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OBJECTIVE: Siblings of childhood cancer patients experience social challenges. The results presented in this article are part of a larger qualitative study aiming to generate empirical knowledge about social consequences of childhood cancer from the family's perspective.METHODS: Data were collected through interviews, observational studies, and questionnaires. The study included 68 childhood cancer patients, 39 siblings, and 39 parents from a total of 78 families. Grounded theory informed the data analysis.RESULTS: Major life changes caused by childhood cancer entail an emotional hierarchy regarding the accommodation of each family member's need for help. This study identified a dynamic three-variable, four-adaption model for adaption strategies among siblings towards their parents, based on the sibling's perspective: (1) receives help without asking; (2) receives help after asking; (3) receives no help despite asking; and 4) receives no help and does not ask. Three variables are elaborative to understand the dynamic in adaption strategies: the patient's prognosis, the course of the disease, and the current situation of the diagnosed child. Even though the adaptions are reported by siblings, both patients and parents are aware of and concerned about the siblings' challenges.CONCLUSIONS: These results have implications for practice and have the potential to improve social and healthcare professionals' awareness and ability to offer support and information needed by the families and the siblings. The knowledge presented in this article should be considered basic health care information in line with other information such as treatment protocols.

Kv2.1 is a major delayed-rectifier voltage-gated potassium channel widely expressed in neurons of the CNS. Kv2.1 localizes in high-density cell-surface clusters in the soma and proximal dendrites as well as in the axon initial segment (AIS). Given the crucial roles of both of these compartments in integrating signal input and then generating output, this localization of Kv2.1 is ideal for regulating the overall excitability of neurons. Here we used fluorescence recovery after photobleaching imaging, mutagenesis, and pharmacological interventions to investigate the molecular mechanisms that control the localization of Kv2.1 in these two different membrane compartments in cultured rat hippocampal neurons of mixed sex. Our data uncover a unique ability of Kv2.1 channels to use two molecularly distinct trafficking pathways to accomplish this. Somatodendritic Kv2.1 channels are targeted by the conventional secretory pathway, whereas axonal Kv2.1 channels are targeted by a nonconventional trafficking pathway independent of the Golgi apparatus. We further identified a new AIS trafficking motif in the C-terminus of Kv2.1, and show that putative phosphorylation sites in this region are critical for the restricted and clustered localization in the AIS. These results indicate that neurons can regulate the expression and clustering of Kv2.1 in different membrane domains independently by using two distinct localization mechanisms, which would allow neurons to precisely control local membrane excitability.SIGNIFICANCE STATEMENTOur study uncovered a novel mechanism that targets the Kv2.1 voltage-gated potassium channel to two distinct trafficking pathways and two distinct subcellular destinations: the somatodendritic plasma membrane and that of the axon initial segment. We also identified a distinct motif, including putative phosphorylation sites, that is important for the AIS localization. This raises the possibility that the destination of a channel protein can be dynamically regulated via changes in post-translational modification, which would impact the excitability of specific membrane compartments.

Calcium (Ca2+)-activated potassium (K+) channels (KCa) in epithelia serve important functions in fluid and salt secretion and may be attractive targets for drug development for epithelial disorders, such as cystic fibrosis, diarrhoea, COPD, polycystic kidney disease, and glaucoma. Two very different types of KCa channels are generally found in epithelia: the big conductance, Ca2+-activated K+ channel (BK, KCa1.1), and the intermediate conductance, Ca2+-activated K+ channel (IK, KCa3.1). These channels are differentially expressed in various cells and tissues and serve different physiological and potentially also pathophysiological functions in epithelia.

I dette kapitel beskæftiger vi os med analyser med fokus på tekst: Hvilke analytiske greb kan bringes i anvendelse i forhold til tekstmateriale? Vi besvarer spørgsmålet ved at gennemgå udvalgte elementer i forskellige strategier på tværs af dokumentanalyse, interviewanalyse, analyse af feltnoter fra observationer samt af udskrifter af optagelser fra interaktioner, idet datamaterialet i alle disse tilfælde fremtræder som tekst. Kapitlet rummer overvejelser om tekst som datamateriale samt en ramme for analyse af tekst. I tre hovedafsnit gennemgår vi Grounded Theory, narrativ analyse og sociologisk samtaleanalyse. Sigtet hermed er at eksemplificere valget af og anvendelsen af analytiske greb, ikke en udtømmende behandling af valgmulighederne. Analysens konkrete udformning beror på, hvad der skal til for at drage slutninger i det konkrete projekt, ikke et spørgsmål om at følge standardiserede procedurer. Eksemplerne er derfor i egentlig forstand eksempler på konkrete fremgangsmåder – ikke standarder. Kapitlet rundes af med refleksioner over udfordringerne i analyser af tekst.

The two-pore domain potassium (K(+)) channel TWIK-1 (or K2P1.1) contributes to background K(+) conductance in diverse cell types. TWIK-1, encoded by the KCNK1 gene, is present in the human heart with robust expression in the atria, however its physiological significance is unknown. To evaluate the cardiac effects of TWIK-1 deficiency, we studied zebrafish embryos after knockdown of the two KCNK1 orthologues, kcnk1a and kcnk1b. Knockdown of kcnk1a or kcnk1b individually caused bradycardia and atrial dilation (p

Background and Purpose: Coronary artery disease leads to ischaemic heart disease and ultimately myocardial infarction. Thus, it is important to determine the factors that regulate coronary blood flow. Ca2+‐activated chloride channels contribute to the regulation of arterial tone; however, their role in coronary arteries is unknown. The aim of this study was to investigate the expression and function of the main molecular correlate of Ca2+‐activated chloride channels, TMEM16A, in rat coronary arteries. Experimental Approach: We performed mRNA and protein analysis, electrophysiological studies of coronary artery myocytes, and functional studies of coronary artery contractility and coronary perfusion, using novel inhibitors of TMEM16A. Furthermore, we assessed whether any changes in expression and function occurred in coronary arteries from spontaneously hypertensive rats (SHRs). Key Results: TMEM16A was expressed in rat coronary arteries. The TMEM16A‐specific inhibitor, MONNA, hyperpolarised the membrane potential in U46619. MONNA, T16Ainh‐A01, and Ani9 attenuated 5‐HT/U46619‐induced contractions. MONNA and T16Ainh‐A01 also increased coronary flow in Langendorff perfused rat heart preparations. TMEM16A mRNA was increased in coronary artery smooth muscle cells from SHRs, and U46619 and 5‐HT were more potent in arteries from SHRs than in those from normal Wistar rats. MONNA diminished this increased sensitivity to U46619 and 5‐HT. Conclusions and Implications: In conclusion, TMEM16A is a key regulator of coronary blood flow and is implicated in the altered contractility of coronary arteries from SHRs.

Aim: We investigated the effect of variants in genes encoding sodium channel modifiers SNTA1 and GPD1L found in early onset atrial fibrillation (AF) patients. Patients & methods: Genetic screening in patients with early onset lone AF revealed three variants in GPD1L and SNTA1 in three AF patients. Functional analysis was performed by patch-clamp electrophysiology. Results: Co-expression of GPD1L or its p.A326E variant with NaV1.5 did not alter INa density or current kinetics. SNTA1 shifted the peak-current by -5 mV. The SNTA1-p.A257G variant significantly increased INa. SNTA1-p.P74L did not produce functional changes. Conclusion: Although genetic variation of sodium channel modifiers may contribute to development of AF at a molecular level, it is unlikely a monogenic cause of the disease.

Background and Purpose KCNQ-encoded voltage-dependent potassium channels (Kv7) are involved in the regulation of vascular tone. In this study we evaluated the influence of Kv7 channel activation on smooth muscle relaxation in rat penile arteries and corpus cavernosum from normal and spontaneously hypertensive, heart failure-prone (SHHF) rats – a rat model of human metabolic syndrome. Experimental Approach Quantitative PCR and immunohistochemistry were used to determine the expression of KCNQ isoforms in penile tissue. Isometric tension was measured in intracavernous arterial rings and corpus cavernosum strips isolated from normal and SHHF rats. Key Results Transcripts for KCNQ3, KCNQ4 and KCNQ5 were detected in penile arteries and corpus cavernosum. KCNQ1 was only found in corpus cavernosum. Immunofluorescence signals to Kv7.4 and Kv7.5 were found in penile arteries, penile veins and corpus cavernosum. The Kv7.2–7.5 activators, ML213 and BMS204352, relaxed pre-contracted penile arteries and corpus cavernosum independently of nitric oxide synthase or endothelium-derived hyperpolarization. Relaxations to sildenafil, a PDE5 inhibitor, and sodium nitroprusside (SNP), an nitric oxide donor, were reduced by blocking Kv7 channels with linopirdine in penile arteries and corpus cavernosum. In SHHF rat penile arteries and corpus cavernosum, relaxations to ML213 and BMS204352 were attenuated, and the blocking effect of linopirdine on sildenafil-induced and SNP-induced relaxations reduced. KCNQ3, KCNQ4 and KCNQ5 were down-regulated, and KCNQ1 was up-regulated in corpus cavernosum from SHHF rats. KCNQ1–5 transcripts remained unchanged in penile arteries from SHHF rats. Conclusions and Implications These data suggest that Kv7 channels play a role in erectile function and contribute to the pathophysiology of erectile dysfunction, an early indicator of cardiovascular disease.

The voltage-gated K+channels Kv7.2 and Kv7.3 are located at the axon initial segment (AIS) and exert strong control over action potential generation. Therefore, changes in their localization or cell surface numbers are likely to influence neuronal signaling. However, nothing is known about the cell surface dynamics of Kv7.2/7.3 at steady state or during short-term neuronal stimulation. This is primarily attributable to their membrane topology, which hampers extracellular epitope tagging. Here we circumvent this limitation by fusing an extra phluorin-tagged helix to the N terminus of human Kv7.3. This seven transmembrane chimera, named super ecliptic phluorin (SEP)–TAC-7.3, functions and traffics as a wild-type (WT) channel. We expressed SEP–TAC-7.3 in dissociated rat hippocampal neurons to examine the lateral mobility, surface numbers, and localization of AIS Kv7.2/7.3 heteromers using live imaging. We discovered that they are extraordinarily stable and exhibit a very low surface mobility both during steady state and neuronal stimulation. In the latter case, we also found that neither localization nor cell surface numbers were changed. However, at high glutamate loads, we observed a rapid irreversible endocytosis of Kv7.2/7.3, which required the activation of NR2B-containing NMDA receptors, Ca2+influx, and calpain activation. This excitotoxic mechanism may be specific to ankyrin G-bound AIS proteins because Nav1.2 channels, but not AIS GABAAreceptors, were also endocytosed. In conclusion, we have, for the first time, characterized the cell surface dynamics of a full-length Kv7 channel using a novel chimeric strategy. This approach is likely also applicable to other Kv channels and thus of value for the additional characterization of this ion channel subfamily.SIGNIFICANCE STATEMENTThe voltage-gated K+channels Kv7.2 and Kv7.3 exert strong control over action potential generation, but little is known about their cell surface dynamics. Using a novel phluorin-based approach, we here show that these channels are highly stable at steady state and different types of neuronal stimulation. However, at high glutamate loads, they undergo a rapid calpain-dependent endocytosis that likely represents an early response during excitotoxic states.

In isolated human atrial cardiomyocytes, inhibition of K2P3.1 K(+) channels results in action potential (action potential duration (APD)) prolongation. It has therefore been postulated that K2P3.1 (KCNK3), together with K2P9.1 (KCNK9), could represent novel drug targets for the treatment of atrial fibrillation (AF). However, it is unknown whether these findings in isolated cells translate to the whole heart. The purposes of this study were to investigate the expression levels of KCNK3 and KCNK9 in human hearts and two relevant rodent models and determine the antiarrhythmic potential of K2P3.1 inhibition in isolated whole-heart preparations. By quantitative PCR, we found that KCNK3 is predominantly expressed in human atria whereas KCNK9 was not detectable in heart human tissue. No differences were found between patients in AF or sinus rhythm. The expression in guinea pig heart resembled humans whereas rats displayed a more uniform expression of KCNK3 between atria and ventricle. In voltage-clamp experiments, ML365 and A293 were found to be potent and selective inhibitors of K2P3.1, but at pH 7.4, they failed to prolong atrial APD and refractory period (effective refractory period (ERP)) in isolated perfused rat and guinea pig hearts. At pH 7.8, which augments K2P3.1 currents, pharmacological channel inhibition produced a significant prolongation of atrial ERP (11.6 %, p = 0.004) without prolonging ventricular APD but did not display a significant antiarrhythmic effect in our guinea pig AF model (3/8 hearts converted on A293 vs 0/7 hearts in time-matched controls). These results suggest that when K2P3.1 current is augmented, K2P3.1 inhibition leads to atrial-specific prolongation of ERP; however, this ERP prolongation did not translate into significant antiarrhythmic effects in our AF model.

Key points KCNE4 alters the biophysical properties and cellular localization of voltage-gated potassium channel Kv7.4. KCNE4 is expressed in a variety of arteries and, in mesenteric arteries, co-localizes with Kv7.4, which is important in the control of vascular contractility. Knockdown of KCNE4 leads to reduced Kv7.4 membrane abundance, a depolarized membrane potential and an augmented response to vasoconstrictors. KCNE4 is a key regulator of the function and expression of Kv7.4 in vascular smooth muscle. Abstract The KCNE ancillary subunits (KCNE1–5) significantly alter the expression and function of voltage-gated potassium channels; however, their role in the vasculature has yet to be determined. The present study aimed to investigate the expression and function of the KCNE4 subunit in rat mesenteric arteries and to determine whether it has a functional impact on the regulation of arterial tone by Kv7 channels. In HEK cells expressing Kv7.4, co-expression of KCNE4 increased the membrane expression of Kv7.4 and significantly altered Kv7.4 current properties. Quantitative PCR analysis of different rat arteries found that the KCNE4 isoform predominated and proximity ligation experiments showed that KCNE4 co-localized with Kv7.4 in mesenteric artery myocytes. Morpholino-induced knockdown of KCNE4 depolarized mesenteric artery smooth muscle cells and resulted in their increased sensitivity to methoxamine being attenuated (mean ± SEM EC50 decreased from 5.7 ± 0.63 μm to 1.6 ± 0.23 μm), which coincided with impaired effects of Kv7 modulators. When KCNE4 expression was reduced, less Kv7.4 expression was found in the membrane of the mesenteric artery myocytes. These data show that KCNE4 is consistently expressed in a variety of arteries, and knockdown of the expression product leads to reduced Kv7.4 membrane abundance, a depolarized membrane potential and an augmented response to vasoconstrictors. The present study is the first to demonstrate an integral role of KCNE4 in regulating the function and expression of Kv7.4 in vascular smooth muscle.

The potassium channel Kv7.1 plays critical physiological roles in both heart and epithelial tissues. In heart, Kv7.1 and the accessory subunit KCNE1 forms the slowly activating delayed-rectifier potassium current current, which is enhanced by protein kinase A (PKA)-mediated phosphorylation. The observed current increase requires both phosphorylation of Kv7.1 and the presence of KCNE1. However, PKA also stimulates Kv7.1 currents in epithelial tissues, such as colon, where the channel does not coassemble with KCNE1. Here, we demonstrate that PKA activity significantly impacts the subcellular localization of Kv7.1 in Madin-Darby canine kidney cells. While PKA inhibition reduced the fraction of channels at the cell surface, PKA activation increased it. We show that PKA inhibition led to intracellular accumulation of Kv7.1 in late endosomes/lysosomes. By mass spectroscopy we identified eight phosphorylated residues on Kv7.1, however, none appeared to play a role in the observed response. Instead, we found that PKA acted by regulating endocytic trafficking involving the ubiquitin ligase Nedd4-2. We show that a Nedd4-2-resistant Kv7.1-mutant displayed significantly reduced intracellular accumulation upon PKA inhibition. Similar effects were observed upon siRNA knockdown of Nedd4-2. However, although Nedd4-2 is known to regulate Kv7.1 by ubiquitylation, biochemical analyses demonstrated that PKA did not influence the amount of Nedd4-2 bound to Kv7.1 or the ubiquitylation level of the channel. This suggests that PKA influences Nedd4-2-dependent Kv7.1 transport though a different molecular mechanism. In summary, we identify a novel mechanism whereby PKA can increase Kv7.1 current levels, namely by regulating Nedd4-2-dependent Kv7.1 transport.

Inherited ion channelopathies and electrical remodeling in heart disease alter the cardiac action potential with important consequences for excitation-contraction coupling. Potassium channel-interacting protein 2 (KChIP2) is reduced in heart failure and interacts under physiological conditions with both Kv4 to conduct the fast-recovering transient outward K+ current ( Ito,f) and with CaV1.2 to mediate the inward L-type Ca2+ current ( ICa,L). Anesthetized KChIP2−/− mice have normal cardiac contraction despite the lower ICa,L, and we hypothesized that the delayed repolarization could contribute to the preservation of contractile function. Detailed analysis of current kinetics shows that only ICa,L density is reduced, and immunoblots demonstrate unaltered CaV1.2 and CaVβ2 protein levels. Computer modeling suggests that delayed repolarization would prolong the period of Ca2+ entry into the cell, thereby augmenting Ca2+-induced Ca2+ release. Ca2+ transients in disaggregated KChIP2−/− cardiomyocytes are indeed comparable to wild-type transients, corroborating the preserved contractile function and suggesting that the compensatory mechanism lies in the Ca2+-induced Ca2+ release event. We next functionally probed dyad structure, ryanodine receptor Ca2+ sensitivity, and sarcoplasmic reticulum Ca2+ load and found that increased temporal synchronicity of the Ca2+ release in KChIP2−/− cardiomyocytes may reflect improved dyad structure aiding the compensatory mechanisms in preserving cardiac contractile force. Thus the bimodal effect of KChIP2 on Ito,f and ICa,L constitutes an important regulatory effect of KChIP2 on cardiac contractility, and we conclude that delayed repolarization and improved dyad structure function together to preserve cardiac contraction in KChIP2−/− mice.

KCNQ1 Mutations in Early-Onset Lone AF Introduction Atrial fibrillation (AF) is the most frequent cardiac arrhythmia. The potassium current IKs is essential for cardiac repolarization. Gain-of-function mutation in KCNQ1, the gene encoding the pore-forming α-subunit of the IKs channel (KV7.1), was the first ion channel dysfunction to be associated with familial AF. We hypothesized that early-onset lone AF is associated with a high prevalence of mutations in KCNQ1. Methods and Results We bidirectionally sequenced the entire coding sequence of KCNQ1 in 209 unrelated patients with early-onset lone AF (

The chapter focuses on some mostly unnoticed, but crucial, aspects of professional practice among welfare professions. One such aspect comprises of the informal and strategic forms of negotiations occurring within professional practice and how they relate to various forms of complexity. The text illustrates informal and strategic negotiations by using examples from empirical research on different groups of service users (e.g. people with dementia, unemployed, families with complex problems). A central assertion is that knowledge about such negotiations is vital in terms of understanding how professional practice is actually conducted and works. Such knowledge can enhance professional creativity, confidence and capability to act.

Large-conductance Ca(2+)-activated K(+) channels (BK, KCa1.1, MaxiK) are important regulators of urinary bladder function and may be an attractive therapeutic target in bladder disorders. In this study, we established a high-throughput fluorometric imaging plate reader-based screening assay for BK channel activators and identified a small-molecule positive modulator, NS19504 (5-[(4-bromophenyl)methyl]-1,3-thiazol-2-amine), which activated the BK channel with an EC50 value of 11.0 ± 1.4 µM. Hit validation was performed using high-throughput electrophysiology (QPatch), and further characterization was achieved in manual whole-cell and inside-out patch-clamp studies in human embryonic kidney 293 cells expressing hBK channels: NS19504 caused distinct activation from a concentration of 0.3 and 10 µM NS19504 left-shifted the voltage activation curve by 60 mV. Furthermore, whole-cell recording showed that NS19504 activated BK channels in native smooth muscle cells from guinea pig urinary bladder. In guinea pig urinary bladder strips, NS19504 (1 µM) reduced spontaneous phasic contractions, an effect that was significantly inhibited by the specific BK channel blocker iberiotoxin. In contrast, NS19504 (1 µM) only modestly inhibited nerve-evoked contractions and had no effect on contractions induced by a high K(+) concentration consistent with a K(+) channel-mediated action. Collectively, these results show that NS19504 is a positive modulator of BK channels and provide support for the role of BK channels in urinary bladder function. The pharmacologic profile of NS19504 indicates that this compound may have the potential to reduce nonvoiding contractions associated with spontaneous bladder overactivity while having a minimal effect on normal voiding.

The Role of KChIP2 in Heart Failure Introduction KV4 together with KV Channel-Interacting Protein 2 (KChIP2) mediate the fast recovering transient outward potassium current (Ito,f) in the heart. KChIP2 is downregulated in human heart failure (HF), potentially underlying the loss of Ito,f. We investigated remodeling associated with HF hypothesizing that KChIP2 plays a central role in the modulation of outward K+ currents in HF. Methods and Results HF was induced by aortic banding in wild-type (WT) and KChIP2 deficient (KChIP2–/–) mice, evaluated by echocardiography. Action potentials were measured by floating microelectrodes in intact hearts. Ventricular cardiomyocytes were isolated and whole-cell currents were recorded by patch clamp. Left ventricular action potentials in KChIP2–/– mice were prolonged in a rate dependent manner, consistent with patch-clamp data showing loss of a fast recovering outward K+ current and upregulation of the slow recovering Ito,s and IKur. HF decreased all outward K+ currents in WT mice and did not change the relative contribution of Ito,f in WT mice. Compared to WT HF, KChIP2–/– HF had a larger reduction of K+-current density. However, the relative APD prolongation caused by HF was shorter for KChIP2–/– compared with WT, and the APs of the 2 HF mouse types were indistinguishable. Conclusion Ito,f is just one of many K+ currents being downregulated in murine HF. The downregulation of repolarizing currents in HF is accentuated in KChIP2–/– mice. However, the prolongation of APs associated with HF is less in KChIP2–/– compared to WT, suggesting other compensatory mechanism(s) in the KChIP2–/– mouse.

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.

Key points • Previous studies have suggested that the K+ channel auxiliary subunit K+ channel-interacting protein 2 (KChIP2) serves as a regulator of cardiac remodelling leading to heart failure and increased risk of arrhythmias. • The results presented here show that the progression of cardiac remodelling and heart failure induced by transverse aortic constriction follows a similar time course in wild-type and KChIP2−/− mice. • Protein expression analysis shows that ventricular KChIP2 is significantly downregulated in heart failure in wild-type mice. • The electrophysiological analysis reveals enlarged J and T wave amplitudes and lower vulnerability to pacing-induced ventricular arrhythmias in KChIP2−/− control mice compared to wild-type control mice. Heart failure in wild-type and KChIP2−/− mice prompted comparable prolongation of QT intervals and ventricular effective refractory periods. • Collectively, these results demonstrate that KChIP2 does not influence the structural and functional development of heart failure. Moreover, in contrast to previously reported data, downregulation of KChIP2 expression in heart failure may reduce the risk of cardiac arrhythmia. Abstract Abnormal ventricular repolarization in ion channelopathies and heart disease is a major cause of ventricular arrhythmias and sudden cardiac death. K+ channel-interacting protein 2 (KChIP2) expression is significantly reduced in human heart failure (HF), contributing to a loss of the transient outward K+ current (Ito). We aim to investigate the possible significance of a changed KChIP2 expression on the development of HF and proarrhythmia. Transverse aortic constrictions (TAC) and sham operations were performed in wild-type (WT) and KChIP2−/− mice. Echocardiography was performed before and every 2 weeks after the operation. Ten weeks post-surgery, surface ECG was recorded and we paced the heart in vivo to induce arrhythmias. Afterwards, tissue from the left ventricle was used for immunoblotting. Time courses of HF development were comparable in TAC-operated WT and KChIP2−/− mice. Ventricular protein expression of KChIP2 was reduced by 70% after 10 weeks TAC in WT mice. The amplitudes of the J and T waves were enlarged in KChIP2−/− control mice. Ventricular effective refractory period, RR, QRS and QT intervals were longer in mice with HF compared to sham-operated mice of either genotype. Pacing-induced ventricular tachycardia (VT) was observed in 5/10 sham-operated WT mice compared with 2/10 HF WT mice with HF. Interestingly, and contrary to previously published data, sham-operated KChIP2−/− mice were resistant to pacing-induced VT resulting in only 1/10 inducible mice. KChIP2−/− with HF mice had similar low vulnerability to inducible VT (1/9). Our results suggest that although KChIP2 is downregulated in HF, it is not orchestrating the development of HF. Moreover, KChIP2 affects ventricular repolarization and lowers arrhythmia susceptibility. Hence, downregulation of KChIP2 expression in HF may be antiarrhythmic in mice via reduction of the fast transient outward K+ current.

Aims The purpose of this study was to investigate the functional role of G-protein-coupled inward rectifier potassium (GIRK) channels in the cardiac ventricle. Methods and results Immunofluorescence experiments demonstrated that GIRK4 was localized in outer sarcolemmas and t-tubules in GIRK1 knockout (KO) mice, whereas GIRK4 labelling was not detected in GIRK4 KO mice. GIRK4 was localized in intercalated discs in rat ventricle, whereas it was expressed in intercalated discs and outer sarcolemmas in rat atrium. GIRK4 was localized in t-tubules and intercalated discs in human ventricular endocardium and epicardium, but absent in mid-myocardium. Electrophysiological recordings in rat ventricular tissue ex vivo showed that the adenosine A1 receptor agonist N 6-cyclopentyladenosine (CPA) and acetylcholine (ACh) shortened action potential duration (APD), and that the APD shortening was reversed by either the GIRK channel blocker tertiapin-Q, the adenosine A1 receptor antagonist DPCPX or by the muscarinic M2 receptor antagonist AF-DX 116. Tertiapin-Q prolonged APD in the absence of the exogenous receptor activation. Furthermore, CPA and ACh decreased the effective refractory period and the effect was reversed by either tertiapin-Q, DPCPX or AF-DX 116. Receptor activation also hyperpolarized the resting membrane potential, an effect that was reversed by tertiapin-Q. In contrast, tertiapin-Q depolarized the resting membrane potential in the absence of the exogenous receptor activation. Conclusion Confocal microscopy shows that among species GIRK4 is differentially localized in the cardiac ventricle, and that it is heterogeneously expressed across human ventricular wall. Electrophysiological recordings reveal that GIRK current may contribute significantly to ventricular repolarization and thereby to cardiac electrical stability.

G protein-coupled inwardly rectifying K⁺ channels (GIRK) are important in the regulation of heart rate and atrial electrophysiology. GIRK channels are activated by G protein-coupled receptors, including muscarinic M₂ receptors and adenosine A₁ receptors. The aim of this study was to characterize and compare the electrophysiological effects of acetylcholine (ACh) and adenosine on GIRK channels in rat atria. Action potential duration at 90% repolarization (APD₉₀), effective refractory period (ERP), and resting membrane potential (RMP) were investigated in isolated rat atria by intracellular recordings. Both the adenosine analog N6-cyclopentyladenosine (CPA) and ACh profoundly shortened APD₉₀ and ERP and hyperpolarized the RMP. No additive or synergistic effect of CPA and ACh coapplication was observed. To antagonize GIRK channel activation, the specific inhibitor rTertiapin Q (TTQ) was applied. The coapplication of TTQ reversed the CPA and ACh-induced effects. When TTQ was applied without exogenous receptor activator, both APD₉₀ and ERP were prolonged and RMP was depolarized, confirming a basal activity of the GIRK current. The results reveal that activation of A₁ and M₂ receptors has a profound and equal effect on the electrophysiology in rat atrium. This effect is to a major extent mediated through GIRK channels. Furthermore, these results support the notion that atrial GIRK currents from healthy hearts have a basal component and additional activation can be mediated via at least 2 different receptor mechanisms.

Acetylcholine (ACh) release from the vagus nerve slows heart rate and atrioventricular conduction. ACh stimulates a variety of receptors and channels, including an inward rectifying current [ACh-dependent K+ current ( IK,ACh)]. The effect of ACh in the ventricle is still debated. We compared the effect of ACh on action potentials in canine atria, Purkinje, and ventricular tissue as well as on ionic currents in isolated cells. Action potentials were recorded from ventricular slices, Purkinje fibers, and arterially perfused atrial preparations. Whole cell currents were recorded under voltage-clamp conditions, and unloaded cell shortening was determined on isolated cells. The effect of ACh (1–10 μM) as well as ACh plus tertiapin, an IK,ACh-specific toxin, was tested. In atrial tissue, ACh hyperpolarized the membrane potential and shortened the action potential duration (APD). In Purkinje and ventricular tissues, no significant effect of ACh was observed. Addition of ACh to atrial cells activated a large inward rectifying current (from −3.5 ± 0.7 to −23.7 ± 4.7 pA/pF) that was abolished by tertiapin. This current was not observed in other cell types. A small inhibition of Ca2+ current ( ICa) was observed in the atria, endocardium, and epicardium after ACh. ICa inhibition increased at faster pacing rates. At a basic cycle length of 400 ms, ACh (1 μM) reduced ICa to 68% of control. In conclusion, IK,ACh is highly expressed in atria and is negligible/absent in Purkinje, endocardial, and epicardial cells. In all cardiac tissues, ACh caused rate-dependent inhibition of ICa.

In several European countries there is an increasing trend within social and labour market policy to implement employment-oriented policies. In some countries, this turn towards employment has opened up a new professional arena for social workers, so called employment-oriented social work. The chapter examines the challenges that this development has brought to the social work profession, especially in regard to how knowledge and decision-making is negotiated in relation to clients and other professions within the same policy arena. The chapter is based on Danish, Italian and Austrian data including register data, semi-structured interviews, observations and sound recordings.

Supplemental Digital Content is available in the text., Background Evidence has emerged that small-conductance Ca2+-activated K+ (SK) channels constitute a new target for treatment of atrial fibrillation (AF). SK channels are predominantly expressed in the atria as compared with the ventricles. Various marketed antiarrhythmic drugs are limited by ventricular adverse effects and efficacy loss as AF progresses. Methods and Results A total of 43 pigs were used for the studies. AF reversion in conscious long-term tachypaced pigs: Pigs were subjected to atrial tachypacing (7 Hz) until they developed sustained AF that could not be reverted by vernakalant 4 mg/kg (18.8±3.3 days of atrial tachypacing). When the SK channel inhibitor AP14145 was tested in these animals, vernakalant-resistant AF was reverted to sinus rhythm, and reinduction of AF by burst pacing (50 Hz) was prevented in 8 of 8 pigs. Effects on refractory period and AF duration in open chest pigs: The effects of AP14145 and vernakalant on the effective refractory periods and acute burst pacing-induced AF were examined in anaesthetized open chest pigs. Both vernakalant and AP14145 significantly prolonged atrial refractoriness and reduced AF duration without affecting the ventricular refractoriness or blood pressure in pigs subjected to 7 days atrial tachypacing, as well as in sham-operated control pigs. Conclusions SK currents play a role in porcine atrial repolarization, and pharmacological inhibition of these with AP14145 demonstrates antiarrhythmic effects in a vernakalant-resistant porcine model of AF. These results suggest SK channel blockers as potentially interesting anti-AF drugs.

The voltage-gated potassium channel KV7.1 is regulated by non-pore forming regulatory KCNE β-subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current IKs. However, where the subunits assemble and which of the subunits determines localization of the IKs-complex has not been unequivocally resolved yet. We employed trafficking-deficient KV7.1 and KCNE1 mutants to investigate IKs trafficking using the polarized Madin-Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post-endoplasmic reticulum compartment. We demonstrate that KV7.1 targets the IKs-complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical KV7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that KV7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of KV7.1/KCNE complexes.

Aims Atrial fibrillation (AF) is the most common cardiac arrhythmia, and early-onset lone AF has been linked to mutations in genes encoding ion channels. Mutations in the pore forming subunit KV4.3 leading to an increase in the transient outward potassium current ( I to) have previously been associated with the Brugada Syndrome. Here we aim to determine if mutations in KV4.3 or in the auxiliary subunit K+ Channel-Interacting Protein (KChIP) 2 are associated with early-onset lone AF. Methods and results Two hundred and nine unrelated early-onset lone AF patients (

Calcium-activated potassium channels of small (K(Ca)2, SK) and intermediate (K(Ca)3.1, IK) conductance are involved in endothelium-dependent relaxation of pulmonary arteries. We hypothesized that the function and expression of K(Ca)2 and K(Ca)3.1 increase as a compensatory mechanism to counteract hypoxia-induced pulmonary hypertension in rats. For functional studies, pulmonary arteries were mounted in microvascular myographs for isometric tension recordings. The K(Ca) channel expression was evaluated by immunoblotting and quantitative PCR. Although ACh induced similar relaxations, the ACh-induced relaxations were abolished by the combined inhibition of nitric oxide synthase (by L-nitro-arginine, L-NOARG), cyclo-oxygenase (by indomethacin) and soluble guanylate cyclase (by ODQ) in pulmonary arteries from hypoxic rats, whereas 20 ± 6% (n = 8) maximal relaxation in response to ACh persisted in arteries from normoxic rats. Inhibiting Na(+),K(+)-ATPase with ouabain or blocking K(Ca)2 and K(Ca)3.1 channels reduced the persisting ACh-induced relaxation. In the presence of L-NOARG and indomethacin, a novel K(Ca)2 and K(Ca)3.1 channel activator, NS4591, induced concentration- and endothelium-dependent relaxations, which were markedly reduced in arteries from chronically hypoxic rats compared with arteries from normoxic rats. The mRNA levels of K(Ca)2.3 and K(Ca)3.1 were unaltered, whereas K(Ca)2.3 protein expression was upregulated and K(Ca)3.1 protein expression downregulated in pulmonary arteries from rats exposed to hypoxia. In conclusion, endothelium-dependent relaxation was conserved in pulmonary arteries from chronically hypoxic rats, while endothelium-derived hyperpolarization (EDH)-type relaxation was impaired in chronically hypoxic pulmonary small arteries despite upregulation of K(Ca)2.3 channels. Since impaired EDH-type relaxation was accompanied by K(Ca)3.1 channel protein downregulation, these findings suggest that K(Ca)3.1 channels are important for the maintenance of EDH-type relaxation.

Cardiac experimental electrophysiology is in need of a well-defined Minimum Information Standard for recording, annotating, and reporting experimental data. As a step towards establishing this, we present a draft standard, called Minimum Information about a Cardiac Electrophysiology Experiment (MICEE). The ultimate goal is to develop a useful tool for cardiac electrophysiologists which facilitates and improves dissemination of the minimum information necessary for reproduction of cardiac electrophysiology research, allowing for easier comparison and utilisation of findings by others. It is hoped that this will enhance the integration of individual results into experimental, computational, and conceptual models. In its present form, this draft is intended for assessment and development by the research community. We invite the reader to join this effort, and, if deemed productive, implement the Minimum Information about a Cardiac Electrophysiology Experiment standard in their own work. © 2011 Elsevier Ltd.

We investigated whether Brugada syndrome (BrS)-associated variants identified in the general population have an effect on J-point elevation as well as whether carriers of BrS variants were more prone to experience syncope and malignant ventricular arrhythmia and had increased mortality compared with noncarriers. All BrS-associated variants were identified using the Human Gene Mutation Database (HGMD). Individuals were randomly selected from a general population study using whole-exome sequencing data (n = 870) and genotype array data (n = 6,161) and screened for BrS-associated variants. Electrocardiograms (ECG) were analyzed electronically, and data on syncope, ventricular arrhythmias, and mortality were obtained from administrative health-care registries. In HGMD, 382 BrS-associated genetic variants were identified. Of these, 28 variants were identified in the study cohort. None of the carriers presented with type 1 BrS ECG pattern. Mean J-point elevation in V1 and V2 were within normal guideline limits for carriers and noncarriers. There was no difference in syncope susceptibility (carriers 8/624; noncarriers 98/5,562; P = 0.51), ventricular arrhythmia (carriers 4/620; noncarriers 9/5,524; P = 0.24), or overall mortality (hazard ratio 0.93, 95% CI 0.63–1.4). Our data indicate that a significant number of BrS-associated variants are not the monogenic cause of BrS. Genet Med advance online publication 06 October 2016

Retigabine is a first in class anticonvulsant that has recently undergone clinical trials to test its efficacy in epileptic patients. Retigabine's novel mechanism of action – activating Kv7 channels – suppresses neuronal activity to prevent seizure generation by hyperpolarizing the membrane potential and suppressing depolarizing surges. However, Kv7 channels are not expressed exclusively in neurones and data generated over the last decade have shown that Kv7 channels play a key role in various smooth muscle systems of the body. This review discusses the potential of targeting Kv7 channels in the smooth muscle to treat diseases such as hypertension, bladder instability, constipation and preterm labour.

Det var mere end kosmetik, da arbejdsmarkedspolitikken i 2001 tog navneforandring til 'beskæftigelsespolitik'. Man kan tale om, at politikområdet på såvel indholds- som organiseringssiden er blevet reformeret. Reformeringen har, ifølge medarbejderne, medført fundamentale skift i arbejdets indhold og organisering. Det indbefatter bla. et skift fra klientorienteret arbejde til administrativt arbejde, et skift i sammensætningen af medarbejdere samt i oplevelsen af autonomien i arbejdet og endelig et skift i udførelsen af arbejdet, der for visse medarbejdere har betydet dilemmaer af etisk og faglig karakter. Der er således tale om forandringer, der har haft stor betydning for den faglige praksis og kvalificeringsproblematikken i jobcentrene.

ObjectivesThis study investigated whether an individual's risk of developing lone atrial fibrillation (AF) before age 60 years is associated with lone AF in relatives.BackgroundGenetic factors may play a role in the development of lone AF.MethodsUsing Danish national registers, a cohort was established of ∼4 million persons born between 1950 and 2008, and those with a family history of lone AF (AF without preceding cardiovascular/endocrine diagnoses) were identified. Individuals were followed up until the first diagnosis of lone AF. Poisson regression was used to estimate incidence rate ratios (IRRs).ResultsIn ∼92 million person-years of follow-up, 9,507 persons were identified as having lone AF. The IRRs for lone AF given an affected first- or second-degree relative were 3.48 (95% confidence interval [CI]: 3.08 to 3.93) and 1.64 (95% CI: 1.04 to 2.59), respectively. IRRs were higher for men than for women but were not associated with the affected relative's sex. IRR for lone AF was 6.24 (95% CI: 2.59 to 15.0), given at least 2 first-degree relatives affected with lone AF. The IRR for lone AF in persons aged

Background— Atrial fibrillation (AF) is the most common cardiac arrhythmia. The cardiac sodium channel, Na V 1.5, plays a pivotal role in setting the conduction velocity and the initial depolarization of the cardiac myocytes. We hypothesized that early-onset lone AF was associated with genetic variation in SCN5A . Methods and Results— The coding sequence of SCN5A was sequenced in 192 patients with early-onset lone AF. Eight nonsynonymous mutations (T220I, R340Q, T1304M, F1596I, R1626H, D1819N, R1897W, and V1951M) and 2 rare variants (S216L in 2 patients and F2004L) were identified. Of 11 genopositive probands, 6 (3.2% of the total population) had a variant previously associated with long QT syndrome type 3 (LQTS3). The prevalence of LQTS3-associated variants in the patients with lone AF was much higher than expected, compared with the prevalence in recent exome data (minor allele frequency, 1.6% versus 0.3%; P =0.003), mainly representing the general population. The functional effects of the mutations were analyzed by whole cell patch clamp in HEK293 cells; for 5 of the mutations previously associated with LQTS3, patch-clamp experiments showed an increased sustained sodium current, suggesting a mechanistic overlap between LQTS3 and early-onset lone AF. In 9 of 10 identified mutations and rare variants, we observed compromised biophysical properties affecting the transient peak current. Conclusions— In a cohort of patients with early-onset lone AF, we identified a high prevalence of SCN5A mutations previously associated with LQTS3. Functional investigations of the mutations revealed both compromised transient peak current and increased sustained current.

Introduction: Remodeling of ion channel expression is well established in heart failure (HF). Loss of transient outward current (Ito) during HF contributes to repolarization abnormalities. We determined the extent to which Ito is reduced in tachypacing-induced HF and assessed the ability of an Ito activator (NS5806) to recover this current. Methods: Whole cell patch clamp was used to record Ito in epicardial (Epi) ventricular myocytes. Epi- and endocardial action potentials were recorded from left ventricular (LV) wedge preparations. Results: Rapid pacing resulted in a reduction in the size of Ito in Epi myocytes (Control= 22.13±1.9 pA/pF vs 16.12±1.4 after 2-weeks and 10.69±1.4 pA/pF after 5-weeks, +50 mV). HF also slowed the recovery of Ito. Recovery of Ito showed a fast and slow phase as follows: i) 1=28.4±2.54 ms and 2= 177.5±7.82 ms for Control Epi, ii) 1=56.6±5.92 ms and 2=337.8±28.9 ms for 2-week Epi, and iii) 1=73.4±3.52 ms and 2=546.3±38.6ms for 5-week Epi. In 2-week paced dogs, NS5806 increased Ito (at +50 mV) from 16.12±1.4 pA/pF to 23.85±2.1 pA/pF (p

KCNQ4-encoded voltage-dependent potassium (Kv7.4) channels are important regulators of vascular tone that are severely compromised in models of hypertension. However, there is no information as to the role of these channels in responses to endogenous vasodilators. We used a molecular knockdown strategy, as well as pharmacological tools, to examine the hypothesis that Kv7.4 channels contribute to β-adrenoceptor–mediated vasodilation in the renal vasculature and underlie the vascular deficit in spontaneously hypertensive rats. Quantitative PCR and immunohistochemistry confirmed gene and protein expression of KCNQ1, KCNQ3, KCNQ4, KCNQ5, and Kv7.1, Kv7.4, and Kv7.5 in rat renal artery. Isoproterenol produced concentration-dependent relaxation of precontracted renal arteries and increased Kv7 channel currents in isolated smooth muscle cells. Application of the Kv7 blocker linopirdine attenuated isoproterenol-induced relaxation and current. Isoproterenol-induced relaxations were also reduced in arteries incubated with small interference RNAs targeted to KCNQ4 that produced a ≈60% decrease in Kv7.4 protein level. Relaxation to isoproterenol and the Kv7 activator S-1 were abolished in arteries from spontaneously hypertensive rats, which was associated with ≈60% decrease in Kv7.4 abundance. This study provides the first evidence that Kv7 channels contribute to β-adrenoceptor–mediated vasodilation in the renal vasculature and that abrogation of Kv7.4 channels is strongly implicated in the impaired β-adrenoceptor pathway in spontaneously hypertensive rats. These findings may provide a novel pathogenic link between arterial dysfunction and hypertension.

Kv7.1 (KCNQ1) channels are regulators of several physiological processes including vasodilatation, repolarization of cardiomyocytes, and control of secretory processes. A number of Kv7.1 pore mutants are sensitive to extracellular potassium. We hypothesized that extracellular potassium also modulates wild-type Kv7.1 channels. The Kv7.1 currents were measured in Xenopus laevis oocytes at different concentrations of extracellular potassium (1–50 mM). As extracellular potassium was elevated, Kv7.1 currents were reduced significantly more than expected from theoretical calculations based on the Goldman-Hodgkin-Katz flux equation. Potassium inhibited the steady-state current with an IC50 of 6.0 ± 0.2 mM. Analysis of tail-currents showed that potassium increased the fraction of channels in the inactivated state. Similarly, the recovery from inactivation was slowed by potassium, suggesting that extracellular potassium stabilizes an inactivated state in Kv7.1 channels. The effect of extracellular potassium was absent in noninactivating Kv7.1/KCNE1 and Kv7.1/KCNE3 channels, further supporting a stabilized inactivated state as the underlying mechanism. Interestingly, coexpression of Kv7.1 with KCNE2 did not attenuate the inhibition by potassium. In a number of other Kv channels, including Kv1.5, Kv4.3, and Kv7.2–5 channels, currents were only minimally reduced by an increase in extracellular potassium as expected. These results show that extracellular potassium modulates Kv7.1 channels and suggests that physiological changes in potassium concentrations may directly control the function of Kv7.1 channels. This may represent a novel regulatory mechanism of excitability and of potassium transport in tissues expressing Kv7.1 channels.

Widely used murine models of adrenergic-induced cardiomyopathy offer little insight into electrical derangements seen in human heart failure owing to profound differences in the characteristics of ventricular repolarization in mice and rats compared with humans. We therefore sought to determine whether sustained adrenergic activation may produce a clinically relevant heart failure phenotype in the guinea-pig, an animal species whose ventricular action potential shape and restitution properties resemble those determined in humans. Isoprenaline (ISO), a β-adrenoceptor agonist, was infused at variable dosage and duration using either subcutaneously implanted osmotic minipumps or daily injections, in an attempt to establish the relevant treatment protocol. We found that 3 months of daily ISO injections (final dose of 1 mg kg(-1), i.p.) promote heart failure evidenced by cardiac hypertrophy [increased cardiac weights, left ventricular (LV) posterior wall thickness, myocyte cross-sectional area and LV protein content], cardiac dilatation (increased LV internal diameters), basal systolic dysfunction (reduced LV fractional shortening determined by echocardiography and flattened LV systolic pressure-volume and stress-strain relationships assessed in isolated, perfused heart preparations), reduced contractile reserve in the presence of acute β-adrenoceptor stimulation, and pulmonary oedema (increased lung weights). These changes were associated with prolongation of LV epicardial action potential, effective refractory period and QT interval, an upward shift of the electrical restitution curve determined over a wide range of diastolic intervals, and reduced maximal restitution slope. The physiological right ventricular-to-LV difference in action potential duration was eliminated in ISO-treated hearts, thereby contributing to impaired activation-to-repolarization coupling and reversed right ventricular-to-LV difference in repolarization time. In summary, we establish the guinea-pig model of ISO-induced cardiomyopathy, which enables the correlation of detrimental structural and contractile changes with repolarization abnormalities typically seen in human heart failure.

In this article we want to contribute theoretically and methodologically towards a qualitative approach to effects of social work interventions. We develop a relational understanding of effects, based on actor-network theory combined with narrative analysis. The central actor-network theory concepts, actant and translation, are introduced, arguing that social work interventions and their consequences are evolving through current adaption (translation) in networks where human actors and non-human entities (actants) are ascribed agency in narrative accounts. This is done with reference to an empirical study of the content and consequences of employment efforts, seen from the perspective of cash benefit recipients. In this study, short narratives were used to trace changes in cash benefit recipients’ experience of work identity when they were participating in employment efforts. We argue that our approach to the effects of social work interventions is not limited to studies on employment efforts. It can also...

Background KCNQ1 (Kv7.1), together with its KCNE β subunits, plays a pivotal role both in the repolarization of cardiac tissue and in water and salt transport across epithelial membranes. Nedd4/Nedd4-like (neuronal precursor cell–expressed developmentally downregulated 4) ubiquitin–protein ligases interact with the KCNQ1 potassium channel through a PY motif located in the C terminus of KCNQ1. This interaction induces ubiquitylation of KCNQ1, resulting in a reduced surface density of the channel. It was reported recently that the epithelial sodium channel is regulated by the reverse process—deubiquitylation—mediated by USP2 (ubiquitin-specific protease 2). Objective In this article, we investigated whether deubiquitylation may regulate KCNQ1 channel complexes. Methods In this study, we used electrophysiology, biochemistry, and confocal microscopy. Results Electrophysiological investigations of KCNQ1/KCNE1 proteins coexpressed with USP2-45 or USP2-69 isoforms and Nedd4-2 in Xenopus laevis oocytes and mammalian cells revealed that both USP2 isoforms counter the Nedd4-2–specific downregulation of I Ks . Biochemical studies showed that the total and surface-expressed KCNQ1 protein was more abundant when coexpressed with USP2 and Nedd4-2 as compared with Nedd4-2 alone. Western blotting revealed partial protection against covalent attachment of ubiquitin moieties on KCNQ1 when USP2 was coexpressed with Nedd4-2. Coimmunoprecipitation assays suggested that USP2 can bind to KCNQ1 independently of the PY motif. Immunocytochemistry confirmed that USP2 restores the membrane localization of KCNQ1. Conclusion These results demonstrate that USP2 can be a potent regulator of KCNQ1 surface density. USP2, which is well expressed in many tissues, may therefore be important in controlling the KCNQ1 channel dynamics in vivo.

KCNQ gene expression was previously shown in various rodent blood vessels, where the products of KCNQ4 and KCNQ5, Kv7.4 and Kv7.5 potassium channel subunits, respectively, have an influence on vascular reactivity. The aim of this study was to determine if small cerebral resistance arteries of the rat express KCNQ genes and whether Kv7 channels participate in the regulation of myogenic control of diameter. Quantitative reverse transcription polymerase chain reaction (QPCR) was undertaken using RNA isolated from rat middle cerebral arteries (RMCAs) and immunocytochemistry was performed using Kv7 subunit-specific antibodies and freshly isolated RMCA myocytes. KCNQ4 message was more abundant than KCNQ5 = KCNQ1, but KCNQ2 and KCNQ3 message levels were negligible. Kv7.1, Kv7.4 and Kv7.5 immunoreactivity was present at the sarcolemma of freshly isolated RMCA myocytes. Linopirdine (1 microm) partially depressed, whereas the Kv7 activator S-1 (3 and/or 20 microm) enhanced whole-cell Kv7.4 (in HEK 293 cells), as well as native RMCA myocyte Kv current amplitude. The effects of S-1 were voltage-dependent, with progressive loss of stimulation at potentials of >15 mV. At the concentrations employed linopirdine and S-1 did not alter currents due to recombinant Kv1.2/Kv1.5 or Kv2.1/Kv9.3 channels (in HEK 293 cells) that are also expressed by RMCA myocytes. In contrast, another widely used Kv7 blocker, XE991 (10 microm), significantly attenuated native Kv current and also reduced Kv1.2/Kv1.5 and Kv2.1/Kv9.3 currents. Pressurized arterial myography was performed using RMCAs exposed to intravascular pressures of 10-100 mmHg. Linopirdine (1 microm) enhanced the myogenic response at 20 mmHg, whereas the activation of Kv7 channels with S-1 (20 microm) inhibited myogenic constriction at >20 mmHg and reversed the increased myogenic response produced by suppression of Kv2-containing channels with 30 nm stromatoxin (ScTx1). These data reveal a novel contribution of KCNQ gene products to the regulation of myogenic control of cerebral arterial diameter and suggest that Kv7 channel activating drugs may be appropriate candidates for the development of an effective therapy to ameliorate cerebral vasospasm.

Background and purpose The compound NS5806 increases the transient outward current (I(to)) in canine ventricular cardiomyocytes and slows current decay. In human and canine ventricle, I(to) is thought to be mediated by K(V)4.3 and various ancillary proteins, yet, the exact subunit composition of I(to) channels is still debated. Here we characterize the effect of NS5806 on heterologously expressed putative I(to) channel subunits and other potassium channels. Experimental approach Cloned K(V)4 channels were co-expressed with KChIP2, DPP6, DPP10, KCNE2, KCNE3 and K(V)1.4 in Xenopus laevis oocytes or CHO-K1 cells. Key results NS5806 increased K(V)4.3/KChIP2 peak current amplitudes with an EC(50) of 5.3 +/- 1.5microM and significantly slowed current decay. KCNE2, KCNE3, DPP6 and DPP10 modulated K(V)4.3 currents and the response to NS5806, but current decay was slowed only in complexes containing KChIP2. The effect of NS5806 on K(V)4.2 was similar to that on K(V)4.3, and current decay was only slowed in presence of KChIP2. However, for K(V)4.1, the slowing of current decay by NS5806 was independent of KChIP2. K(V)1.4 was strongly inhibited by 10 microM NS5806 and K(V)1.5 was inhibited to a smaller extent. Effects of NS5806 on kinetics of currents generated by K(V)4.3/KChIP2/DPP6 with K(V)1.4 in oocytes could reproduce those on cardiac I(to) in canine ventricular myocytes. K(V)7.1, K(V)11.1 and K(ir)2 currents were unaffected by NS5806. Conclusion and implications NS5806 modulated K(V)4 channel gating depending on the presence of KChIP2, suggesting that NS5806 can potentially be used to address the molecular composition as well as the physiological role of cardiac I(to).

Large-conductance Ca2+-activated potassium (BK) channels play an important role in regulating the function and activity of urinary bladder smooth muscle (UBSM), and the loss of BK channel function has been shown to increase UBSM excitability and contractility. However, it is not known whether activation of BK channels has the converse effect of reducing UBSM excitability and contractility. Here, we have sought to investigate this possibility by using the novel BK channel opener NS11021. NS11021 (3 μM) caused an approximately threefold increase in both single BK channel open probability ( Po) and whole cell BK channel currents. The frequency of spontaneous action potentials in UBSM strips was reduced by NS11021 from a control value of 20.9 ± 5.9 to 10.9 ± 3.7 per minute. NS11021 also reduced the force of UBSM spontaneous phasic contractions by ∼50%, and this force reduction was blocked by pretreatment with the BK channel blocker iberiotoxin. NS11021 (3 μM) had no effect on contractions evoked by nerve stimulation. These findings indicate that activating BK channels reduces the force of UBSM spontaneous phasic contractions, principally through decreasing the frequency of spontaneous action potentials.