MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between -60 and -95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a beta subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.     

Targeted Deletion of Kcne2 Impairs HCN Channel Function in Mouse Thalamocortical Circuits

Background

Hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels generate the pacemaking current, I_h, which regulates neuronal excitability, burst firing activity, rhythmogenesis, and synaptic integration. The physiological consequence of HCN activation depends on regulation of channel gating by endogenous modulators and stabilization of the channel complex formed by principal and ancillary subunits. KCNE2 is a voltage-gated potassium channel ancillary subunit that also regulates heterologously expressed HCN channels; whether KCNE2 regulates neuronal HCN channel function is unknown.

Methodology/Principal Findings

We investigated the effects of Kcne2 gene deletion on I_h properties and excitability in ventrobasal (VB) and cortical layer 6 pyramidal neurons using brain slices prepared from Kcne2 ^+/+ and Kcne2 ^−/− mice. Kcne2 deletion shifted the voltage-dependence of I_h activation to more hyperpolarized potentials, slowed gating kinetics, and decreased I_h density. Kcne2 deletion was associated with a reduction in whole-brain expression of both HCN1 and HCN2 (but not HCN4), although co-immunoprecipitation from whole-brain lysates failed to detect interaction of KCNE2 with HCN1 or 2. Kcne2 deletion also increased input resistance and temporal summation of subthreshold voltage responses; this increased intrinsic excitability enhanced burst firing in response to 4-aminopyridine. Burst duration increased in corticothalamic, but not thalamocortical, neurons, suggesting enhanced cortical excitatory input to the thalamus; such augmented excitability did not result from changes in glutamate release machinery since miniature EPSC frequency was unaltered in Kcne2 ^−/− neurons.

Conclusions/Significance

Loss of KCNE2 leads to downregulation of HCN channel function associated with increased excitability in neurons in the cortico-thalamo-cortical loop. Such findings further our understanding of the normal physiology of brain circuitry critically involved in cognition and have implications for our understanding of various disorders of consciousness.     

Modulation of functional properties of KCNQ1 channel by association of KCNE1 and KCNE2

The KCNE proteins (KCNE1 through KCNE5) function as beta-subunits of several voltage-gated K(+) channels. Assembly of KCNQ1 K(+) channel alpha-subunits and KCNE1 underlies cardiac I(Ks), while KCNQ1 interacts with all other members of KCNE forming complexes with different properties. Here we investigated synergic actions of KCNE1 and KCNE2 on functional properties of KCNQ1 heterologously expressed in COS7 cells. Patch-clamp recordings from cells expressing KCNQ1 and KCNE1 exhibited the slowly activating current, while co-expression of KCNQ1 with KCNE2 produced a practically time-independent current. When KCNQ1 was co-expressed with both of KCNE1 and KCNE2, the membrane current exhibited a voltage- and time-dependent current whose characteristics differed substantially from those of the KCNQ1/KCNE1 current. The KCNQ1/KCNE1/KCNE2 current had a more depolarized activation voltage, a faster deactivation kinetics, and a less sensitivity to activation by mefenamic acid. These results suggest that KCNE2 can functionally couple to KCNQ1 even in the presence of KCNE1.     

KCNE2 protein is more abundant in ventricles than in atria and can accelerate hERG protein degradation in a phosphorylation-dependent manner

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.     

拉莫三嗪对GHB 致失神发作大鼠脑内HCN1、HCN2表达的影响

目的:观察拉莫三嗪对γ-羟丁酸(GHB)致失神发作大鼠脑电及脑内超极化激活环核苷酸门控阳离子通道(HCN)的亚型HCN1、HCN2表达变化的影响,探讨拉莫三嗪抗失神癫痫的可能作用机制。方法:健康成年雄性SD大鼠,随机分为空白对照组,模型组,拉莫三嗪治疗组(低剂量组为8 mg/(kg·d)、中剂量组为12 mg/(kg·d)、高剂量组为24 mg/(kg·d)),每组7只。空白对照组及模型组每日应用0.25%的甲基纤维素钠溶液灌胃,治疗组每日应用0.25%的甲基纤维素钠溶液配制的浓度为2 mg/mL的拉莫三嗪混悬液灌胃。手术埋置皮层脑电电极。腹腔注射GHB的前体γ-丁内酯(GBL)200 mg/kg制作大鼠失神发作模型,并监测脑电。免疫组化法检测皮层HCN1及丘脑HCN2的表达。结果:皮层脑电图拉莫三嗪治疗组比模型组失神发作的潜伏期延长,最高波幅降低(P0.05)。模型组皮质HCN1比空白对照组表达减少,而拉莫三嗪高、中剂量组皮质HCN1比模型组表达增加(P0.05)。模型组丘脑HCN2表达减少,与空白对照组及治疗组相比,差异有统计学意义。结论:拉莫三嗪可以改善GHB致失神发作模型脑电图的异常表现;拉莫三嗪抗失神癫痫作用可能与调节HCN表达有关。     

Biophysical properties of mutant KCNQ1 S277L channels linked to hereditary long QT syndrome with phenotypic variability

Hereditary long QT syndrome (LQTS) is associated with ventricular torsade de pointes tachyarrhythmias and sudden cardiac death. Mutations in a cardiac voltage-gated potassium channel, KCNQ1, induce the most frequent variant of LQTS. We identified a KCNQ1 missense mutation, KCNQ1 S277L, in a patient presenting with recurrent syncope triggered by emotional stress (QTc = 528 ms). This mutation is located in the conserved S5 transmembrane region of the KCNQ1 channel. Using in vitro electrophysiological testing in the Xenopus oocyte expression system, the S277L mutation was found to be non-functional and to suppress wild type currents in dominant-negative fashion in the presence and in the absence of the regulatory ß-subunit, KCNE1. In addition, expression of S277L and wild type KCNQ1 with KCNE1 resulted in a shift of the voltage-dependence of activation by − 8.7 mV compared to wild type I_Ks, indicating co-assembly of mutant and wild type subunits. The electrophysiological phenotype corresponds well with the severe clinical phenotype of the index patient. However, investigation of family members revealed three patients that exhibit asymptomatic QT interval prolongation (QTc = 493-518 ms). In conclusion, this study emphasizes the value of biophysical testing to provide mechanistic evidence for pathogenicity of ion channel mutations identified in LQTS patients. The inconsistent association of the KCNQ1 S277L mutation with the clinical presentation suggests that additional genetic, epigenetic, or environmental factors play a role in defining the individual clinical LQTS phenotype.     

HCN2在大鼠外周神经病理性疼痛发生中的作用

目的：初步探讨超极化激活的环核苷酸门控通道2型（HCN2）在外周神经病理性疼痛发生中的作用。方法：将24只健康成年大鼠进行随机分组（n=12）：假手术组（Sham）大鼠仅分离左侧L4、L5脊神经,模型组（SNL）分离脊神经后进行相应的结扎处理,手术7 d后用行为学方法进行模型评价;将造模成功的大鼠进行随机分组（n=6）：①阴性对照组（Saline）,左侧足底注射生理盐水;②阳性对照组（GBPT）,腹腔注射加巴喷丁;③实验组（ZD7288）,左侧足底注射HCN非特异性阻断剂ZD7288。在给药前以及给药后1 h、4 h、24 h、48 h用疼痛行为学实验检测其对神经病理性疼痛的作用;分别取手术前对照组（Control）、假手术组（Sham）和模型组（SNL）大鼠的背根神经节（DRG）（n=6）,利用qPCR和Western blot的方法研究造模前后大鼠DRG内HCN2的表达的变化情况。结果：①成功建立大鼠神经痛模型;②与Saline组比较,GBPT组和ZD7288组在注射1 h后,均能明显的减轻大鼠神经病理性疼痛的症状（P<0.01）,而GBPT组和ZD7288组之间比较则无差异;③与Control组和Sham组相比较,SNL组大鼠DRG内的HCN2 mRNA表达量明显增加（P<0.01）;与Control组和Sham组相比较,SNL组大鼠DRG内的HCN2通道蛋白表达量显著增加（P<0.05）。结论：HCN2参与外周神经病理性疼痛的发生,并有可能成为治疗神经病理性疼痛一个潜在的新靶点。     

HCN1 and HCN2 in Rat DRG Neurons: Levels in Nociceptors and Non-Nociceptors,NT3-Dependence and Influence of CFA-Induced Skin Inflammation on HCN2 and NT3 Expression

I_h, which influences neuronal excitability, has recently been measured in vivo in sensory neuron subtypes in dorsal root ganglia (DRGs). However, expression levels of HCN (hyperpolarization-activated cyclic nucleotide-gated) channel proteins that underlie I_h were unknown. We therefore examined immunostaining of the most abundant isoforms in DRGs, HCN1 and HCN2 in these neuron subtypes. This immunostaining was cytoplasmic and membrane-associated (ring). Ring-staining for both isoforms was in neurofilament-rich A-fiber neurons, but not in small neurofilament-poor C-fiber neurons, although some C-neurons showed cytoplasmic HCN2 staining. We recorded intracellularly from DRG neurons in vivo, determined their sensory properties (nociceptive or low-threshold-mechanoreceptive, LTM) and conduction velocities (CVs). We then injected fluorescent dye enabling subsequent immunostaining. For each dye-injected neuron, ring- and cytoplasmic-immunointensities were determined relative to maximum ring-immunointensity. Both HCN1- and HCN2-ring-immunointensities were positively correlated with CV in both nociceptors and LTMs; they were high in Aβ-nociceptors and Aα/β-LTMs. High HCN1 and HCN2 levels in Aα/β-neurons may, via I_h, influence normal non-painful (e.g. touch and proprioceptive) sensations as well as nociception and pain. HCN2-, not HCN1-, ring-intensities were higher in muscle spindle afferents (MSAs) than in all other neurons. The previously reported very high I_h in MSAs may relate to their very high HCN2. In normal C-nociceptors, low HCN1 and HCN2 were consistent with their low/undetectable I_h. In some C-LTMs HCN2-intensities were higher than in C-nociceptors. Together, HCN1 and HCN2 expressions reflect previously reported I_h magnitudes and properties in neuronal subgroups, suggesting these isoforms underlie I_h in DRG neurons. Expression of both isoforms was NT3-dependent in cultured DRG neurons. HCN2-immunostaining in small neurons increased 1 day after cutaneous inflammation (CFA-induced) and recovered by 4 days. This could contribute to acute inflammatory pain. HCN2-immunostaining in large neurons decreased 4 days after CFA, when NT3 was decreased in the DRG. Thus HCN2-expression control differs between large and small neurons.     

KCNE2 and the K+ channel: The tail wagging the dog

KCNE2, originally designated MinK-related peptide 1 (MiRP1), belongs to a five-strong family of potassium channel ancillary (β) subunits that, despite the diminutive size of the family and its members, has loomed large in the field of ion channel physiology. KCNE2 dictates K⁺ channel gating, conductance, α subunit composition, trafficking and pharmacology, and also modifies functional properties of monovalent cation-nonselective HCN channels. The Kcne2^−/− mouse exhibits cardiac arrhythmia and hypertrophy, achlorhydria, gastric neoplasia, hypothyroidism, alopecia, stunted growth and choroid plexus epithelial dysfunction, illustrating the breadth and depth of the influence of KCNE2, mutations which are also associated with human cardiac arrhythmias. Here, the modus operandi and physiological roles of this potent regulator of membrane excitability and ion secretion are reviewed with particular emphasis on the ability of KCNE2 to shape the electrophysiological landscape of both excitable and non-excitable cells.     

Upregulation of KCNQ1/KCNE1 K+ channels by Klotho

Klotho is a transmembrane protein expressed primarily in kidney, parathyroid gland, and choroid plexus. The extracellular domain could be cleaved off and released into the systemic circulation. Klotho is in part effective as β-glucuronidase regulating protein stability in the cell membrane. Klotho is a major determinant of aging and life span. Overexpression of Klotho increases and Klotho deficiency decreases life span. Klotho deficiency may further result in hearing loss and cardiac arrhythmia. The present study explored whether Klotho modifies activity and protein abundance of KCNQ1/KCNE1, a K⁺ channel required for proper hearing and cardiac repolarization. To this end, cRNA encoding KCNQ1/KCNE1 was injected in Xenopus oocytes with or without additional injection of cRNA encoding Klotho. KCNQ1/KCNE1 expressing oocytes were treated with human recombinant Klotho protein (30 ng/ml) for 24 h. Moreover, oocytes which express both KCNQ1/KCNE1 and Klotho were treated with 10 µM DSAL (D-saccharic acid-1,4-lactone), a β-glucuronidase inhibitor. The KCNQ1/KCNE1 depolarization-induced current (I_Ks) was determined utilizing dual electrode voltage clamp, while KCNQ1/KCNE1 protein abundance in the cell membrane was visualized utilizing specific antibody binding and quantified by chemiluminescence. KCNQ1/KCNE1 channel activity and KCNQ1/KCNE1 protein abundance were upregulated by coexpression of Klotho. The effect was mimicked by treatment with human recombinant Klotho protein (30 ng/ml) and inhibited by DSAL (10 µM). In conclusion, Klotho upregulates KCNQ1/KCNE1 channel activity by 'mainly' enhancing channel protein abundance in the plasma cell membrane, an effect at least partially mediated through the β-glucuronidase activity of Klotho protein.     

The protective effect of peony extract on acute myocardial infarction in rats

To investigate the protective effects, and the mechanisms involved, of an extract of the medicinal herb radix paeoniae rubra (PE) on cardiovascular disease, acute myocardial infarction (AMI) was induced by ligation of the left coronary artery in Sprague Dawley rats. Animals were randomly divided into six groups: control, sham-operated, AMI, AMI + PE low dose, AMI + PE high dose, and AMI + positive control. Myocardial enzymes, cytokines, oxidative stress, blood coagulation times, a marker for early stage apoptosis, caspase-3 activity, and expression levels of bax, bcl-2 and fas in isolated primary cardiomyocytes were examined. In contrast with control and sham groups, significant increases in the following parameters were measured in the blood of AMI group animals: activities of cardiac enzymes including glutamic oxaloacetic transaminase, creatine kinase, creatine kinase-MB, lactate dehydrogenase, α-hydroxybutyric dehydrogenase, and levels of IL-10, TNFα, and lipid peroxidation. Under the same conditions, superoxide dismutase activity, thrombin time and activated partial thromboplastin time decreased significantly. PE showed a dose-dependent protection against AMI-induced alterations in cardiac enzymes, cytokines, oxidative stress, and coagulation. In AMI cardiomyocytes, compared with control and sham groups, the left ventricular end-diastolic pressure, early stage apoptosis, caspase-3 activity and expression levels of bax, bcl-2 and fas significantly increased, while the ratio bcl-2/bax decreased. PE showed dose-dependent protection. These results suggest that PE is an effective agent for protecting against AMI; possible mechanisms may include the regulation of cardiac enzymes, cytokines, oxidative stress, coagulation and apoptosis.     

Voltage-dependent opening of HCN channels: Facilitation or inhibition by the phytoestrogen, genistein, is determined by the activation status of the cyclic nucleotide gating ring

Investigation of the mechanistic bases and physiological importance of cAMP regulation of HCN channels has exploited an arginine to glutamate mutation in the nucleotide-binding fold, an approach critically dependent on the mutation selectively lowering the channel's nucleotide affinity. In apparent conflict with this, in intact Xenopus oocytes, HCN and HCN-RE channels exhibit qualitatively and quantitatively distinct responses to the tyrosine kinase inhibitor, genistein — the estrogenic isoflavonoid strongly depolarizes the activation mid-point of HCN1-R538E, but not HCN1 channels (+ 9.8 mV ± 0.9 versus + 2.2 mV ± 0.6) and hyperpolarizes gating of HCN2 (− 4.8 mV ± 1.0) but depolarizes gating of HCN2-R591E (+ 13.2 mV ± 2.1). However, excised patch recording, X-ray crystallography and modeling reveal that this is not due to either a fundamental effect of the mutation on channel gating per se or of genistein acting as a mutation-sensitive partial agonist at the cAMP site. Rather, we find that genistein equivalently moves both HCN and HCN-RE channels closer to the open state (rendering the channels inherently easier to open but at a cost of decreasing the coupling energy of cAMP) and that the anomaly reflects a balance of these energetic effects with the isoform-specific inhibition of activation by the nucleotide gating ring and relief of this by endogenous cAMP. These findings have specific implications with regard to findings based on HCN-RE channels and kinase antagonists and general implications with respect to interpretation of drug effects in mutant channel backgrounds.     

Properties of hyperpolarization-activated pacemaker current defined by coassembly of HCN1 and HCN2 subunits and basal modulation by cyclic nucleotide

Members of the HCN channel family generate hyperpolarization-activated cation currents (Ih) that are directly regulated by cAMP and contribute to pacemaker activity in heart and brain. The four HCN isoforms show distinct but overlapping patterns of expression in different tissues. Here, we report that HCN1 and HCN2, isoforms coexpressed in neocortex and hippocampus that differ markedly in their biophysical properties, coassemble to generate heteromultimeric channels with novel properties. When expressed in Xenopus oocytes, HCN1 channels activate 5-10-fold more rapidly than HCN2 channels. HCN1 channels also activate at voltages that are 10-20 mV more positive than those required to activate HCN2. In cell-free patches, the steady-state activation curve of HCN1 channels shows a minimal shift in response to cAMP (+4 mV), whereas that of HCN2 channels shows a pronounced shift (+17 mV). Coexpression of HCN1 and HCN2 yields Ih currents that activate with kinetics and a voltage dependence that tend to be intermediate between those of HCN1 and HCN2 homomers, although the coexpressed channels do show a relatively large shift by cAMP (+14 mV). Neither the kinetics, steady-state voltage dependence, nor cAMP dose-response curve for the coexpressed Ih can be reproduced by the linear sum of independent populations of HCN1 and HCN2 homomers. These results are most simply explained by the formation of heteromeric channels with novel properties. The properties of these heteromeric channels closely resemble the properties of I(h) in hippocampal CA1 pyramidal neurons, cells that coexpress HCN1 and HCN2. Finally, differences in Ih channel properties recorded in cell-free patches versus intact oocytes are shown to be due, in part, to modulation of Ih by basal levels of cAMP in intact cells.     

Differential association between HERG and KCNE1 or KCNE2

The small proteins encoded by KCNE1 and KCNE2 have both been proposed as accessory subunits for the HERG channel. Here we report our investigation into the cell biology of the KCNE-HERG interaction. In a co-expression system, KCNE1 was more readily co-precipitated with co-expressed HERG than was KCNE2. When forward protein trafficking was prevented (either by Brefeldin A or engineering an ER-retention/retrieval signal onto KCNE cDNA) the intracellular abundance of KCNE2 and its association with HERG markedly increased relative to KCNE1. HERG co-localized more completely with KCNE1 than with KCNE2 in all the membrane-processing compartments of the cell (ER, Golgi and plasma membrane). By surface labeling and confocal immunofluorescence, KCNE2 appeared more abundant at the cell surface compared to KCNE1, which exhibited greater co-localization with the ER-marker calnexin. Examination of the extracellular culture media showed that a significant amount of KCNE2 was extracellular (both soluble and membrane-vesicle-associated). Taken together, these results suggest that during biogenesis of channels HERG is more likely to assemble with KCNE1 than KCNE2 due to distinctly different trafficking rates and retention in the cell rather than differences in relative affinity. The final channel subunit constitution, in vivo, is likely to be determined by a combination of relative cell-to-cell expression rates and differential protein processing and trafficking.