Periodicals LLC.)

Transient outward potassium currents were first described nearly 60 years ago, since then major strides have been made in understanding their molecular basis and physiological roles. From the large family of voltage-gated potassium channels members of 3 subfamilies can produce such fast-inactivating A-type potassium currents. Each subfamily gives rise to currents with distinct biophysical properties and pharmacological profiles and a simple workflow is provided to aid the identification of channels mediating A-type currents in native cells. Their unique properties and regulation enable A-type K + channels to perform varied roles in excitable cells including repolarisation of the cardiac action potential, controlling spike and synaptic timing, regulating dendritic integration and long-term potentiation as well as being a locus of neural plasticity., (© 2021. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The present study investigates the physiological role of Kvβ1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvβ1.1 knockout (KO) or wild-type (WT) mice and established that Kvβ1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvβ1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvβ1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvβ1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvβ1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvβ1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvβ1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart. NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvβ1.1 subunit. In the present study, we identify Kvβ1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart., (Copyright © 2017 the American Physiological Society.)

KCNE2 functions as an auxiliary subunit in voltage-gated K and HCN channels in the heart. Genetic variations in KCNE2 have been linked to long QT syndrome. The underlying mechanisms are not entirely clear. One of the issues is whether KCNE2 protein is expressed in ventricles. We use adenovirus-mediated genetic manipulations of adult cardiac myocytes to validate two antibodies (termed Ab1 and Ab2) for their ability to detect native KCNE2 in the heart. Ab1 faithfully detects native KCNE2 proteins in spontaneously hypertensive rat and guinea pig hearts. In both cases, KCNE2 protein is more abundant in ventricles than in atria. In both ventricular and atrial myocytes, KCNE2 protein is preferentially distributed on the cell surface. Ab1 can detect a prominent KCNE2 band in human ventricular muscle from nonfailing hearts. The band intensity is much fainter in atria and in failing ventricles. Ab2 specifically detects S98 phosphorylated KCNE2. Through exploring the functional significance of S98 phosphorylation, we uncover a novel mechanism by which KCNE2 modulates the human ether-a-go-go related gene (hERG) current amplitude: by accelerating hERG protein degradation and thus reducing the hERG protein level on the cell surface. S98 phosphorylation appears to be required for this modulation, so that S98 dephosphorylation leads to an increase in hERG/rapid delayed rectifier current amplitude. Our data confirm that KCNE2 protein is expressed in the ventricles of human and animal models. Furthermore, KCNE2 can modulate its partner channel function not only by altering channel conductance and/or gating kinetics, but also by affecting protein stability.

Globus pallidus (GP) neurons fire rhythmically in the absence of synaptic input, suggesting that they may encode their inputs as changes in the phase of their rhythmic firing. Action potential afterhyperpolarization (AHP) enhances precision of firing by ensuring that the ion channels recover from inactivation by the same amount on each cycle. Voltage-clamp experiments in slices showed that the longest component of the GP neuron's AHP is blocked by apamin, a selective antagonist of calcium-activated SK channels. Application of 100 nm apamin also disrupted the precision of firing in perforated-patch and cell-attached recordings. SK channel blockade caused a small depolarization in spike threshold and made it more variable, but there was no reduction in the maximal rate of rise during an action potential. Thus, the firing irregularity was not caused solely by a reduction in voltage-gated Na(+) channel availability. Subthreshold voltage ramps triggered a large outward current that was sensitive to the initial holding potential and had properties similar to the A-type K(+) current in GP neurons. In numerical simulations, the availability of both Na(+) and A-type K(+) channels during autonomous firing were reduced when SK channels were removed, and a nearly equal reduction in Na(+) and K(+) subthreshold-activated ion channel availability produced a large decrease in the neuron's slope conductance near threshold. This change made the neuron more sensitive to intrinsically generated noise. In vivo, this change would also enhance the sensitivity of GP neurons to small synaptic inputs.

Our previous study indicated that TGF-beta1 induced the expression of a transient outward K+ channel (A-type) during the phenotypic transformation of vascular fibroblasts to myofibroblasts. Here, we studied the relevant signal transduction pathway using whole cell recording and a quantitative RT-PCR technique. Results indicate that the protein kinase C (PKC) agonist phorbol-12-myristate-13-acetate (PMA, 1 microM) could mimic the effect of TGF-beta1 (20 ng/ml) on the expression of an A-type K+ channel and induced a similar A-type K+ current. Moreover, a PKC inhibitor, bisindolylmaleimide I (1 microM), could abrogate the effect of TGF-beta1 on K(V)4.2 expression. This result suggests that a PKC pathway may be involved in the expression of an A-type K+ channel induced by TGF-beta1 in rat vascular myofibroblasts.

n-Butyl-p-aminobenzoate (BAB; butamben) is a long-duration anesthetic used for the treatment of chronic pain. Epidural administration of BAB is thought to reduce the electrical excitability of dorsal root nociceptor fibers by inhibiting voltage-gated ion channels. To further investigate this mechanism, we examined the effects of BAB on the potassium currents of acutely dissociated neurons from the rat dorsal root ganglion (DRG). These neurons express a rapidly inactivating A-type K(+) current (I(A)) that is resistant to tetraethylammonium (20 mM) but inhibited by 4-aminopyridine (5 mM). At low concentrations, BAB (< or =1 microM) selectively inhibited the I(A) component of DRG K(+) current. The voltage dependence of activation and inactivation, kinetics of recovery from inactivation, and the pharmacology of the DRG I(A) were similar to those of the Kv4 family of K(+) channels. Reverse transcription-polymerase chain reaction was used to establish that the messages encoding for all three of the mammalian Kv4 channel subunits (Kv4.1-Kv4.3) were present in the rat DRG. BAB produced a high-affinity, partial inhibition of heterologously expressed Kv4.2 channels (K(D) = 59 nM) but did not alter the kinetics or voltage sensitivity of gating. Substituting polar threonines for conserved hydrophobic residues of the S6 segment weakened BAB binding but did not alter the voltage-dependent gating of the Kv4.2 channel. At physiological pH, BAB is uncharged, suggesting that hydrophobic interactions may contribute to drug binding. The data support a mechanism in which BAB binds near the narrow cytoplasmic entrance of Kv4 channels and inhibits current by a pore blocking mechanism.

Gating of voltage-dependent K(+) channels involves movements of membrane-spanning regions that control the opening of the pore. Much less is known, however, about the contributions of large intracellular channel domains to the conformational changes that underlie gating. Here, we investigated the functional role of intracellular regions in Kv4 channels by probing relevant cysteines with thiol-specific reagents. We find that reagent application to the intracellular side of inside-out patches results in time-dependent irreversible inhibition of Kv4.1 and Kv4.3 currents. In the absence or presence of Kv4-specific auxiliary subunits, mutational and electrophysiological analyses showed that none of the 14 intracellular cysteines is essential for channel gating. C110, C131, and C132 in the intersubunit interface of the tetramerization domain (T1) are targets responsible for the irreversible inhibition by a methanethiosulfonate derivative (MTSET). This result is surprising because structural studies of Kv4-T1 crystals predicted protection of the targeted thiolate groups by constitutive high-affinity Zn(2+) coordination. Also, added Zn(2+) or a potent Zn(2+) chelator (TPEN) does not significantly modulate the accessibility of MTSET to C110, C131, or C132; and furthermore, when the three critical cysteines remained as possible targets, the MTSET modification rate of the activated state is approximately 200-fold faster than that of the resting state. Biochemical experiments confirmed the chemical modification of the intact alpha-subunit and the purified tetrameric T1 domain by MTS reagents. These results conclusively demonstrate that the T1--T1 interface of Kv4 channels is functionally active and dynamic, and that critical reactive thiolate groups in this interface may not be protected by Zn(2+) binding.

Dipeptidyl-aminopeptidase-like protein 6 (DPPX) was recently shown in the brain to modulate the kinetics of transient A-type currents by accelerating inactivation and recovery from inactivation. Since the kinetics of human cardiac transient outward current (I(to)) are not mimicked by coexpression of the alpha-subunit Kv4.3 with its known beta-subunit KChIP2, we have tested the hypothesis that DPPX may serve as an additional beta-subunit in the human heart. With quantitative real-time RT-PCR strong mRNA expression of DPPX was detected in human ventricles and was verified at the protein level in human but not in rat heart by a DPPX-specific antibody. Co-expression of DPPX with Kv4.3 in Chinese hamster ovary cells produced I(to)-like currents, but compared with expression of KChIP2a and Kv4.3, the time constant of inactivation was faster, the potential of half-maximum steady-state inactivation was more negative and recovery from inactivation was delayed. Co-expression of DPPX in addition to Kv4.3 and KChIP2a produced similar current kinetics as in human ventricular myocytes. We therefore propose that DPPX is an essential component of the native cardiac I(to) channel complex in human heart.

During pregnancy, the heart develops a reversible physiological hypertrophic growth in response to mechanical stress and increased cardiac output; however, underlying molecular mechanisms remain unknown. Here, we investigated pregnancy-related changes in heart structure, function, and gene expression of known markers of pathological hypertrophy and cell stretching in mice hearts. In late pregnancy, hearts show eccentric hypertrophy, as expected for a response to volume overload, with normal left ventricular diastolic function and a moderate reduction in systolic function. Pregnancy-related physiological heart hypertrophy does not induce expression changes of known markers of pathological hypertrophy like: alpha- and beta-myosin heavy chain, atrial natriuretic factor, phospholamban, and sarcoplasmic reticulum Ca2+-ATPase. Instead, it induces the remodeling of Kv4.3 channel and increased c-Src tyrosine kinase activity, a stretch-responsive kinase. Cardiac Kv4.3 channel gene expression was downregulated by approximately 3- to 5-fold, both at the mRNA and protein levels, and was paralleled by a reduction in transient outward K+ currents, a longer action potential and by prolongation of the QT interval. Downregulation of cardiac Kv4.3 transcripts was mimicked by estrogen treatment in ovariectomized mice, and was prevented by the estrogen receptor antagonist ICI 182,780. c-Src activity increased by approximately 2-fold in late pregnancy and after estrogen treatment. We propose that, in addition to mechanical stress, the rise of estrogen toward the end of pregnancy contributes to pregnancy-related heart hypertrophy by increased c-Src activity and that the rise of estrogen is one factor that down regulates cardiac Kv4.3 gene expression providing a molecular correlate for a longer QT interval in pregnancy.

An asthma-related peptidase homologue (DPP10) may act as an auxiliary subunit of Kv4 channels, similar to DPPX. Here we show that DPP10 preferentially binds to Kv4 channel proteins to increase current density and alter channel gating. DPP10 also forms complexes by themselves and with DPPX in the absence of Kv4 channels. DPP10 mRNA is abundantly expressed in nodose and dorsal root ganglia, suggesting that DPP10 participates in controlling airway reactivity and mechanosensation. The region from the N-terminus to the end of the transmembrane of DPP10 mediates its association with the channel, whereas the S1-S2 portion of the channel is sufficient for complex formation. This N-terminal portion of DPP10 also confers all the gating effects produced by the peptidase homologue. Thus, interaction between transmembranes of DPP10/DPPX and Kv4 channel mediates functional complex formation. We call this protein DPPY, instead of DPP10, because of its revealed role as a Kv4 channel regulator.

Heart failure (HF) is characterized by marked prolongation of action potential duration and reduction in cellular repolarization reserve. These changes are caused in large part by HF-induced K(+) current downregulation. Molecular mechanisms underlying these changes remain unclear. We determined whether downregulation of K(+) currents in a canine model of tachycardia-induced HF is caused by altered expression of underlying K(+) channel alpha- and beta-subunits encoding these currents. K(+) channel subunit expression was quantified in normal and failing dogs at the mRNA and protein levels in epicardial (Epi), midmyocardial (Mid), and endocardial (Endo) layers of left ventricle. Analysis of mRNA and protein levels of candidate genes encoding the transient outward K(+) current (I(to)) revealed marked reductions in canine cKv4.3 expression in HF in Epi (44% mRNA, 39% protein), Mid (52% mRNA, 34% protein), and Endo (49% mRNA, 73% protein) layers and a paradoxical enhancement (41% Epi, 97% Mid, 113% Endo) in cKv1.4 protein levels, without significant changes in Kv channel-interacting protein cKChIP2 expression. Expression of cKir2.1, the gene underlying inward rectifier K(+) current (I(K1)), was unaffected by HF at mRNA and protein levels despite significant reduction in I(K1), whereas canine ether-a-go-go-related gene (cERG), which encodes the rapidly activating component of the delayed rectifier current (I(K)), exhibited increased protein expression. HF was not accompanied by significant changes in cKvLQT1 or cMinK mRNA and protein levels. These data indicate that 1) downregulation of I(to) in HF is associated with decreased cKv4.3 and not cKv1.4 or cKChIP2, and 2) alterations in both the rapidly activating and slowly activating components of I(K) as well as I(K1) in nonischemic dilated cardiomyopathy are not caused by changes in either transcript or immunoreactive protein levels of relevant channel subunits, which suggests posttranslational modification of these currents by HF.

A new member of a family of proteins characterized by structural similarity to dipeptidyl peptidase (DPP) IV known as DPP10 was recently identified and linked to asthma susceptibility; however, the cellular functions of DPP10 are thus far unknown. DPP10 is highly homologous to subfamily member DPPX, which we previously reported as a modulator of Kv4-mediated A-type potassium channels (Nadal, M. S., Ozaita, A., Amarillo, Y., Vega-Saenz de Miera, E., Ma, Y., Mo, W., Goldberg, E. M., Misumi, Y., Ikehara, Y., Neubert, T. A., and Rudy, B. (2003) Neuron. 37, 449-461). We studied the ability of DPP10 protein to modulate the properties of Kv4.2 channels in heterologous expression systems. We found DPP10 activity to be nearly identical to DPPX activity and significantly different from DPPIV activity. DPPX and DPP10 facilitated Kv4.2 protein trafficking to the cell membrane, increased A-type current magnitude, and modified the voltage dependence and kinetic properties of the current such that they resembled the properties of A-type currents recorded in neurons in the central nervous system. Using in situ hybridization, we found DPP10 to be prominently expressed in brain neuronal populations that also express Kv4 subunits. Furthermore, DPP10 was detected in immunoprecipitated Kv4.2 channel complexes from rat brain membranes, confirming the association of DPP10 proteins with native Kv4.2 channels. These experiments suggest that DPP10 contributes to the molecular composition of A-type currents in the central nervous system. To dissect the structural determinants of these integral accessory proteins, we constructed chimeras of DPPX, DPP10, and DPPIV lacking the extracellular domain. Chimeras of DPPX and DPP10, but not DPPIV, were able to modulate the properties of Kv4.2 channels, highlighting the importance of the intracellular and transmembrane domains in this activity.

RNA interference (RNAi) is a powerful technique for gene silencing, in which the downregulation of mRNA is triggered by short RNAs complementary to a target mRNA sequence, with consequent reduction of the encoded protein. The aim of this study was to test the effects of silencing the expression of the cardiac potassium channel Kv4.3 in a heterologous expression system, in order to investigate the effect of RNAi on channel properties. A Chinese hamster ovary cell line stably expressing Kv4.3 and the accessory beta-subunit KChIP2 was transfected with small-interfering RNAs (siRNAs) targeting Kv4.3. Effects of RNAi were monitored at the mRNA, protein, and functional levels. Real-time PCR and immunofluorescence staining revealed significant reduction of Kv4.3 mRNA and protein expression. These results were confirmed by functional patch-clamp measurements of the transient outward current (I(to)) which was reduced up to 80% by RNAi. We conclude that the use of siRNAs reagents for post-transcriptional gene silencing is a new effective method for the reduction of the expression and function of different ionic channels which may be adapted for studying their role also in native cells.

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

A-type K(+) channels belonging to the Shal subfamily are found in various receptor and neuronal cells. Although their kinetics and cell surface expression are regulated by auxiliary subunits, little is known about the proteins that may interact with Kv4 during development. A yeast two-hybrid screening of a cDNA library made from the sensory epithelium of embryonic chick cochlea revealed a novel association of Kv4.2 with a protein containing a pentraxin domain (PPTX). Sequence analysis shows that PPTX is a member of the long pentraxin family, is 53% identical to mouse PTX3, and has a signal peptide at the N terminus. Studies with chick cochlear tissues reveal that Kv4.2 coprecipitates PPTX and that both proteins are colocalized to the sensory and ganglion cells. A yeast two-hybrid assay demonstrated that the last 22 amino acids of the PPTX C terminus interact with the N terminus of Kv4.2. Chinese hamster ovary cells transfected with recombinant PPTX reveal secretory products in both non-truncated and truncated forms. Among the secreted variants are several blocked by Brefeldin A, suggesting export via a classical pathway. PPTX is soluble in the presence of sodium carbonate, suggesting localization to the cytosolic side of the plasmalemma. Immunohistochemical studies show that Kv4.2 and PPTX colocalize in the region of the plasmalemma of Chinese hamster ovary cells; however, both are locked in the endoplasmic reticulum of COS-7 cells, suggesting that PPTX does not act as a shuttle protein. Reverse transcription-PCR demonstrates that PPTX mRNA is found in tissues that include brain, eye, heart, and blood vessels.

Potassium channels are key determinants of neuronal excitability. We recently identified KChIPs as a family of calcium binding proteins that coassociate and colocalize with Kv4 family potassium channels in mammalian brain (An et al. [2000] Nature 403:553). Here, we used light microscopic immunohistochemistry and multilabel immunofluorescence labeling, together with transmission electron microscopic immunohistochemistry, to examine the subcellular distribution of KChIPs and Kv4 channels in adult rat cerebellum. Light microscopic immunohistochemistry was performed on 40-microm free-floating sections using a diaminobenzidine labeling procedure. Multilabel immunofluorescence staining was performed on free-floating sections and on 1-microm ultrathin cryosections. Electron microscopic immunohistochemistry was performed using an immunoperoxidase pre-embedding labeling procedure. By light microscopy, immunoperoxidase labeling showed that Kv4.2, Kv4.3, and KChIPs 1, 3, and 4 (but not KChIP2) were expressed at high levels in cerebellar granule cells (GCs). Kv4.2 and KChIP1 were highly expressed in GCs in rostral cerebellum, whereas Kv4.3 was more highly expressed in GCs in caudal cerebellum. Immunofluorescence labeling revealed that KChIP1 and Kv4.2 are concentrated in somata of cerebellar granule cells and in synaptic glomeruli that surround synaptophysin-positive mossy fiber axon terminals. Electron microscopic analysis revealed that KChIP1 and Kv4.2 immunoreactivity is concentrated along the plasma membrane of cerebellar granule cell somata and dendrites. In synaptic glomeruli, KChIP1 and Kv4.2 immunoreactivity is concentrated along the granule cell dendritic membrane, but is not concentrated at postsynaptic densities. Taken together, these data suggest that A-type potassium channels containing Kv4.2 and KChIP1, and perhaps also KChIP3 and 4, play a critical role in regulating postsynaptic excitability at the cerebellar mossy-fiber/granule cell synapse., (Copyright 2005 Wiley-Liss, Inc.)

The human cardiac transient outward potassium current I(to) is formed by co-assembly of voltage-dependent K(+) channel (Kv 4.3) pore-forming alpha-subunits with differently spliced K channel interacting protein (KChIP) accessory proteins. I(to) is of considerable importance for the normal course of the cardiac ventricular action potential. The present study was performed to determine whether isoforms of the serum- and glucocorticoid-inducible kinase (SGK) family influence Kv 4.3/KChIP2b channel activity in the Xenopus laevis heterologous expression system. Co-expression of SGK1, but not of SGK2 or SGK3, increased Kv 4.3/KChIP2b channel currents. The up-regulation of the current was not due to changes in the activation curve or changes of channel inactivation. The currents in oocytes expressing Kv 4.3 alone were smaller than those in Kv 4.3/KChIP2b expressing oocytes, but were still stimulated by SGK1. The effect of wild-type SGK1 was mimicked by constitutively active SGK1 (SGK1 S422D) but not by an inactive mutant (SGK1 K127N). The current amplitude increase mediated by SGK1 was not dependent on NEDD4.2 or RAB5, nor did it reflect increased cell surface expression. In conclusion, SGK1 stimulates Kv 4.3 potassium channels expressed in Xenopus oocytes by a novel mechanism distinct from the known NEDD4.2-dependent pathway.

Kv4 voltage-gated K(+) channels are responsible for transient K(+) currents in the central nervous system and in the heart. HpTx2 is a peptide toxin that selectively inhibits these currents; making it a useful probe for understanding Kv4 channel structure and drug binding. Therefore, we developed a method to produce large amounts of recombinant HpTx2. Recombinant toxin inhibits all three Kv4 isoforms to the same degree; however, the voltage-dependence of inhibition is less apparent for Kv4.1 than for Kv4.3. Similarly, recombinant HpTx2(GS) effects gating characteristics of both channels, but Kv4.1 to a much lesser degree. The toxin lacks affinity for Kv1.4, Kv2.1, and Kv3.4. To locate the binding site, the amino acids linking the third and forth membrane spanning segments of Kv4.3 were replaced with analogous amino acids of Kv1.4. The chimeric K(+) channel was completely insensitive to block by rHpTx2, suggesting that its binding site is near the channel's voltage sensor. These data show that rHpTx2(GS) is a gating modifier toxin that binds to a site remote from the pore.

Voltage-gated K+ (Kv) channel accessory (beta) subunits associate with pore-forming Kv alpha subunits and modify the properties and/or cell surface expression of Kv channels in heterologous expression systems. There is very little presently known, however, about the functional role(s) of Kv beta subunits in the generation of native cardiac Kv channels. Exploiting mice with a targeted disruption of the Kvbeta1 gene (Kvbeta1-/-), the studies here were undertaken to explore directly the role of Kvbeta1 in the generation of ventricular Kv currents. Action potential waveforms and peak Kv current densities are indistinguishable in myocytes isolated from the left ventricular apex (LVA) of Kvbeta1-/- and wild-type (WT) animals. Analysis of Kv current waveforms, however, revealed that mean+/-SEM I(to,f) density is significantly (P< or =0.01) lower in Kvbeta1-/- (21.0+/-0.9 pA/pF; n=68), than in WT (25.3+/-1.4 pA/pF; n=42), LVA myocytes, and that mean+/-SEM I(K,slow) density is significantly (P< or =0.01) higher in Kvbeta1-/- (19.1+/-0.9 pA/pF; n=68), compared with WT (15.9+/-0.7 pA/pF; n=42), LVA cells. Pharmacological studies demonstrated that the TEA-sensitive component of I(K,slow), I(K,slow2,) is selectively increased in Kvbeta1-/- LVA myocytes. In parallel with the alterations in I(to,f) and I(K,slow2) densities, Kv4.3 expression is decreased and Kv2.1 expression is increased in Kvbeta1-/- ventricles. Taken together, these results demonstrate that Kvbeta1 differentially regulates the functional cell surface expression of myocardial I(to,f) and I(K,slow2) channels.

This study characterized functional ion channels in cultured undifferentiated human mesenchymal stem cells (hMSCs) from bone marrow with whole-cell patch clamp and reverse transcription polymerase chain reaction (RT-PCR) techniques. Three types of outward currents were found in hMSCs, including a noise-like rapidly activating outward current inhibited by the large conductance Ca(2+)-activated K(+) channel (I(KCa)) blocker iberiotoxin, a transient outward K(+) current (I(to)) suppressed by 4-aminopyridine (4-AP), and a delayed rectifier K(+) current (IK(DR))-like ether-à-go-go (eag) K(+) channel. In addition, tetrodotoxin-sensitive sodium current (I(Na.TTX)) and nifedipine-sensitive L-type Ca(2+) current (I(Ca.L)) were also detected in 29% and 15% hMSCs, respectively. Moreover, RT-PCR revealed the molecular evidence of high levels of mRNA for the functional ionic currents, including human MaxiK for I(KCa), Kv4.2 and Kv1.4 for I(to), heag1 for IK(DR), hNE-Na for I(Na.TTX), and CACNAIC for I(Ca.L). These results demonstrate that multiple functional ion channel currents--that is, I(KCa), I(to), heag1, I(Na.TTX), and I(Ca.L)--are expressed in hMSCs from bone marrow.

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

The voltage-dependent K+ channel 4.3 (Kv4.3) is one of the major molecular correlates encoding a class of rapidly inactivating K+ currents, including the transient outward current in the heart (Ito) and A currents (IA) in neuronal and smooth muscle preparations. Recent studies have shown that Ito in human atrial myocytes and IA in murine colonic myocytes are modulated by Ca2+/calmodulin-dependent protein kinase II (CaMKII); however, the molecular target of CaMKII in these studies has not been elucidated. We performed experiments to investigate whether CaMKII could regulate Kv4.3 currents directly. Inclusion of the autothiophosphorylated form of CaMKII in the patch pipette (10 nM) prolonged Kv4.3 currents such that the time required to reach 50% inactivation from peak more than doubled, with positive shifts in voltage dependence of both activation and inactivation. In contrast, the rate of recovery from inactivation was accelerated under these conditions. CaMKII-inhibitory peptide or KN-93 produced effects opposite to that above; thus the rate of inactivation was increased, and recovery from inactivation decreased. A number of mutagenesis experiments were conducted on the three candidate CaMKII consensus sequence sites on the channel. Mutations at S550A, located at the COOH-terminal region of the channel, resulted in currents that inactivated more rapidly but recovered from inactivation at a slower rate than that of wild-type controls. In addition, these currents were unaffected by dialysis with either autothiophosphorylated CaMKII or the specific inhibitory peptide of CaMKII, suggesting that CaMKII slows the inactivation and accelerates the rate of recovery from inactivation of Kv4.3 currents by a direct effect at S550A, located at the COOH-terminal region of the channel.

Voltage-dependent potassium (Kv) channels in the CNS are involved in regulation of subthreshold membrane potentials, and thus reception and integration of synaptic signals. Although such features are particularly important for induction of hippocampal synaptic plasticity, relatively little is known about their subcellular localization. Here we analyzed the detailed distribution of Kv4.2 potassium channels in the mouse hippocampal region using confocal and electron microscopy. At the light microscopic level, the Kv4.2 immunoreactivity occurred in a punctate fashion in the whole area of the hippocampal region. In the hippocampus proper, most of the Kv4.2-positive puncta were small, and they were abundant at the dendritic compartments of pyramidal neurons. High-resolution confocal microscopy revealed that there was no apparent association between Kv4.2-positive puncta with major synaptic markers, such as vesicular glutamate transporters and glutamic acid decarboxylase. In the subicular complex and dentate gyrus, we encountered large distinct Kv4.2-positive puncta at the perimeter of somata and proximal dendrites of principal cells. These puncta were often in contact with glutamic acid decarboxylase-positive boutons, but showed no apparent association with vesicular glutamate transporters. The glutamic acid decarboxylase-positive boutons apposing to Kv4.2-positive puncta were parvalbumin-positive. Quantitative image analysis showed that approximately half of Kv4.2-positive puncta were closely apposed to glutamic acid decarboxylase-positive boutons in the parasubiculum and dentate gyrus. Electron microscopic examination substantiated the presence of large Kv4.2-positive patches at postsynaptic sites of symmetric synapses and small patches at extrasynaptic sites. No presynaptic terminals were labeled. The present findings indicate targeted clustering of Kv4.2 potassium channels at postsynaptic sites of GABAergic synapses and extrasynaptic sites, and provide some key to understand their role in the hippocampal region.

Background: Long QT syndrome (LQTS) is a hereditary cardiac arrhythmogenic disorder characterized by prolongation of the QT interval in the electrocardiogram, torsades de pointes arrhythmia, and syncopes and sudden death. LQTS is caused by mutations in ion channel genes. However, only in half of the families is it possible to identify mutations in one of the seven known LQTS genes, why further genetic heterogeneity is expected. The genes KCND2 and KCND3, encoding the alpha-subunits of the voltage-gated potassium channels Kv4.2 and Kv4.3 conducting the fast transient outward current (I(TO,f)) of the cardiac action potential (AP) in the myocardium, have been associated with prolongation of AP duration and QT prolongation in murine models., Methods: KCND2 and KCND3 were examined for mutations using single-strand conformation polymorphism (SSCP) analysis in 43 unrelated LQTS patients, where mutations in the coding regions of known LQTS genes had been excluded., Results: Seven single nucleotide polymorphismsm (SNPs) were found, two exonic SNPs in KCND2 and three exonic and two intronic in KCND3. None of the five exonic SNPs had coding effect. All seven SNPs are considered normal variants., Conclusion: The data suggest that mutations in KCND2 and KCND3 are not a frequent cause of long QT syndrome.

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

The transient outward current (I(to)), an important contributor to transmural electrophysiological heterogeneity, is significantly remodelled in congestive heart failure (CHF). The molecular bases of transmural I(to) gradients and CHF-dependent ionic remodelling are incompletely understood. To elucidate these issues, we studied mRNA and protein expression of Kv4.3 and KChIP2, the principal alpha and beta subunits believed to form I(to), in epicardial and endocardial tissues and in isolated cardiomyocytes from control dogs and dogs with CHF induced by 240 beats min(-1) ventricular tachypacing. CHF decreased I(to) density in both epicardium and endocardium (by 73 and 55% at +60 mV, respectively), without a significant change in relative current density (endocardium/epicardium 0.11 control, 0.17 CHF). There were transmural gradients in mRNA expression of both Kv4.3 (endocardium/epicardium ratio 0.3 under control conditions) and KChIP2 (endocardium/epicardium ratio 0.2 control), which remained in the presence of CHF (Kv4.3 endocardium/epicardium ratio 0.4; KChIP2 0.4). There were qualitatively similar protein expression gradients in human and canine cardiac tissues and isolated canine cardiomyocytes; however, the KChIP2 gradient was only detectable with a highly selective monoclonal antibody and closely approximated the I(to) density gradient. Kv4.3 mRNA expression was reduced by CHF, but KChIP2 mRNA was not significantly changed. CHF decreased Kv4.3 protein expression in canine cardiac tissues and cardiomyocytes, as well as in terminally failing human heart tissue samples, but KChIP2 protein was not down-regulated in any of the corresponding sample sets. We conclude that both Kv4.3 and KChIP2 may contribute to epicardial-endocardial gradients in I(to), and that I(to) down-regulation in human and canine CHF appears due primarily to changes in Kv4.3.

Background: Inhibition of cardiac K channels by local anesthetic may contribute to QTc interval prolongation of the electrocardiogram and induction of ventricular arrhythmia. The transient outward current Ito has been identified as a toxicologically relevant target of bupivacaine. S(-)-ropivacaine has been developed as a safer alternative to bupivacaine. The effects of S(-)-ropivacaine on Ito have not been investigated. In human ventricular myocardium, Ito is formed by Kv4.3 and KChIP2.2 subunits. Therefore, the aim of this study was to establish the effects of S(-)-ropivacaine on human Kv4.3/KChIP2.2 channels., Methods: Kv4.3/KChIP2.2 complementary DNA cloned from human heart was transiently transfected in Chinese hamster ovary cells. The pharmacologic effects of S(-)-ropivacaine were investigated with the patch clamp method., Results: Ropivacaine inhibited Kv4.3/KChIP2.2 channels in a concentration-dependent, stereospecific, and reversible manner. The IC50 value of S(-)-ropivacaine for inhibition of the charge conducted by Kv4.3/KChIP2.2 channel was 117 +/- 21 microm (n = 30). The local anesthetic accelerated macroscopic current decline with an IC50 value of 77 +/- 11 microm (n = 30). It shifted the midpoint of channel activation into the depolarizing direction, and it slowed recovery from inactivation without altering steady state inactivation. Kv4.3 channels are more sensitive to the inhibitory effect than Kv4.3/KChIP2.2 channels., Conclusions: : The results are consistent with the idea that ropivacaine, by blocking Kv4.3/KChIP2.2 from the open state, interferes with the gating modifying effects of KChIP2.2 on Kv4.3 channels. Inhibition of Kv4.3/KChIP2.2 channels by the local anesthetic may contribute to the deterioration of cardiac function during events of intoxication.

Objective: In the present study we examined the effects of diltiazem, an L-type Ca(2+) channel blocker widely used for the control of the ventricular rate in patients with supraventricular arrhythmias, on hKv1.5 and Kv4.3 channels that generate the cardiac ultrarapid delayed rectifier (I(Kur)) and the 4-aminopyridine sensitive transient outward (I(to)) K(+) currents, respectively., Methods: hKv1.5 and Kv4.3 channels were stably and transiently expressed in mouse fibroblast and Chinese hamster ovary cells, respectively. Currents were recorded using the whole-cell patch clamp., Results: Diltiazem (0.01 nM-500 muM) blocked hKv1.5 channels, in a frequency-dependent manner exhibiting a biphasic dose-response curve (IC(50)=4.8+/-1.5 nM and 42.3+/-3.6 muM). Diltiazem delayed the initial phase of the tail current decline and shifted the midpoint of the activation (Vh=-16.5+/-2.1 mV vs -20.4+/-2.6 mV, P<0.001) and inactivation (Vh=-22.4+/-0.7 mV vs. -28.2+/-1.9 mV, P<0.001) curves to more negative potentials. The analysis of the development of the diltiazem-induced block yielded apparent association (k) and dissociation (P) rate constants of (1.6+/-0.2) x 10(6) M(-1)s(-1) and 46.8+/-4.8 s(-1), respectively. Diltiazem (0.1 nM-100 muM) also blocked Kv4.3 channels in a frequency-dependent manner exhibiting a biphasic dose-response curve (IC(50)=62.6+/-11.1 nM and 109.9+/-12.8 muM). Diltiazem decreased the peak current and, at concentrations > or =0.1 microM, accelerated the inactivation time course. The apparent association and dissociation rate constants resulted (1.7+/-0.2) x 10(6) M(-1)s(-1) and 258.6+/-38.1 s(-1), respectively. Diltiazem, 10 nM, shifted to more negative potentials the voltage-dependence of Kv4.3 channel inactivation (Vh=-33.1+/-2.3 mV vs -38.2+/-3.5 mV, n=6, Plt;0.05) the blockade increasing at potentials at which the amount of inactivated channels increased., Conclusion: The results demonstrated for the first time that diltiazem, at therapeutic concentrations, decreased hKv1.5 and Kv4.3 currents by binding to the open and the inactivated state of the channels.

The N-terminal end of the subunits of the voltage-gated K+ channel Kv1.4 is essential for their rapid N-type inactivation, but removal of the entire Kv4.2 N-terminus slows inactivation only moderately. In this study, we investigated the effect of substituting the Kv4.2 N-terminal for that of Kv1.4 subunits. Despite the minor role of the Kv4.2 N-terminal in Kv4.2 inactivation and the limited degree of amino acid identity between Kv1.4 and Kv4.2 N-terminals, attachment of the Kv4.2 N-terminal to inactivation-deficient, N-terminal-deleted Kv1.4 subunits restored rapid inactivation. The Kv4.2 N-terminal/N-deleted Kv1.4 chimeric construct had inactivation kinetics like those of Kv4.2, inactivation voltage-dependence resembling Kv1.4 and recovery from inactivation substantially faster than wild-type Kv1.4. Acceleration of reactivation appeared to be due to the ability of chimeric channels to recover from inactivation without passing through the open state. Co-expression of wild-type Kv1.4 with the K+ channel interacting protein-2 (KChIP2) did not alter Kvl.4 properties, but co-expression of KChIP2 with Kv4.2 N-terminal/N-deleted Kv1.4 chimeric subunits significantly increased current expression and slowed inactivation without altering the rate of recovery from inactivation. We conclude that substitution of the Kv4.2 N-terminal for that of Kv1.4 transfers a variety of properties of Kv4.2, including inactivation time-dependence, accelerated recovery from inactivation and interaction with KChIP2, to Kv1.4, indicating the ability of Kvl.4 subunits to display these properties and the sufficiency of the Kv4.2 N-terminal to convey them.

The somatodendritic subthreshold A-type K+ current (ISA) in nerve cells is a critical component of the ensemble of voltage-gated ionic currents that determine somatodendritic signal integration. The underlying K+ channel belongs to the Shal subfamily of voltage-gated K+ channels. Most Shal channels across the animal kingdom share a high degree of structural conservation, operate in the subthreshold range of membrane potentials, and exhibit relatively fast inactivation and recovery from inactivation. Mammalian Shal K+ channels (Kv4) undergo preferential closed-state inactivation with features that are generally inconsistent with the classical mechanisms of inactivation typical of Shaker K+ channels. Here, we review (1) the physiological and genetic properties of ISA, 2 the molecular mechanisms of Kv4 inactivation and its remodeling by a family of soluble calcium-binding proteins (KChIPs) and a membrane-bound dipeptidase-like protein (DPPX), and (3) the modulation of Kv4 channels by protein phosphorylation.

Background: Prolongation of the action potential duration (APD) and decreased transient outward K+ current (I(to)) have been consistently observed in cardiac hypertrophy. The relation between electrical remodeling and cardiac hypertrophy in vivo is unknown., Methods and Results: We studied rat hearts subjected to pressure overload by surgical ascending aortic stenosis (AS) and simultaneously infected these hearts with an adenovirus carrying either the Kv4.3 gene (Ad.Kv4.3) or the beta-galactosidase gene (Ad.beta-gal). I(to) density was reduced and APD50 was prolonged (P<0.05) in AS rats compared with sham rats. Kv4.2 and Kv4.3 expressions were decreased by 58% and 51%, respectively (P<0.05). AS rats infected with Ad.beta-gal developed cardiac hypertrophy compared with sham rats, as assessed by cellular capacitance and heart weight-body weight ratio. Associated with the development of cardiac hypertrophy, the expression of calcineurin and its downstream transcription factor nuclear factor of activated T cells (NFAT) c1 was persistently increased by 47% and 36%, respectively (P<0.05) in AS myocytes infected with Ad.beta-gal compared with sham myocytes. In vivo gene transfer of Kv4.3 in AS rats was shown to increase Kv4.3 expression, increase I(to) density, and shorten APD50 by 1.6-fold, 5.3-fold, and 3.6-fold, respectively (P<0.05). Furthermore, AS rats infected with Ad.Kv4.3 showed significant reductions in calcineurin and NFAT expression. (P<0.05)., Conclusions: Downregulation of I(to), APD prolongation, and cardiac hypertrophy occur early after AS, and in vivo gene transfer of Kv4.3 can restore these electrical parameters and abrogate the hypertrophic response via the calcineurin pathway.

We report a novel signal transduction complex of the angiotensin receptor type 1. In this complex the angiotensin receptor type 1 associates with the potassium channel alpha-subunit Kv4.3 and regulates its intracellular distribution and gating properties. Co-localization of Kv4.3 with angiotensin receptor type 1 and fluorescent resonance energy transfer between those two proteins labeled with cyan and yellow-green variants of green fluorescent protein revealed that Kv4.3 and angiotensin receptor type I are located in close proximity to each other in the cell. The angiotensin receptor type 1 also co-immunoprecipitates with Kv4.3 from canine ventricle or when co-expressed with Kv4.3 and its beta-subunit KChIP2 in human embryonic kidney 293 cells. Treatment of the cells with angiotensin II results in the internalization of Kv4.3 in a complex with the angiotensin receptor type 1. When stimulated with angiotensin II, angiotensin receptors type 1 modulate gating properties of the remaining Kv4.3 channels on the cell surface by shifting their activation voltage threshold to more positive values. We hypothesize that the angiotensin receptor type 1 provides its internalization molecular scaffold to Kv4.3 and in this way regulates the cell surface representation of the ion channel.

Increased food intake is a major factor in the development of obesity, and the control of meal size is a valid approach to reduce food intake in humans. Meal termination, or satiety, is thought to be organized within the caudal brainstem where direct signals from the food handling alimentary canal and long-term signals from the forebrain converge in the solitary nucleus. Cholecystokinin (CCK) released from the gut after ingestion of food has been strongly implicated in nucleus tractus solitarius (NTS)-mediated satiation, but the exact cellular and intracellular signaling events are not understood. Using Western blotting and immunohistochemistry with phosphospecific antibodies, we demonstrate here that peripheral administration of CCK in rats leads to rapid activation of the extracellular signal-regulated kinase (ERK) signaling cascade in NTS neurons and that blockade of ERK signaling with microinfusion of a selective mitogen-activated ERK kinase inhibitor into the fourth ventricle attenuates the capacity of CCK to suppress food intake. In addition, we show that CCK-induced activation of ERK results in phosphorylation of the voltage-dependent potassium channel Kv4.2 and the nuclear transcription factor CREB (cAMP response element-binding protein). The results demonstrate that ERK signaling is necessary for exogenous CCK to suppress food intake in deprived rats and suggest that this pathway may also be involved in natural satiation and the period of satiety between meals through coupling of ERK activation to both cytosolic and nuclear effector mechanisms that have the potential to confer acute and long-term changes in neuronal functioning.

It has recently been reported that dipeptidyl aminopeptidase X (DPPX) interacts with the voltage-gated potassium channel Kv4 and that co-expression of DPPX together with Kv4 pore forming alpha-subunits, and potassium channel interacting proteins (KChIPs), reconstitutes properties of native A-type potassium channels in vitro. Here we report the X-ray crystal structure of the extracellular domain of human DPPX determined at 3.0A resolution. This structure reveals the potential for a surface electrostatic change based on the protonation state of histidine. Subtle changes in extracellular pH might modulate the interaction of DPPX with Kv4.2 and possibly with other proteins. We propose models of DPPX interaction with the voltage-gated potassium channel complex. The dimeric structure of DPPX is highly homologous to the related protein DPP-IV. Comparison of the active sites of DPPX and DPP-IV reveals loss of the catalytic serine residue but the presence of an additional serine near the "active" site. However, the arrangement of residues is inconsistent with that of canonical serine proteases and DPPX is unlikely to function as a protease (dipeptidyl aminopeptidase).

Focal activation of glutamate receptors in distal dendrites of hippocampal pyramidal cells triggers voltage-dependent Ca(2+) channel-mediated plateau potentials that are confined to the stimulated dendrite. We examined the role of dendritic K(+) conductances in determining the amplitude, duration, and spatial compartmentalization of plateau potentials. Manipulations that blocked SK-type Ca(2+)-activated K(+) channels, including apamin and BAPTA dialysis, increased the duration of plateau potentials without affecting their amplitude or compartmentalization. Manipulations that blocked Kv4.2 A-type K(+) channels, including a dominant-negative Kv4.2 construct and 4-aminopyridine, increased the amplitude of plateau potentials by allowing them to recruit neighboring dendrites. Prolongation of plateau potentials or block of Kv4.2 channels at branch points facilitated the ability of dendritic excitation to trigger fast action potentials. SK channels thus underlie repolarization of dendritic plateau potentials, whereas Kv4.2 channels confine these potentials to single dendritic branches, and both act in concert to regulate synaptic integration.

The dipeptidyl aminopeptidase-like protein DPPX (DPP6) associates with Kv4 potassium channels, increasing surface trafficking and reconstituting native neuronal ISA-like properties. Dipeptidyl peptidase 10 (DPP10) shares with DPP6 a high amino acid identity, lack of enzymatic activity, and expression predominantly in the brain. We used a two-electrode voltage-clamp and oocyte expression system to determine if DPP10 also interacts with Kv4 channels and modulates their expression and function. Kv4.2 coimmunoprecipitated with HA/DPP10 from extracts of oocytes heterologously expressing both proteins. Coexpression with DPP10 and HA/DPP10 enhanced Kv4.2 current by approximately fivefold without increasing protein level. DPP10 also remodeled Kv4.2 kinetic and steady-state properties by accelerating time courses of inactivation and recovery (taurec: WT = 200 ms, +DPP10 = 78 ms). Furthermore, DPP10 introduced hyperpolarizing shifts in the conductance-voltage relationship (approximately 19 mV) as well as steady-state inactivation (approximately 7 mV). The effects of DPP10 on Kv4.1 were similar to Kv4.2; however, distinct biophysical differences were observed. Additional experiments suggested that the cytoplasmic N-terminal domain of DPP10 determines the acceleration of inactivation. In summary, DPP10 is a potent modulator of Kv4 expression and biophysical properties and may be a critical component of somatodendritic ISA channels in the brain., (Copyright 2004 Biophysical Society)

Background: Automatic implantable cardioverter-defibrillator therapy is considered the only effective treatment for high-risk patients with Brugada syndrome. Quinidine depresses I(to) current, which may play an important role in the arrhythmogenesis of this disease., Methods and Results: The effects of quinidine bisulfate (mean dose, 1483+/-240 mg) on the prevention of inducible and spontaneous ventricular fibrillation (VF) were prospectively evaluated in 25 patients (24 men, 1 woman; age, 19 to 80 years) with Brugada syndrome. There were 15 symptomatic patients (including 7 cardiac arrest survivors and 7 patients with unexplained syncope) and 10 asymptomatic patients. All 25 patients had inducible VF at baseline electrophysiological study. Quinidine prevented VF induction in 22 of the 25 patients (88%). After a follow-up period of 6 months to 22.2 years, all patients are alive. Nineteen patients were treated with quinidine for 6 to 219 months (mean+/-SD, 56+/-67 months). None had an arrhythmic event, although 2 had non-arrhythmia-related syncope. Administration of quinidine was associated with a 36% incidence of side effects that resolved after drug discontinuation., Conclusions: Quinidine effectively prevents VF induction in patients with Brugada syndrome. Our data suggest that quinidine also suppresses spontaneous arrhythmias and could prove to be a safe alternative to automatic implantable cardioverter-defibrillator therapy for a substantial proportion of patients with Brugada syndrome. Randomized studies comparing these two therapies seem warranted.

The K(+) channel interacting proteins (KChIPs1-4) interact with and modulate activity and trafficking of Kv4 potassium channels. We report here the distribution of KChIPs in adult mouse brain. KChIP1 was expressed in a subpopulation of neurons widely distributed in brain and enriched in Purkinje cells of the cerebellum and in the reticular thalamic and medial habenular nuclei. KChIP2 transcripts were highly expressed in layer IV of the cerebral cortex and in striatum and hippocampus, but expressed at low levels in cerebellum. KChIP3 transcripts were detected primarily in the layer V and deep layer VI of the cerebral cortex, the hippocampus, and the entire cerebellum. KChIP4 was highly expressed by neurons in layers II-IV of cortex and in hippocampus, thalamus and the Purkinje cell layer of the cerebellum. Collectively, different KChIPs appear to be expressed by selected neuronal populations in different brain regions where expression of Kv4.2 and Kv4.3 have been reported. These findings support the likelihood of functional interactions between KChIPs and Kv4 K(+) channels in brain.

The transient outward K+ current (Ito) modulates transmembrane Ca2+ influx into cardiomyocytes, which, in turn, might act on Ito. Here, we investigated whether Ca2+ modifies functional expression of Ito. Whole-cell Ito were recorded using the patch clamp technique in single right ventricular myocytes isolated from adult rats and incubated for 24 h at 37 degrees C in a serum-free medium containing various Ca2+ concentrations ([Ca2+]o). Increasing the [Ca2+]o from 0.5 to 1.0 and 2.5 mM produced a gradual decrease in Ito density without change in current kinetics. Quantitativereverse transcriptase-PCR showed that a decrease of the Kv4.2 mRNA could account for this decrease. In the acetoxymethyl ester form of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA-AM)-loaded myocytes (a permeant Ca2+ chelator), Ito density increased significantly when cells were exposed for 24 h to either 1 or 2.5 mM [Ca2+]o. Moreover, 24-h exposure to the Ca2+ channel agonist, Bay K8644, in 1 mM [Ca2+]o induced a decrease in Ito density, whereas the Ca2+ channel antagonist, nifedipine, blunted Ito decrease in 2.5 mM [Ca2+]o. The decrease of Ito in 2.5 mM [Ca2+]o was also prevented by co-incubation with either the calmodulin inhibitor W7 or the calcineurin inhibitors FK506 or cyclosporin A. Furthermore, in myocytes incubated for 24 h with 2.5 mM [Ca2+]o, calcineurin activity was significantly increased compared with 1 mM [Ca2+]o. Our data suggest that modulation of [Ca2+]i via L-type Ca2+ channels, which appears to involve the Ca2+/calmodulin-regulated protein phosphatase calcineurin, down-regulates the functional expression of Ito. This effect might be involved in many physiological and pathological modulations of Ito channel expression in cardiac cells, as well other cell types., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)

Voltage-gated potassium (Kv) channels from the Kv4, or Shal-related, gene family underlie a major component of the A-type potassium current in mammalian central neurons. We recently identified a family of calcium-binding proteins, termed KChIPs (Kv channel interacting proteins), that bind to the cytoplasmic N termini of Kv4 family alpha subunits and modulate their surface density, inactivation kinetics, and rate of recovery from inactivation (An et al., 2000). Here, we used single and double-label immunohistochemistry, together with circumscribed lesions and coimmunoprecipitation analyses, to examine the regional and subcellular distribution of KChIPs1-4 and Kv4 family alpha subunits in adult rat brain. Immunohistochemical staining using KChIP-specific monoclonal antibodies revealed that the KChIP polypeptides are concentrated in neuronal somata and dendrites where their cellular and subcellular distribution overlaps, in an isoform-specific manner, with that of Kv4.2 and Kv4.3. For example, immunoreactivity for KChIP1 and Kv4.3 is concentrated in the somata and dendrites of hippocampal, striatal, and neocortical interneurons. Immunoreactivity for KChIP2, KChIP4, and Kv4.2 is concentrated in the apical and basal dendrites of hippocampal and neocortical pyramidal cells. Double-label immunofluorescence labeling revealed that throughout the forebrain, KChIP2 and KChIP4 are frequently colocalized with Kv4.2, whereas in cortical, hippocampal, and striatal interneurons, KChIP1 is frequently colocalized with Kv4.3. Coimmunoprecipitation analyses confirmed that all KChIPs coassociate with Kv4 alpha subunits in brain membranes, indicating that KChIPs 1-4 are integral components of native A-type Kv channel complexes and are likely to play a major role as modulators of somatodendritic excitability.

Previous studies in pigs and goats have demonstrated that AVE0118 prolongs atrial refractoriness without any effect on the QT-interval. The purpose of the present study was to investigate the effect of the compound on various cardiac ion channels. AVE0118 blocked the pig Kv1.5 and the human Kv1.5 expressed in Xenopus oocytes with IC(50) values of 5.4+/-0.7 microM and 6.2+/-0.4 microM respectively. In Chinese hamster ovary (CHO) cells, AVE0118 decreased the steady-state hKv1.5 current with an IC(50) of 1.1+/-0.2 microM. The hKv4.3/KChIP2.2 current in CHO cells was blocked by AVE0118 by accelerating the apparent time-constant of inactivation ( tau(inact)), and the integral current was inhibited with an IC(50) of 3.4+/-0.5 microM. At 10 microM AVE0118 tau(inact) decreased from 9.3+/-0.6 ms ( n=8, control) to 3.0+/-0.3 ms ( n=8). The K(ACh) current was investigated in isolated pig atrial myocytes by application of 10 microM carbachol. At a clamp potential of -100 mV the I(KACh) was half-maximally blocked by 4.5+/-1.6 microM AVE0118. In the absence of carbachol, AVE0118 had no effect on the inward current recorded at -100 mV. Effects on the I(Kr) current were investigated on HERG channels expressed in CHO cells. AVE0118 blocked this current half-maximally at approximately 10 microM. Comparable results were obtained in isolated guinea pig ventricular myocytes, where half-maximal inhibition of the I(Kr) tail current occurred at a similar concentration of AVE0118. Other ionic currents, like the I(Ks), I(KATP) (recorded in guinea pig ventricular myocytes), and L-type Ca(2+) (recorded in pig atrial myocytes) were blocked by 10 microM AVE0118 by 10+/-3% ( n=6), 28+/-7% ( n=4), and 22+/-13% ( n=5) respectively. In summary, AVE0118 preferentially inhibits the atrial K(+) channels I(Kur), I(to) and I(KACH). This profile may explain the selective prolongation of atrial refractoriness described previously in pigs and goats.

To prove heteromeric assembly of KChIP proteins, the present study is carried out. The results of chemical crosslinking and pull down assay revealed that KChIP1, KChIP2.1, and KChIP2.2 could form homo- as well as hetero-oligomer, and this oligomerization exhibited a Ca(2+)-dependent manner. Moreover, homomeric and heteromeric assembly of KChIPs did not perturb their interaction with Kv4.2 K(+) channel, indicating that the region associated with oligomerization of KChIPs was distinct from that for binding with Kv4.2. Together with previous findings that the net effects of KChIP proteins on the molecular properties and trafficking of Kv channel were different, these observations open a fascinating possibility that the electrophysiological properties of Kv channel may be differently regulated by homomeric and heteromeric assembly of KChIPs.

Background: Cardiac hypertrophy underlies arrhythmias and sudden death, for which mineralocorticoid receptor (MR) activity has recently been implicated. We sought to establish the sequence of ionic events that link the initiating insult and MR to hypertrophy development., Methods and Results: Using whole-cell, patch-clamp and quantitative reverse transcription-polymerase chain reaction techniques on right ventricular myocytes of a myocardial infarction (MI) rat model, we examined the cellular response over time. One week after MI, no sign of cellular hypertrophy was found, but action potential duration (APD) was lengthened. Both an increase in Ca2+ current (I(Ca)) and a decrease in K+ transient outward current (I(to)) underlay this effect. Consistently, the relative expression of mRNA coding for the Ca2+ channel alpha1C subunit (Ca(v)1.2) increased, and that of the K+ channel K(v)4.2 subunit decreased. Three weeks after MI, AP prolongation endured, whereas cellular hypertrophy developed. I(Ca) density, Ca(v)1.2, and K(v)4.2 mRNA levels regained control levels, but I(to) density remained reduced. Long-term treatment with RU28318, an MR antagonist, prevented this electrical remodeling. In a different etiologic model of abdominal aortic constriction, we confirmed that APD prolongation and modifications of ionic currents precede cellular hypertrophy., Conclusions: Electrical remodeling, which is triggered at least in part by MR activation, is an initial, early cellular response to hypertrophic insults.

Background: Calcium-insensitive transient outward current (I(to)) is important to the development of cardiac memory (CM), which itself reflects the capacity of the heart to remodel electrophysiologically. We used cardiac pacing to test the hypothesis that CM evolution can be explained by developmental maturation of I(to)., Methods and Results: Acutely anesthetized dogs from 1 day old to adult were paced from the left ventricle (VP, n=29) or left atrial appendage (AP, n=12) to induce CM. T-wave vector displacement (TVD) obtained during VP was greater than with AP (adults, 0.39+/-0.06 mV; neonates, 0.04+/-0.01 mV; P<0.05). TVD began to increase at approximately 40 days of age, reaching adult levels by approximately 200 days. Microelectrode studies performed in 18 dogs (ages 3 to 94 days) after completing the CM protocol and 20 additional dogs (1 day old to adult) revealed that the epicardial action potential notch was absent in neonates, became apparent in the young, and was deepest in adults. The relationship between TVD and epicardial notch was such that as notch magnitude increased, TVD increased (r=-0.65, P<0.05). KChIP2 and Kv4.3 mRNA (measured via reverse transcription-polymerase chain reaction) also increased with age., Conclusions: The inducibility of CM gradually increases with age in association with evolution of the epicardial action potential notch and mRNA expression for KChIP2 and Kv4.3. This suggests that the capacity of the heart to remodel electrophysiologically and to manifest memory during development depends in part on evolution of the determinants of I(to).

Inherited channelopathies are at the origin of many neurological disorders. Here we report a form of channelopathy that is acquired in experimental temporal lobe epilepsy (TLE), the most common form of epilepsy in adults. The excitability of CA1 pyramidal neuron dendrites was increased in TLE because of decreased availability of A-type potassium ion channels due to transcriptional (loss of channels) and posttranslational (increased channel phosphorylation by extracellular signal-regulated kinase) mechanisms. Kinase inhibition partly reversed dendritic excitability to control levels. Such acquired channelopathy is likely to amplify neuronal activity and may contribute to the initiation and/or propagation of seizures in TLE.

Direct protein-protein interaction between Taiwan cobra cardiotoxin3 (CTX3) and potassium channel-interacting proteins (KChIPs) was investigated in the present study. It was found that KChIPs bound with CTX3, in which KChIP and CTX3 formed a 1:1 complex as evidenced by the results of chemical cross-linking. Pull-down assay revealed that the intact EF-hands 3 and 4 of KChIP1 were critical for CTX3-binding. Likewise, removal of EF-hands 3 and 4 distorted the ability of KChIP1 to bind with Kv4.2 N-terminal fragment (KvN) as well as fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS). In contrast to the interaction between KChIP1 and KvN, the binding of CTX3 to KChIP1 showed a Ca(2+)-independent manner. Fluorescence measurement revealed that CTX3 affected the binding of ANS to Ca(2+)-bound KChIP1, but not Ca(2+)-free KChIP1. Alternatively, KChIP1 simultaneously bound with KvN and CTX3, and the interaction between KChIP1 and KvN was enhanced by CTX3. In terms of the fact that KChIPs regulate the electrophysiological properties of Kv K(+) channel, the potentiality of CTX for this biomedical application could be considered.

Potassium channel dysfunction has been implicated in Alzheimer's disease. In the present study, the expression of voltage-gated potassium channel (Kv) subunits in rat brain was measured after a single intracerebroventricular injection of beta-amyloid peptide 25-35 (Abeta(25-35)). After injection of Abeta, the spatial memory of rats was significantly impaired in the Morris water maze. Expression of five main Kv channel subunits (Kv1.5, Kv2.1, Kv1.4, Kv4.2, and Kv4.3) in mRNA level was assessed by using reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels of Kv2.1 and Kv1.4 were increased by 72% and 67%, respectively, in hippocampus, and Kv4.2 mRNA was increased by 58% in cortex. No other significant mRNA expression changes were found in Abeta-treated rats. The protein expression of Kv2.1, Kv1.4, and Kv4.2 was detected by using Western blotting. Kv2.1 and Kv1.4 protein levels were increased by 48% and 50%, respectively, in hippocampus of Abeta-treated rats, and Kv4.2 protein was increased by 42% in cerebral cortex. This study indicates that the expression up-regulation of Kv1.4, Kv2.1, and Kv4.2 in Abeta-induced cognitive impairment might play an important role in the pathogenesis of Alzheimer's disease., (Copyright 2004 Wiley-Liss, Inc.)

Shal-type (Kv4.x) K(+) channels are expressed in a variety of tissue, with particularly high levels in the brain and heart. These channels are the primary subunits that contribute to transient, voltage-dependent K(+) currents in the nervous system (A currents) and the heart (transient outward current). Recent studies have revealed an enormous degree of complexity in the regulation of these channels. In this review, we describe the surprisingly large number of ancillary subunits and scaffolding proteins that can interact with the primary subunits, resulting in alterations in channel trafficking and kinetic properties. Furthermore, we discuss posttranslational modification of Kv4.x channel function with an emphasis on the role of kinase modulation of these channels in regulating membrane properties. This concept is especially intriguing as Kv4.2 channels may integrate a variety of intracellular signaling cascades into a coordinated output that dynamically modulates membrane excitability. Finally, the pathophysiology that may arise from dysregulation of these channels is also reviewed.