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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.

The function of the KCNE5 (KCNE1-like) protein has not previously been described. Here we show that KCNE5 induces both a time- and voltage-dependent modulation of the KCNQ1 current. Interaction of the KCNQ1 channel with KCNE5 shifted the voltage activation curve of KCNQ1 by more than 140 mV in the positive direction. The activation threshold of the KCNQ1+KCNE5 complex was +40 mV and the midpoint of activation was +116 mV. The KCNQ1+KCNE5 current activated slowly and deactivated rapidly as compared to the KCNQ1+KCNE1 at 22 degrees C; however, at physiological temperature, the activation time constant of the KCNQ1+KCNE5 current decreased fivefold, thus exceeding the activation rate of the KCNQ1+KCNE1 current. The KCNE5 subunit is specific for the KCNQ1 channel, as none of other members of the KCNQ-family or the human ether a-go-go related channel (hERG1) was affected by KCNE5. Four residues in the transmembrane domain of the KCNE5 protein were found to be important for the control of the voltage-dependent activation of the KCNQ1 current. We speculate that since KCNE5 is expressed in cardiac tissue it may here along with the KCNE1 beta-subunit regulate KCNQ1 channels. It is possible that KCNE5 shapes the I(Ks) current in certain parts of the mammalian heart.

KCNQ1 and its β-subunit KCNE1 form the delayed rectifier potassium current IKs, playing an important role in repolarisation of the cardiac tissue and in water and salt transport across epithelial tissues. In the heart IKs is partly responsible for terminating the cardiac action potential. Malfunctions in this channel can result in arrhythmias leading to cardiac arrest.In heart physiology proper function and regulation of IKs current is essential. It has been reported that one of the mechanisms controlling the membrane density of KCNQ1 channels is mediated by ubiquitylation. IKs was shown to be down-regulated by Nedd4/Nedd4-like ubiquitin-protein ligases and this interaction was dependent on the PY-motif on the C-terminal of KCNQ1. Recently it was also discovered that epithelial sodium channel ENaC is regulated by the reverse process - de-ubiquitylation, mediated by an enzyme USP2 (ubiquitin-specific protease 2), which is one of the best described de-ubiquitylases. Therefore the aim of the work was to investigate whether a similar mechanism is valid for KCNQ1/E1 channel complex.The effect of USP2-mediated de-ubiquitylation on IKs channel was investigated using electrophysiology and biochemistry. We observed that when KCNQ1/E1 was co-expressed with USP2-45 or USP2-69 isoform and Nedd4-2 in oocytes, USP2 counteracted the Nedd4-2-specific down-regulation of IKs. It resulted in a rescue of the current amplitude, which was then comparable to the one of IKs expressed alone. Biochemical studies of transfected HEK293 cells confirmed this observation as both total and surface expressed KCNQ1 protein was more abundant when co-expressed with USP2-45/-69 and Nedd4-2 as compared to Nedd4-2 alone. Co-immunoprecipitation assay suggested that USP2 can bind to KCNQ1 independently of the PY-motif and the presence of Nedd4-2.These results point towards an interplay between ubiquitylating enzymes and de-ubiquitylases acting on IKs channel complex in vitro.

Background: The Brugada syndrome (BrS) is characterized by elevated ST segments in the right precordial leads, ventricular tachycardia and sudden death. The syndrome has been linked to loss-of-function of sodium and calcium channels, however the transient outward potassium current (Ito) is thought to be central in the pathogenesis of BrS. We assessed the effects of Ito augmentation in a mammalian model using a novel activator of Ito, NS5806. Methods and Results: Voltage-clamp experiments were performed on midmyocardial cells isolated from the canine left ventricular wall. At 40mV NS5806 (10 μM) increased peak Ito by 79±4 % and the time-course of inactivation was slowed (from Tau=12.6±3.2 ms to 20.3±2.9 ms). We next assessed the effect of increased Ito in the development of BrS phenotype using canine ventricular wedge preparations. NS5806 increased the epicardial action potential (AP) phase 1 magnitude, whereas the APs of endocardial cells were largely unaffected. The accentuated epicardial notch was associated with an accentuated J-wave on the ECG. Loss of the epicardial AP dome at some sites but not others, led to development of phase-2-reentry and polymorphic ventricular tachycardia. NS5806 was able to induce the BrS phenotype in wedges from both right and left ventricles of the canine heart; at 15 μM NS5806 BrS developed in 4/6 right ventricular preparations compared to 2/10 left ventricular preparations. Conclusion: The Ito agonist NS5806 recapitulates the electrographic and arrhythmic manifestation of BrS, providing evidence in support of its pivotal role in the genesis of the disease. Our findings suggest that a genetic defect leading to a prominent gain of function of Ito could explain variants of BrS in which ST segment elevation are evident in both right and left ECG leads.

Background: In human and canine ventricles the transient outward current (Ito) is mediated by Kv4.3 and various accessory proteins. However, the exact molecular composition constituting Ito is still debated. We used the NeuroSearch compound NS5806 known to increase Ito in canine ventricular myocytes as a tool to investigate the molecular components comprising Ito. Methods and Results: The effect of NS5806 was investigated on cloned Kv4.3 channels heterologously expressed together with various β-subunits in Xenopus laevis oocytes. NS5806 was found to inhibit Kv4.3 currents with an IC50 of 6.2 μM, whereas it caused a dose-dependent increase in the current carried by Kv4.3 in complex with KChIP2. These results were confirmed in mammalian CHO-K1 cells. The effect of the compound was further tested on Kv4.3/KChIP2 channels in the presence of the accessory proteins KCNE2, KCNE3, DPP6 and DPP10, which were all found to modulate the effect of NS5806. Of the various combinations of subunits investigated, we found that a complex of Kv4.3, KChIP2 and DPP6 resemble the native Ito current in canine cardiomyocytes most with regard to the effect of NS5806. The NS5806 effect on Kv4.1 and Kv4.2 was similar to the effect found for Kv4.3 both in presence and absence of KChIP2, whereas the compound had minor effect on Kv1.4 and Kv1.5 channels independent of KChIP2.Conclusion The effect of the novel Ito activator NS5806 on Kv4.3 channels was found to be strongly modulated by the presence of accessory proteins suggesting that NS5806 can be used as a valuable tool compound for addressing the molecular compositions of Ito in the heart and of IA in neuronal tissue in addition to its applicability for testing the physiological role of these currents.

This work demonstrates cell swelling as a new regulatory mechanism for the cloned hyperpolarization-activated, cyclic nucleotide-gated channel 2 (HCN2). HCN2 channels were coexpressed with aquaporin1 in Xenopus laevis oocytes and currents were monitored using a two-electrode voltage-clamp. HCN2 channels were activated by hyperpolarization to −100mV and the currents were measured before and during hypoosmotic cell swelling. Cell swelling increased HCN2 currents by 30% without changing the kinetics of the currents. Injection of 50nl intracellular solution resulted in a current increase of 20%, indicating that an increase in cell volume also under isoosmotic conditions may lead to activation of HCN2. In the absence of aquaporin1 only negligible changes in oocyte cell volume occur during exposure to hypoosmotic media and no significant change in HCN2 channel activity was observed during perfusion with hypoosmotic media. This indicates that cell swelling and not a change in ionic strength of the media, caused the observed swelling-induced increase in current. The increase in HCN2 current induced by cell swelling could be abolished by cytochalasin D treatment, indicating that an intact F-actin cytoskeleton is a prerequisite for the swelling-induced current.

K+ movement across mitochondrial membranes is involved in volume regulation and may play a role in cardioprotection. The Ca2+-dependent K+ (KCa) channel has been proposed as a contributor to mitochondrial K+ uniport activity, but its functional role is not well understood. To investigate the impact of KCa channels on mitochondrial energetics, we measured K+ fluxes in parallel with ΔΨm and light scattering in isolated mitochondria from guinea pig hearts. We first analyzed the role of different anions on K+ fluxes. Mitochondria loaded with the K+-sensitive fluorescent probe PBFI were incubated with 5mM glutamate-Na+/malate-Na+ in isotonic sucrose medium and subjected to pulses containing different concentrations of KCl, KAc or KH2PO4. K+ fluxes saturated at ≈10mM regardless of the anion, attaining maximal rates (nmol K+/min/mg) of 172±17 (KCl), 84±2.4 (KAc), and 74±3.8 (KH2PO4), with similar K0.5 in all three cases. We then analyzed the effect of NS11021, a novel activator of KCa channels, on the maximal K+ uptake rate. In the presence of KH2PO4 or KAc, 20-50nM of NS11021 increased mitochondrial volume and K+ flux by ∼2.5-fold whereas KCl increased K+ uptake by 30% with little change in volume. ΔΨm was minimally affected in this concentration range. The NS11021 effect was blocked by 200nM charybdotoxin, a KCa channel blocker. At 50nM NS11021, the respiratory control ratio of the mitochondria increased 2.5-fold in the presence of KH2PO4, but not KCl, indicating that a regulatory volume increase is required to improve oxidative phosphorylation. At higher concentrations of the compound (≥1μM) substantial effects on ΔΨm and state 4 respiration were observed, which were not inhibited by Chtx. The findings indicate that activation of K+ fluxes through KCa channels, coupled with swelling without loss of ΔΨm, improves mitochondrial energetic performance.