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Angiogenesis is largely driven by motile endothelial tip-cells capable of invading avascular tissue domains and enabling new vessel formation. Highly responsive to Vascular Endothelial Growth-Factor-A (VEGFA), endothelial tip-cells also suppress angiogenic sprouting in adjacent stalk cells, and thus have been a primary therapeutic focus in addressing neovascular pathologies. Surprisingly, however, there remains a paucity of specific endothelial tip-cell markers. Here, we employ transcriptional profiling and alacZreporter allele to identifyKcne3as an early and selective endothelial tip-cell marker in multiple angiogenic contexts. In development,Kcne3expression initiates during early phases of angiogenesis (E9) and remains specific to endothelial tip-cells, often adjacent to regions expressing VEGFA. Consistently,Kcne3activation is highly responsive to exogenous VEGFA but maintains tip-cell specificity throughout normal retinal angiogenesis. We also demonstrate endothelial tip-cell selectivity ofKcne3in several injury and tumor models. Together, our data show thatKcne3is a unique marker of sprouting angiogenic tip-cells and offers new opportunities for investigating and targeting this cell type.

Tetrameric voltage-gated K+ channels have four identical voltage sensor domains, and they regulate channel gating. KCNQ1 (Kv7.1) is a voltage-gated K+ channel, and its auxiliary subunit KCNE proteins dramatically regulate its gating. For example, KCNE3 makes KCNQ1 a constitutively open channel at physiological voltages by affecting the voltage sensor movement. However, how KCNE proteins regulate the voltage sensor domain is largely unknown. In this study, by utilizing the KCNQ1-KCNE3-calmodulin complex structure, we thoroughly surveyed amino acid residues on KCNE3 and the S1 segment of the KCNQ1 voltage sensor facing each other. By changing the side-chain bulkiness of these interacting amino acid residues, we found that the distance between the S1 segment and KCNE3 is elaborately optimized to achieve the constitutive activity. In addition, we identified two pairs of KCNQ1 and KCNE3 mutants that partially restored constitutive activity by co-expression. Our work suggests that tight binding of the S1 segment and KCNE3 is crucial for controlling the voltage sensor domains.

BACKGROUND Relaxation of prostate smooth muscle tone is a key strategy for the medical treatment of lower urinary tract symptoms (LUTS) in men. However, potassium channel's physiological role inhuman prostatic smooth muscle (HPrSM) has yet to be determined. OBJECTIVES In this study, we examined the molecular characteristics and physiological roles of Kv7 channels in HPrSM. MATERIALS AND METHODS The expressions of KCNQ1-5 (Kv7 channel pore-forming α-subunits) and KCNE1-5 (β-regulatory subunits) isoforms in HPrSM were examined using real-time PCR. The relaxation effect of ML213 was investigated by cumulatively adding ML213 to the prostate strips. Kv7 currents were recorded using an amphotericin-B perforated patch-clamp technique. RESULTS In HPrSM cells, KCNQ4, KCNQ5, and KCNE4 isoforms were predominantly expressed, while KCNQ1, KCNQ5, and KCNE3 isoforms were the most abundantly expressed in human prostatic tissues. Western blot analysis revealed the protein expression of the Kv7.1, 7.4, and 7.5 channel subtypes in human prostate tissues (n = 3). ML213 (an activator of Kv7.2/7.4/7.5) induced the concentration-dependent relaxation of HPrSM strips (n = 15, p = 0.001), and this effect was attenuated by pretreatment with XE991 (a Kv7.1-7.5 blocker). In electrophysiology studies, ML213 significantly increased the amplitude of whole-cell Kv7 currents in HPrSM cells. ML213-induced outward currents were greater than retigabine (a Kv7.2-7.5 channel activator). The subsequent addition of XE991 completely inhibited the ML213-induced currents (n = 9, p

The function of the voltage-gated KCNQ1 potassium channel is regulated by co-assembly with KCNE auxiliary subunits. KCNQ1-KCNE1 channels generate the slow delayed rectifier current, IKs, which contributes to the repolarization phase of the cardiac action potential. A three amino acid motif (F57-T58-L59, FTL) in KCNE1 is essential for slow activation of KCNQ1-KCNE1 channels. However, how this motif interacts with KCNQ1 to control its function is unknown. Combining computational modeling with electrophysiological studies, we developed structural models of the KCNQ1-KCNE1 complex that suggest how KCNE1 controls KCNQ1 activation. The FTL motif binds at a cleft between the voltage-sensing and pore domains and appears to affect the channel gate by an allosteric mechanism. Comparison with the KCNQ1-KCNE3 channel structure suggests a common transmembrane-binding mode for different KCNEs and illuminates how specific differences in the interaction of their triplet motifs determine the profound differences in KCNQ1 functional modulation by KCNE1 versus KCNE3.

The IKs channel complex is formed by the co-assembly of Kv7.1 (KCNQ1), a voltage-gated potassium channel, with its β-subunit, KCNE1 and the association of numerous accessory regulatory molecules such as PIP2, calmodulin, and yotiao. As a result, the IKs potassium current shows kinetic and regulatory flexibility, which not only allows IKs to fulfill physiological roles as disparate as cardiac repolarization and the maintenance of endolymph K+ homeostasis, but also to cause significant disease when it malfunctions. Here, we review new areas of understanding in the assembly, kinetics of activation and inactivation, voltage-sensor pore coupling, unitary events and regulation of this important ion channel complex, all of which have been given further impetus by the recent solution of cryo-EM structural representations of KCNQ1 alone and KCNQ1+KCNE3. Recently, the stoichiometric ratio of KCNE1 to KCNQ1 subunits has been confirmed to be variable up to a ratio of 4:4, rather than fixed at 2:4, and we will review the results and new methodologies that support this conclusion. Significant advances have been made in understanding differences between KCNQ1 and IKs gating using voltage clamp fluorimetry and mutational analysis to illuminate voltage sensor activation and inactivation, and the relationship between voltage sensor translation and pore domain opening. We now understand that the KCNQ1 pore can open with different permeabilities and conductance when the voltage sensor is in partially or fully activated positions, and the ability to make robust single channel recordings from IKs channels has also revealed the complicated pore subconductance architecture during these opening steps, during inactivation, and regulation by 1−4 associated KCNE1 subunits. Experiments placing mutations into individual voltage sensors to drastically change voltage dependence or prevent their movement altogether have demonstrated that the activation of KCNQ1 alone and IKs can best be explained using allosteric models of channel gating. Finally, we discuss how the intrinsic gating properties of KCNQ1 and IKs are highly modulated through the impact of intracellular signaling molecules and co-factors such as PIP2, protein kinase A, calmodulin and ATP, all of which modulate IKs current kinetics and contribute to diverse IKs channel complex function.

Key points K+ channels are important in intestinal epithelium as they ensure the ionic homeostasis and electrical potential of epithelial cells during anion and fluid secretion. Intestinal epithelium cAMP-activated anion secretion depends on the activity of the (also cAMP dependent) KCNQ1-KCNE3 K+ channel, but the secretory process survives after genetic inactivation of the K+ channel in the mouse. Here we use double mutant mice to investigate which alternative K+ channels come into action to compensate for the absence of KCNQ1-KCNE3 K+ channels. Our data establish that whilst Ca2+ -activated KCa 3.1 channels are not involved, K2P two-pore domain TASK-2 K+ channels are major players providing an alternative conductance to sustain the intestinal secretory process. Work with double mutant mice lacking both TASK-2 and KCNQ1-KCNE3 channels nevertheless points to yet-unidentified K+ channels that contribute to the robustness of the cAMP-activated anion secretion process. Abstract Anion and fluid secretion across the intestinal epithelium, a process altered in cystic fibrosis and secretory diarrhoea, is mediated by cAMP-activated CFTR Cl- channels and requires the simultaneous activity of basolateral K+ channels to maintain cellular ionic homeostasis and membrane potential. This function is fulfilled by the cAMP-activated K+ channel formed by the association of pore-forming KCNQ1 with its obligatory KCNE3 β-subunit. Studies using mice show sizeable cAMP-activated intestinal anion secretion in the absence of either KCNQ1 or KCNE3 suggesting that an alternative K+ conductance must compensate for the loss of KCNQ1-KCNE3 activity. We used double mutant mouse and pharmacological approaches to identify such a conductance. Ca2+ -dependent anion secretion can also be supported by Ca2+ -dependent KCa 3.1 channels after independent CFTR activation, but cAMP-dependent anion secretion is not further decreased in the combined absence of KCa 3.1 and KCNQ1-KCNE3 K+ channel activity. We show that the K2P K+ channel TASK-2 is expressed in the epithelium of the small and large intestine. Tetrapentylammonium, a TASK-2 inhibitor, abolishes anion secretory current remaining in the absence of KCNQ1-KCNE3 activity. A double mutant mouse lacking both KCNQ1-KCNE3 and TASK-2 showed a much reduced cAMP-mediated anion secretion compared to that observed in the single KCNQ1-KCNE3 deficient mouse. We conclude that KCNQ1-KCNE3 and TASK-2 play major roles in the intestinal anion and fluid secretory phenotype. The persistence of an, admittedly reduced, secretory activity in the absence of these two conductances suggests that further additional K+ channel(s) as yet unidentified contribute to the robustness of the intestinal anion secretory process.

Intestinal Cl − secretion is involved in the pathogenesis of secretory diarrheas including cholera. We recently demonstrated that flufenamic acid (FFA) suppressed Vibrio cholerae El Tor variant-induced intestinal fluid secretion via mechanisms involving AMPK activation and NF-κB-suppression. The present study aimed to investigate the effect of FFA on transepithelial Cl − secretion in human intestinal epithelial (T84) cells. FFA inhibited cAMP-dependent Cl − secretion in T84 cell monolayers with IC 50 of ∼8 μM. Other fenamate drugs including tolfenamic acid, meclofenamic acid and mefenamic acid exhibited the same effect albeit with lower potency. FFA also inhibited activities of CFTR, a cAMP-activated apical Cl − channel, and KCNQ1/KCNE3, a cAMP-activated basolateral K + channel. Mechanisms of CFTR inhibition by FFA did not involve activation of its negative regulators. Interestingly, FFA inhibited Ca 2+ -dependent Cl − secretion with IC 50 of ∼10 μM. FFA inhibited activities of Ca 2+ -activated Cl − channels and K Ca 3.1, a Ca 2+ -activated basolateral K + channels, but had no effect on activities of Na + –K + –Cl − cotransporters and Na + –K + ATPases. These results indicate that FFA inhibits both cAMP and Ca 2+ -dependent Cl − secretion by suppressing activities of both apical Cl − channels and basolateral K + channels. FFA and other fenamate drugs may be useful in the treatment of secretory diarrheas.

Dexamethasone produces anti-secretory responses in airway epithelium through the inhibition of basolateral membrane K+ channels [1–3]. We have used the human bronchial epithelial cell line 16HBE14o− to investigate the effects of dexamethasone on the expression of K+ channels and regulatory protein kinases. The data demonstrate the expression of three distinct K+ channel types – KCNQ1:KCNE3, KCNN4 and KATP which are differentially regulated by protein kinase A and protein kinase C. The data also provide evidence for rapid non-genomic actions of dexamethasone on PKC and PKA phosphorylation and their association with the various K+ channel sub-types. Biotinylation experiments provide data on the effects of dexamethasone on membrane expression of the K+ channels. Antibody co-immunoprecipitation, rtPCR and western blotting data are given for the non-genomic dexamethasone transcription-cell signaling pathway involving Gi-protein coupled receptor, PKC, adenylyl cyclase Type IV, cAMP, PKA and ERK1/2 activation. Keywords: Dexamethasone, Non-genomic, Airway epithelium, KCNQ1, KCNN4, KATP channels, Protein kinases

The KCNQ1 channel is a voltage-dependent potassium channel and is ubiquitously expressed throughout the human body including the heart, lung, kidney, pancreas, intestine and inner ear. Gating properties of the KCNQ1 channel are modulated by KCNE auxiliary subunits. For example, the KCNQ1-KCNE1 channel produces a slowly-activating potassium current, while KCNE3 makes KCNQ1 a voltage-independent, constitutively open channel. Thus, physiological functions of KCNQ1 channels are greatly dependent on the type of KCNE protein that is co-expressed in that organ. It has long been debated how the similar single transmembrane KCNE proteins produce quite different gating behaviors. Recent applications of voltage-clamp fluorometry (VCF) for the KCNQ1 channel have shed light on this question. The VCF is a quite sensitive method to detect structural changes of membrane proteins and is especially suitable for tracking the voltage sensor domains of voltage-gated ion channels. In this short review, I will introduce how the VCF technique can be applied to detect structural changes and what have been revealed by the recent VCF applications to the gating modulation of KCNQ1 channels by KCNE proteins.

KCNQ1, also known as Kv7.1, is a voltage-dependent K+ channel that regulates gastric acid secretion, salt and glucose homeostasis, and heart rhythm. Its functional properties are regulated in a tissue-specific manner through co-assembly with beta subunits KCNE1-5. In non-excitable cells, KCNQ1 forms a complex with KCNE3, which suppresses channel closure at negative membrane voltages that otherwise would close it. Pore opening is regulated by the signaling lipid PIP2. Using cryoelectron microscopy (cryo-EM), we show that KCNE3 tucks its single-membrane-spanning helix against KCNQ1, at a location that appears to lock the voltage sensor in its depolarized conformation. Without PIP2, the pore remains closed. Upon addition, PIP2 occupies a site on KCNQ1 within the inner membrane leaflet, which triggers a large conformational change that leads to dilation of the pore's gate. It is likely that this mechanism of PIP2 activation is conserved among Kv7 channels.

Hypokalemic periodic paralysis type 1 (OMIM; HOKPP1) and type 2 (OMIM; HOKPP2) are diseases of the muscle characterized by episodes of painless muscle weakness, and is associated with low potassium blood levels. Hyperthyroidism has been associated with thyrotoxic periodic paralysis (TTPP) (OMIM; TTPP1 and TTPP2), and genetic susceptibility has been implicated. In the present study, the clinical and epidemiological characteristics of patients with TTPP are described, together with their association with genetic variants reported previously in other populations. A prospective and a retrospective search of the medical records of patients who attended the emergency department at the Hospital Universitario ‘Dr. Jose E. Gonzalez’ in Monterrey, Nuevo León, Mexico, and were diagnosed with TTPP was performed. A total of 16 gene variants in the genes MUC1, CACNA1S, KCNE3 and SCN4A, and nine ancestry informative markers (AIMs), were analysed by Multiplex TaqMan™ Open Array assay, and a genetic association study was performed. A total of 11 patients were recruited, comprising nine males and two females (age range, 19-52 years) and 64 control subjects. Only two cases (18%) had a previous diagnosis of hyperthyroidism; the rest were diagnosed subsequently with Graves' disease. Based on the analysis, two DNA variants were found to potentially confer an increased risk for TTPP: S1PR1 rs3737576 [odds ratio (OR), 4.38; 95% confidence interval (CI), 1.08-17.76] and AIM rs2330442 (OR, 4.50; 95% CI, 1.21-16.69), and one variant was suggested to be possibly associated with TTPP, namely MUC1 rs4072037 (OR, 3.08; 95% CI, 0.841-1.38). However, there were no statistically significant associations between any of the 24 DNA variants and TTPP in a population from northeast Mexico.

KCNQ1 (Q1) is a voltage-gated potassium channel that is modulated by members of the KCNE family, the best-characterized being KCNE1 (E1) and KCNE3 (E3). The Q1/E1 complex generates a channel with delayed activation and increased conductance. This complex is expressed in cardiomyocytes where it provides the I(Ks) current that is critical for the repolarization phase of the cardiac action potential. The Q1/E3 complex, on the other hand, is expressed in epithelial cells of the colon and stomach, where it serves as a constitutively active leak channel to help maintain water and ion homeostasis. Studies show the single transmembrane segments (TMS) present in both E1 and E3 are essential to their distinct functions. More specifically, residues FTL located near the middle of the E1 TMS are essential for the delayed activation of Q1, while the corresponding TVG sites in E3 are critical for constitutive activation of the channel. Swapping these three residues leads to the switching of the functional properties for both Q1/E1(FTL➔TVG) and Q1/E3(TVG➔FTL) complexes. This work details the backbone assignments and chemical shifts for the E1(FTL➔TVG) mutant, as determined using a suite of 3D NMR experiments along with specific and inverse amino acid isotopic labeling. The completed assignments are being used, in conjunction with other NMR experiments, to generate a 3D structure of E1(FTL➔TVG). The results of TALOS-N analysis of the chemical shifts are reported here. The E1(FTL➔TVG) structure will be compared to the already available E1 and E3 structures to determine the roles that their TMS triplet motifs play in each protein to dictate their distinct channel-modulatory functions.

Brugada syndrome (BrS) is a rare inherited cardiac arrhythmia associated with a high risk of sudden cardiac death (SCD) due to ventricular fibrillation (VF). BrS is characterized by coved-type ST-segment elevation in the right precordial leads (V1-V3) in the absence of structural heart disease. This pattern is spontaneous, or is unmasked by intravenous administration of Class I antiarrhythmic drugs. The SCN5A-encoded α-subunit of the NaV1.5 cardiac sodium channel has been linked to BrS, and mutations in SCN5A are identified in 15–30% of BrS cases. Genetic testing of BrS patients generally involves sequencing of protein-coding portions and flanking intronic regions of SCN5A, according to recent international guidelines. This excludes the regulatory untranslated regions (5’UTR and 3’UTR) from the routine genetic testing of BrS patients. We here screened the coding sequence, the flanking intronic regions as well as the 5’ and 3’UTR regions of SCN5A gene and further five candidate genes (GPD1L, SCN1B, KCNE3, SCN4B, and MOG1) in a Tunisian family diagnosed with Brugada syndrome.A new Q1000K mutation was identified on the SCN5A gene along with two common polymorphisms (H558R and D1819). Furthermore, multiple genetic variants were identified on the SCN5A 3’UTR, one of which is predicted to create additional microRNA (miRNAs) binding site for miR-1270. Additionally, we identified the hsa-miR-219a rs107822. No relevant coding sequence variant was identified in the remaining studied candidate genes. Although Q1000K is localized in the conserved binding site of MOG1 which predicts a functional consequence, this new mutation along with the additional variants were differentially distributed among the family members without any clear genotype-phenotype concordance. This gives extra evidences about the complexity of the disease and suggests that the occurrence and prognosis of BrS is most likely controlled by a combination of multiple genetic factors and exposures, rather than a single polymorphism/mutation. Most SCN5A polymorphisms were localized in non-coding regions hypothesizing an impact on the miRNA-target complementarities. In this regard, over-expression of miR-1270 led to a significant decrease of luciferase activity suggesting a direct role regulating SCN5A. Therefore, genetic variants that disrupt its binding affinity to SCN5A 3’UTR and/or its expression might cause loss of normal repression control and be associated to BrS.

KCNE β-subunits assemble with and modulate the properties of voltage-gated K+ channels. In the heart, KCNE1 associates with the α-subunit KCNQ1 to generate the slowly activating, voltage-dependent potassium current (IKs) in the heart that controls the repolarization phase of cardiac action potentials. By contrast, in epithelial cells from the colon, stomach, and kidney, KCNE3 coassembles with KCNQ1 to form K+ channels that are voltage-independent K+ channels in the physiological voltage range and important for controlling water and salt secretion and absorption. How KCNE1 and KCNE3 subunits modify KCNQ1 channel gating so differently is largely unknown. Here, we use voltage clamp fluorometry to determine how KCNE1 and KCNE3 affect the voltage sensor and the gate of KCNQ1. By separating S4 movement and gate opening by mutations or phosphatidylinositol 4,5-bisphosphate depletion, we show that KCNE1 affects both the S4 movement and the gate, whereas KCNE3 affects the S4 movement and only affects the gate in KCNQ1 if an intact S4-to-gate coupling is present. Further, we show that a triple mutation in the middle of the transmembrane (TM) segment of KCNE3 introduces KCNE1-like effects on the second S4 movement and the gate. In addition, we show that differences in two residues at the external end of the KCNE TM segments underlie differences in the effects of the different KCNEs on the first S4 movement and the voltage sensor-to-gate coupling.

Background & Aims: Human enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation. Methods: Human duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s). Results: Differentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids. Conclusions: Human enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion. Keywords: Chloride Secretion, Bicarbonate Secretion, DRA, Ion Transport

KCNE3 (MiRP2) forms heteromeric voltage-gated K+ channels with the skeletal muscle-expressed KCNC4 (Kv3.4) α subunit. KCNE3 was the first reported skeletal muscle K+ channel disease gene, but the requirement for KCNE3 in skeletal muscle has been questioned. Here, we confirmed KCNE3 transcript and protein expression in mouse skeletal muscle using Kcne3-/- tissue as a negative control. Whole-transcript microarray analysis (770,317 probes, interrogating 28,853 transcripts) findings were consistent with Kcne3 deletion increasing gastrocnemius oxidative metabolic gene expression and the proportion of type IIa fast-twitch oxidative muscle fibers, which was verified using immunofluorescence. The down-regulated transcript set overlapped with muscle unloading gene expression profiles (≥1.5-fold change; P < 0.05). Gastrocnemius K+ channel α subunit remodeling arising from Kcne3 deletion was highly specific, involving just 3 of 69 α subunit genes probed: known KCNE3 partners KCNC4 and KCNH2 (mERG) were down-regulated, and KCNK4 (TRAAK) was up-regulated (P < 0.05). Functionally, Kcne3-/- mice exhibited abnormal hind-limb clasping upon tail suspension (63% of Kcne3-/- mice ≥10-mo-old vs. 0% age-matched Kcne3+/+ littermates). Whereas 5 of 5 Kcne3+/+ mice exhibited the typical biphasic decline in contractile force with repetitive stimuli of hind-limb muscle, both in vivo and in vitro, this was absent in 6 of 6 Kcne3-/- mice tested. Finally, myoblasts isolated from Kcne3-/- mice exhibit faster-inactivating and smaller sustained outward currents than those from Kcne3+/+ mice. Thus, Kcne3 deletion impairs skeletal muscle function in mice.-King, E. C., Patel, V., Anand, M., Zhao, X., Crump, S. M., Hu, Z., Weisleder, N., Abbott, G. W. Targeted deletion of Kcne3 impairs skeletal muscle function in mice.

Aims: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Disturbances in cardiac potassium conductance are considered as one of the disease mechanisms in AF. We aimed to investigate if mutations in potassium-channel β-subunits KCNE2 and KCNE3 are associated with early-onset lone AF. Methods & results: The coding regions of KCNE2 and KCNE3 were bidirectionally sequenced in 192 unrelated patients diagnosed with early-onset lone AF (V7.1, KV11.1, KV4.3 and KV1.5. A significant gain-of-function effect was observed upon coexpression with KV7.1 and KV7.1 + KCNE1. Confocal imaging found no differences in subcellular localization. No disease-suspected mutations were identified in KCNE3. Conclusion: We identified two KCNE2 gain-of-function missense mutations that seem to increase the susceptibility of early-onset lone AF. These results confirm previous findings indicating that gain-of-function in the slow delayed rectifier potassium current might be involved in the pathogenesis of AF.

Mutations in the human KCNE3 potassium channel ancillary subunit gene are associated with life-threatening ventricular arrhythmias. Most genes underlying inherited cardiac arrhythmias, including KCNE3, are not exclusively expressed in the heart, suggesting potentially complex disease etiologies. Here we investigated mechanisms of KCNE3-linked arrhythmogenesis in Kcne3−/− mice using real-time qPCR, echo- and electrocardiography, ventricular myocyte patch-clamp, coronary artery ligation/reperfusion, blood analysis, cardiac synaptosome exocytosis, microarray and pathway analysis, and multitissue histology. Kcne3 transcript was undetectable in adult mouse atria, ventricles, and adrenal glands, but Kcne3−/− mice exhibited 2.3-fold elevated serum aldosterone (P=0.003) and differentially expressed gene networks consistent with an adrenal-targeted autoimmune response. Furthermore, 8/8 Kcne3−/− mice vs. 0/8 Kcne3+/+ mice exhibited an activated-lymphocyte adrenal infiltration (P=0.0002). Kcne3 deletion also caused aldosterone-dependent ventricular repolarization delay (19.6% mean QTc prolongation in females; P

Human, monkey, and bovine retinal pigment epithelial (RPE) cells exhibit an M-type K+ current, which in many other cell types is mediated by channels composed of KCNQ α-subunits and KCNE auxiliary subunits. Recently, we demonstrated the expression of KCNQ1, KCNQ4, and KCNQ5 in the monkey RPE. Here, we investigated the expression of KCNQ and KCNE subunits in native bovine RPE. RT-PCR analysis revealed the expression of KCNQ1, KCNQ4, and KCNQ5 transcripts in the RPE, but, in Western blot analysis of RPE plasma membranes, only KCNQ5 was detected. Among the five members of the KCNE gene family, transcripts for KCNE1, KCNE2, KCNE3, and KCNE4 were detected in bovine RPE, but only KCNE1 and KCNE2 proteins were detected. Immunohistochemistry of frozen bovine retinal sections revealed KCNE1 expression near the apical and basal membranes of the RPE, in cone outer segments, in the outer nuclear layer, and throughout the inner retina. The localization of KCNE1 in the RPE basal membrane, where KCNQ5 was previously found to be present, suggests that this β-subunit may contribute to M-type K(+) channels in this membrane.

Voltage-gated KCNQ1 (Kv7.1) potassium channels are expressed abundantly in heart but they are also found in multiple other tissues. Differential coassembly with single transmembrane KCNE beta subunits in different cell types gives rise to a variety of biophysical properties, hence endowing distinct physiological roles for KCNQ1–KCNEx complexes. Mutations in either KCNQ1 or KCNE1 genes result in diseases in brain, heart, and the respiratory system. In addition to complexities arising from existence of five KCNE subunits, KCNE1 to KCNE5, recent studies in heterologous systems suggest unorthodox stoichiometric dynamics in subunit assembly is dependent on KCNE expression levels. The resultant KCNQ1–KCNE channel complexes may have a range of zero to two or even up to four KCNE subunits coassembling per KCNQ1 tetramer. These findings underscore the need to assess the selectivity of small-molecule KCNQ1 modulators on these different assemblies. Here we report a unique small-molecule gating modulator, ML277, that potentiates both homomultimeric KCNQ1 channels and unsaturated heteromultimeric (KCNQ1) 4 (KCNE1) n ( n < 4) channels. Progressive increase of KCNE1 or KCNE3 expression reduces efficacy of ML277 and eventually abolishes ML277-mediated augmentation. In cardiomyocytes, the slowly activating delayed rectifier potassium current, or I Ks , is believed to be a heteromultimeric combination of KCNQ1 and KCNE1, but it is not entirely clear whether I Ks is mediated by KCNE-saturated KCNQ1 channels or by channels with intermediate stoichiometries. We found ML277 effectively augments I Ks current of cultured human cardiomyocytes and shortens action potential duration. These data indicate that unsaturated heteromultimeric (KCNQ1) 4 (KCNE1) n channels are present as components of I Ks and are pharmacologically distinct from KCNE-saturated KCNQ1–KCNE1 channels.

Voltage-gated potassium (Kv) channels comprise pore-forming α subunits and a multiplicity of regulatory proteins, including the cardiac-expressed and cardiac arrhythmia-linked transmembrane KCNE subunits. After recently uncovering novel, N-terminally extended (L) KCNE3 and KCNE4 isoforms and detecting their transcripts in human atrium, reported here are their functional effects on human cardiac Kv channel α subunits expressed in Xenopus laevis oocytes. As previously reported for short isoforms KCNE3S and KCNE4S, KCNE3L inhibited hERG; KCNE4L inhibited Kv1.1; neither form regulated the HCN1 pacemaker channel. Unlike KCNE4S, KCNE4L was a potent inhibitor of Kv4.2 and Kv4.3; co-expression of cytosolic β subunit KChIP2, which regulates Kv4 channels in cardiac myocytes, partially relieved Kv4.3 but not Kv4.2 inhibition. Inhibition of Kv4.2 and Kv4.3 by KCNE3L was weaker, and its inhibition of Kv4.2 abolished by KChIP2. KCNE3L and KCNE4L also exhibited subunit-specific effects on Kv4 channel complex inactivation kinetics, voltage dependence and recovery. Further supporting the potential physiological significance of the robust functional effects of KCNE4L on Kv4 channels, KCNE4L protein was detected in human atrium, where it co-localized with Kv4.3. The findings establish functional effects of novel human cardiac-expressed KCNE isoforms and further contribute to our understanding of the potential mechanisms influencing cardiomyocyte repolarization.

KCNE3 modulates the KCNQ1 K+ channel in epithelia by directly stabilizing the voltage-sensor S4 segment in its activated state., The single-span membrane protein KCNE3 modulates a variety of voltage-gated ion channels in diverse biological contexts. In epithelial cells, KCNE3 regulates the function of the KCNQ1 potassium ion (K+) channel to enable K+ recycling coupled to transepithelial chloride ion (Cl−) secretion, a physiologically critical cellular transport process in various organs and whose malfunction causes diseases, such as cystic fibrosis (CF), cholera, and pulmonary edema. Structural, computational, biochemical, and electrophysiological studies lead to an atomically explicit integrative structural model of the KCNE3-KCNQ1 complex that explains how KCNE3 induces the constitutive activation of KCNQ1 channel activity, a crucial component in K+ recycling. Central to this mechanism are direct interactions of KCNE3 residues at both ends of its transmembrane domain with residues on the intra- and extracellular ends of the KCNQ1 voltage-sensing domain S4 helix. These interactions appear to stabilize the activated “up” state configuration of S4, a prerequisite for full opening of the KCNQ1 channel gate. In addition, the integrative structural model was used to guide electrophysiological studies that illuminate the molecular basis for how estrogen exacerbates CF lung disease in female patients, a phenomenon known as the “CF gender gap.”

The 5 human (h)KCNE β subunits each regulate various cation channels and are linked to inherited cardiac arrhythmias. Reported here are previously undiscovered protein-coding regions in exon 1 of hKCNE3 and hKCNE4 that extend their encoded extracellular domains by 44 and 51 residues, which yields full-length proteins of 147 and 221 residues, respectively. Full-length hKCNE3 and hKCNE4 transcript and protein are expressed in multiple human tissues; for hKCNE4, only the longer protein isoform is detectable. Two-electrode voltage-clamp electrophysiology revealed that, when coexpressed in Xenopus laevis oocytes with various potassium channels, the newly discovered segment preserved conversion of KCNQ1 by hKCNE3 to a constitutively open channel, but prevented its inhibition of Kv4.2 and KCNQ4. hKCNE4 slowing of Kv4.2 inactivation and positive-shifted steady-state inactivation were also preserved in the longer form. In contrast, full-length hKCNE4 inhibition of KCNQ1 was limited to 40% at +40 mV vs. 80% inhibition by the shorter form, and augmentation of KCNQ4 activity by hKCNE4 was entirely abolished by the additional segment. Among the genome databases analyzed, the longer KCNE3 is confined to primates; full-length KCNE4 is widespread in vertebrates but is notably absent from Mus musculus. Findings highlight unexpected KCNE gene diversity, raise the possibility of dynamic regulation of KCNE partner modulation via splice variation, and suggest that the longer hKCNE3 and hKCNE4 proteins should be adopted in future mechanistic and genetic screening studies.—Abbott, G. W. Novel exon 1 protein-coding regions N-terminally extend human KCNE3 and KCNE4.

KCNQ1 (Kv7.1) channels are expressed in different tissues and exhibit different gating behaviors in these different tissues. In the heart, KCNQ1 channels are coexpressed with KCNE1 beta subunits and form slowly activating, voltage-gated potassium channels, whereas in epithelial cells KCNQ1 are coexpressed with KCNE2 or KCNE3 beta subunits and form potassium channels that appear voltage-independent. We have used voltage clamp fluorometry (VCF) to elucidate the gating mechanism in KCNQ1 channels and the effects of KCNE beta subunits on KCNQ1 gating. Voltage clamp fluorometry allows us to follow both voltage sensor movement and gate opening simultaneously, thereby better understand the relationship of voltage sensor movement and channel opening in these channels. We show that the voltage sensor S4 moves and the gate closes in KCNQ1/KCNE3 channels, but only at very negative voltages. By decoupling the voltage sensor and the gate, we show that KCNE3 mainly affects the voltage sensor and only indirectly affects the pore through the voltage-sensor-to-gate coupling. The KCNE3 seems to affect the voltage sensor S4 via an electrostatic interactions between negatively charged KCNE3 residues and positively charged S4 residues. Our data suggests that KCNE1 and KCNE3 affect different domains of KCNQ1, thereby resulting in very different modulations of KCNQ1 voltage gating. KCNE1 affects both the voltage-sensing domain and the pore-gate domain, whereas KCNE3 affects mainly the voltage-sensing domain. That different KCNE beta subunits affect different domains of KCNQ1 explains how two highly similar KCNE beta subunits have so different effects on KCNQ1 gating.

The KCNE family contains five single transmembrane-spanning proteins that modulate the voltage-gated potassium channel, KCNQ1 and provide functional diversity to the KCNQ1 channel. For example, KCNE1 modulates the KCNQ1 channel by slowing its activation and increasing its conductance. The KCNE3 protein also increases the conductance, but induces constitutive activation of the channel. In 2001, Melman et al made a series of KCNE1 and KCNE3 chimeras, and identified the transmembrane region as key to distinct functions of KCNE1 and KCNE3. They showed that by swapping three transmembrane residues, T71V72G73, of KCNE3 for KCNE1, F57T58L59, to create a KCNE1/3 chimera they could create a current trace similar to KCNE3 and eliminate KCNE1's ability to delay opening. Thus, KCNE1 with TVG from KCNE3 yields a constitutively active channel without a delay in opening. We hypothesize that this is due to changes in flexibility of the transmembrane-helix of KCNE1 when TVG triple mutation is present. By using NMR spectroscopy, biochemical studies, and computational docking, we aim to look at structural and conformational differences between KCNE1 and KCNE1TVG. We have expressed and purified KCNE1TVG for NMR studies and collected 2D-NMR spectra using a TROSY-based pulse sequence. Partial backbone assignments of KCNE1TVG have been determined by aligning and transfer assignments of the WT KCNE1 previous determined in our lab. Further 3D experiments have been carried out to complete assignments. Further NMR studied will be done to structurally determine KCNE1TVG. Computational models of KCNE1TVG/KCNQ1 in the open and closed state will be created. These models, along with working models for KCNQ1/KCNE1 will provide insight into how KCNE1TVG interacts with the channel differently and similarly than both KCNE1 and KCNE3. This will provide insight into how KCNE1TVG interacts with the channel differently and similarly than both KCNE1 and KCNE3.

Hyperaldosteronism is associated with an increased prevalence of atrial fibrillation (AF). Mutations in KCNE3 have been associated with AF, and Kcne3(-/-) mice exhibit hyperaldosteronism. In this study, we used recently developed Kcne3(-/-) mice to study atrial electrophysiology with respect to development of aldosterone-dependent AF. In invasive electrophysiology studies, Kcne3(-/-) mice displayed a reduced atrial effective refractory period (AERP) and inducible episodes of paroxysmal AF. The cellular arrhythmogenic correlate for AF predisposition was a significant increase in atrial Kv currents generated by the micromolar 4-aminopyridine-sensitive Kv current encoded by Kv1.5. Electrophysiological alterations in Kcne3(-/-) mice were aldosterone dependent and were associated with increased Rab4, -5, and -9-dependent recycling of Kv1.5 channels to the Z-disc/T-tubulus region and lateral membrane via activation of the Akt/AS160 pathway. Treatment with spironolactone inhibited Akt/AS160 phosphorylation, reduced Rab-dependent Kv1.5 recycling, normalized AERP and atrial Kv currents to the wild-type level, and reduced arrhythmia induction in Kcne3(-/-) mice. Kcne3 deletion in mice predisposes to AF by a heretofore unrecognized mechanism-namely, increased aldosterone-dependent Kv1.5 recycling via Rab GTPases. The findings uncover detailed molecular mechanisms underpinning a channelopathy-linked form of AF and emphasize the inevitability of considering extracardiac mechanisms in genetic arrhythmia syndromes.-Lisewski, U., Koehncke, C., Wilck, N., Buschmeyer, B., Pieske, B., Roepke, T. K. Increased aldosterone-dependent Kv1.5 recycling predisposes to pacing-induced atrial fibrillation in Kcne3(-/-) mice.

Background: Kv7.5 (KCNQ5) channels conduct M-type potassium currents in the brain, are expressed in skeletal muscle, and contribute to vascular muscle tone. Methods: We coexpressed Kv7.5 and KCNE1–3 peptides in HEK293 cells and then analyzed their association using electrophysiology and co-immunoprecipitation, assessed localization using confocal microscopy, examined targeting of the oligomeric channels to cholesterol-rich membrane surface microdomains using lipid raft isolation, and evaluated their membrane dynamics using fluorescence recovery after photobleaching (FRAP). Results: Kv7.5 forms oligomeric channels specifically with KCNE1 and KCNE3. The expression of Kv7.5 targeted to cholesterol-rich membrane surface microdomains was very low. Oligomeric Kv7.5/KCNE1 and Kv7.5/KCNE3 channels did not localize to lipid rafts. However, Kv7.5 association impaired KCNE3 expression in lipid raft microdomains. Conclusions: Our results indicate that Kv7.5 contributes to the spatial regulation of KCNE3. This new scenario could greatly assist in determining the physiological relevance of putative KCNE3 interactions in nerve and muscle. Muscle Nerve 45: 48–54, 2012