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The sinoatrial node (SAN) and subsidiary pacemakers in the cardiac conduction system generate spontaneous electrical activity which is indispensable for electrical and therefore contractile function of the heart. The hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is responsible for genesis of the pacemaker "funny" current during diastolic depolarisation. S-palmitoylation, the reversible conjugation of the fatty acid palmitate to protein cysteine sulfhydryls, regulates the activity of key cardiac Na + and Ca 2+ handling proteins, influencing their membrane microdomain localisation and function. We investigated HCN4 palmitoylation and its functional consequences in engineered human embryonic kidney 293T cells as well as endogenous HCN4 in neonatal rat ventricular myocytes. HCN4 was palmitoylated in all experimental systems investigated. We mapped the HCN4 palmitoylation sites to a pair of cysteines in the HCN4 intracellular amino terminus. A double cysteine-to-alanine mutation CC93A/179AA of full length HCN4 caused a ∼67% reduction in palmitoylation in comparison to wild type HCN4. We used whole-cell patch clamp to evaluate HCN4 current (I HCN4 ) in stably transfected 293T cells. Removal of the two N-terminal palmitoylation sites did not significantly alter half maximal activation voltage of I HCN4 or the activation slope factor. I HCN4 was significantly larger in cells expressing wild type compared to non-palmitoylated HCN4 across a range of voltages. Phylogenetic analysis revealed that although cysteine 93 is widely conserved across all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 is restricted to placental mammals. Collectively, we provide evidence for functional regulation of HCN4 via palmitoylation of its amino terminus in vertebrates. We suggest that by recruiting the amino terminus to the bilayer, palmitoylation enhances the magnitude of HCN4-mediated currents, but does not significantly affect the kinetics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Congreve, Main, Butler, Gao, Brown, Du, Choisy, Cheng, Hancox and Fuller.)

The T618I KCNH2-encoded hERG mutation is the most frequently observed mutation in genotyped cases of the congenital short QT syndrome (SQTS), a cardiac condition associated with ventricular fibrillation and sudden death. Most T618I hERG carriers exhibit a pronounced U wave on the electrocardiogram and appear vulnerable to ventricular, but not atrial fibrillation (AF). The basis for these effects is unclear. This study used the action potential (AP) voltage clamp technique to determine effects of the T618I mutation on hERG current (I hERG ) elicited by APs from different cardiac regions. Whole-cell patch-clamp recordings were made at 37 °C of I hERG from hERG-transfected HEK-293 cells. Maximal I hERG during a ventricular AP command was increased ∼4-fold for T618I I hERG and occurred much earlier during AP repolarization. The mutation also increased peak repolarizing currents elicited by Purkinje fibre (PF) APs. Maximal wild-type (WT) I hERG current during the PF waveform was 87.2 ± 4.5% of maximal ventricular repolarizing current whilst for the T618I mutant, the comparable value was 47.7 ± 2.7%. Thus, the T618I mutation exacerbated differences in repolarizing I hERG between PF and ventricular APs; this could contribute to heterogeneity of ventricular-PF repolarization and consequently to the U waves seen in T618I carriers. The comparatively shorter duration and lack of pronounced plateau of the atrial AP led to a smaller effect of the T618I mutation during the atrial AP, which may help account for the lack of reported AF in T618I carriers. Use of a paired ventricular AP protocol revealed an alteration to protective I hERG transients that affect susceptibility to premature excitation late in AP repolarization/early in diastole. These observations may help explain altered arrhythmia susceptibility in this form of the SQTS., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The congenital short QT syndrome (SQTS) is a cardiac condition that leads to abbreviated ventricular repolarization and an increased susceptibility to arrhythmia and sudden death. The SQT3 form of the syndrome is due to mutations to the KCNJ2 gene that encodes Kir2.1, a critical component of channels underlying cardiac inwardly rectifying K + current, I K1 . The first reported SQT3 KCNJ2 mutation gives rise to the D172N Kir2.1 mutation, the consequences of which have been studied on recombinant channels in vitro and in ventricular cell and tissue simulations. The aim of this study was to establish the effects of the D172N mutation on ventricular repolarization through real-time replacement of I K1 using the dynamic clamp technique. Whole-cell patch-clamp recordings were made from adult guinea-pig left ventricular myocytes at physiological temperature. Action potentials (APs) were elicited at 1 Hz. Intrinsic I K1 was inhibited with a low concentration (50 µM) of Ba 2+ ions, which led to AP prolongation and triangulation, accompanied by a ∼6 mV depolarization of resting membrane potential. Application of synthetic I K1 through dynamic clamp restored AP duration, shape and resting potential. Replacement of wild-type (WT) I K1 with heterozygotic (WT-D172N) or homozygotic (D172N) mutant formulations under dynamic clamp significantly abbreviated AP duration (APD 90 ) and accelerated maximal AP repolarization velocity, with no significant hyperpolarization of resting potential. Across stimulation frequencies from 0.5 to 3 Hz, the relationship between APD 90 and cycle length was downward shifted, reflecting AP abbreviation at all stimulation frequencies tested. In further AP measurements at 1 Hz from hiPSC cardiomyocytes, the D172N mutation produced similar effects on APD and repolarization velocity; however, resting potential was moderately hyperpolarized by application of mutant I K1 to these cells. Overall, the results of this study support the major changes in ventricular cell AP repolarization with the D172N predicted from prior AP modelling and highlight the potential utility of using adult ventricular cardiomyocytes for dynamic clamp exploration of functional consequences of Kir2.1 mutations., Competing Interests: RR and GB are founders and owners of Cytocybernetics Inc. BF was employed by the company Cytocybernetics Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Du, Rasmusson, Bett, Franks, Zhang and Hancox.)

The lipophilic polycyclic aromatic hydrocarbon (PAH) phenanthrene is relatively abundant in polluted air and water and can access and accumulate in human tissue. Phenanthrene has been reported to interact with cardiac ion channels in several fish species. This study was undertaken to investigate the ability of phenanthrene to interact with hERG (human Ether-à-go-go-Related Gene) encoded Kv11.1 K + channels, which play a central role in human ventricular repolarization. Pharmacological inhibition of hERG can be proarrhythmic. Whole-cell patch clamp recordings of hERG current (I hERG ) were made from HEK293 cells expressing wild-type (WT) and mutant hERG channels. WT I hERG1a was inhibited by phenanthrene with an IC 50 of 17.6 ± 1.7 µM, whilst I hERG1a/1b exhibited an IC 50 of 1.8 ± 0.3 µM. WT I hERG block showed marked voltage and time dependence, indicative of dependence of inhibition on channel gating. The inhibitory effect of phenanthrene was markedly impaired by the attenuated inactivation N588K mutation. Remarkably, mutations of S6 domain aromatic amino acids (Y652, F656) in the canonical drug binding site did not impair the inhibitory action of phenanthrene; the Y652A mutation augmented I hERG block. In contrast, the F557L (S5) and M651A (S6) mutations impaired the ability of phenanthrene to inhibit I hERG , as did the S624A mutation below the selectivity filter region. Computational docking using a cryo-EM derived hERG structure supported the mutagenesis data. Thus, phenanthrene acts as an inhibitor of the hERG K + channel by directly interacting with the channel, binding to a distinct site in the channel pore domain., (© 2021. The Author(s).)

Water-uptakes of pure sodium carbonate (Na 2 CO 3 ), pure β-alanine and internally mixed β-alanine/Na 2 CO 3 aerosol particles with different mole ratios are first monitored using attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) technique. For pure Na 2 CO 3 aerosol particles, combining the absorptions at 877 and 1422 cm -1 with abrupt water loss shows the efflorescence relative humidity (ERH) of 62.9%-51.9%. Upon humidifying, solid Na 2 CO 3 firstly absorbs water to from Na 2 CO 3 ·H 2 O crystal at 72.0% RH and then deliquesces at 84.5% RH (DRH). As for pure β-alanine particles, the crystallization takes place in the range of 42.4%-33.2% RH and becomes droplets at ~88.2% RH. When β-alanine is mixed with Na 2 CO 3 at various mole ratios, it shows no efflorescence of Na 2 CO 3 when β-alanine to Na 2 CO 3 mole ratio (OIR) is 2:1. For 1:1 and 1:2 β-alanine/Na 2 CO 3 aerosols, the ERHs of Na 2 CO 3 are 51.8%-42.3% and 57.1%-42.3%, respectively. While β-alanine crystal appears from 62.7% RH for 2:1 and 59.4% RH for both 1:1 and 1:2 particles and lasts to driest state. On hydration, the DRH is 44.7%-75.2% for Na 2 CO 3 with the OIR of 1:1 and 44.7%-69.0% for 1:2 mixture, and those of β-alanine are 74.8% for 2:1 mixture and 68.9% for two others. After the first dehumidification-humidification, all the water contents decrease despite of constituent fraction. And at ~92% RH, the remaining water contents are 92%, 89% and 82% at ~92% RH, corresponding to OIR of 2:1, 1:1 and 1:2 mixed system, respectively., (Copyright © 2019. Published by Elsevier B.V.)

Rett Syndrome (RTT) is an X-linked neurodevelopmental disorder associated with respiratory abnormalities and, in up to ~40% of patients, with prolongation of the cardiac QT c interval. QT c prolongation calls for cautious use of drugs with a propensity to inhibit hERG channels. The STARS trial has been undertaken to investigate the efficacy of sarizotan, a 5-HT 1A receptor agonist, at correcting RTT respiratory abnormalities. The present study investigated whether sarizotan inhibits hERG potassium channels and prolongs ventricular repolarization. Whole-cell patch-clamp measurements were made at 37 °C from hERG-expressing HEK293 cells. Docking analysis was conducted using a recent cryo-EM structure of hERG. Sarizotan was a potent inhibitor of hERG current (I hERG ; IC 50 of 183 nM) and of native ventricular I Kr from guinea-pig ventricular myocytes. 100 nM and 1 μM sarizotan prolonged ventricular action potential (AP) duration (APD 90 ) by 14.1 ± 3.3% (n = 6) and 29.8 ± 3.1% (n = 5) respectively and promoted AP triangulation. High affinity I hERG inhibition by sarizotan was contingent upon channel gating and intact inactivation. Mutagenesis experiments and docking analysis implicated F557, S624 and Y652 residues in sarizotan binding, with weaker contribution from F656. In conclusion, sarizotan inhibits I Kr /I hERG , accessing key binding residues on channel gating. This action and consequent ventricular AP prolongation occur at concentrations relevant to those proposed to treat breathing dysrhythmia in RTT. Sarizotan should only be used in RTT patients with careful evaluation of risk factors for QT c prolongation., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cardiac potassium channels encoded by human ether-à-go-go-related gene ( hERG ) are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high-affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug-binding site of the hERG pore. hERG current ( I hERG ) recorded at physiological temperature from HEK293 cells was inhibited with an IC 50 of 35.6 nm with time and voltage dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold, respectively, whereas T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold, respectively). The S5 helix F557L mutation decreased potency 10-fold, and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on Phe-656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize Phe-656 side chain orientations compatible with high-affinity block., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)

Introduction: Short QT Syndrome (SQTS) is a rare but dangerous condition characterised by abbreviated repolarisation, atrial and ventricular arrhythmias and risk of sudden death. Implantable cardioverter defibrillators (ICDs) are a first line protection against sudden death, but adjunct pharmacology is beneficial and desirable. Areas covered: The genetic basis for genotyped SQTS variants (SQT1-SQT8) and evidence for arrhythmia substrates from experimental and simulation studies are discussed. The main ion channel/transporter targets for antiarrhythmic pharmacology are considered in respect of potential genotype-specific and non-specific treatments for the syndrome. Expert opinion: Potassium channel blockade is valuable for restoring repolarisation and QT interval, though genotype-specific limitations exist in the use of some K + channel inhibitors. A combination of K + current inhibition during the action potential plateau, with sodium channel inhibition that collectively result in delaying repolarisation and post-repolarisation refractoriness is likely to be valuable in prolonging effective refractory period and wavelength for re-entry. Genotype-specific K + channel inhibition is limited by a lack of targeted inhibitors in clinical use, though experimentally available selective inhibitors now exist. The relatively low proportion of successfully genotyped cases justifies an exome or genome sequencing approach, to reveal new mediators and targets, as demonstrated recently for SLC4A3 in SQT8.

Serine/arginine-protein kinase 1 (SRPK1) regulates alternative splicing of VEGF-A to pro-angiogenic isoforms and SRPK1 inhibition can restore the balance of pro/antiangiogenic isoforms to normal physiological levels. The lack of potency and selectivity of available compounds has limited development of SRPK1 inhibitors, with the control of alternative splicing by splicing factor-specific kinases yet to be translated. We present here compounds that occupy a binding pocket created by the unique helical insert of SRPK1, and trigger a backbone flip in the hinge region, that results in potent (<10 nM) and selective inhibition of SRPK1 kinase activity. Treatment with these inhibitors inhibited SRPK1 activity and phosphorylation of serine/arginine splicing factor 1 (SRSF1), resulting in alternative splicing of VEGF-A from pro-angiogenic to antiangiogenic isoforms. This property resulted in potent inhibition of blood vessel growth in models of choroidal angiogenesis in vivo. This work identifies tool compounds for splice isoform selective targeting of pro-angiogenic VEGF, which may lead to new therapeutic strategies for a diversity of diseases where dysfunctional splicing drives disease development.

Potassium channels encoded by human ether-à-go-go-related gene (hERG) mediate the cardiac rapid delayed rectifier K(+) current (IKr), which participates in ventricular repolarization and has a protective role against unwanted premature stimuli late in repolarization and early in diastole. Ionic current carried by hERG channels (IhERG) is known to exhibit a paradoxical dependence on external potassium concentration ([K(+)]e), but effects of acute [K(+)]e changes on the response of IhERG to premature stimulation have not been characterized. Whole-cell patch-clamp measurements of hERG current were made at 37°C from hERG channels expressed in HEK293 cells. Under conventional voltage-clamp, both wild-type (WT) and S624A pore-mutant IhERG during depolarization to +20 mV and subsequent repolarization to -40 mV were decreased when superfusate [K(+)]e was decreased from 4 to 1 mmol/L. When [K(+)]e was increased from 4 to 10 mmol/L, pulse current was increased and tail IhERG was decreased. Increasing [K(+)]e produced a +10 mV shift in voltage-dependent inactivation of WT IhERG and slowed inactivation time course, while lowering [K(+)]e from 4 to 1 mmol/L produced little change in inactivation voltage dependence, but accelerated inactivation time course. Under action potential (AP) voltage-clamp, lowering [K(+)]e reduced the amplitude of IhERG during the AP and suppressed the maximal IhERG response to premature stimuli. Raising [K(+)]e increased IhERG early during the AP and augmented the IhERG response to premature stimuli. Our results are suggestive that during hypokalemia not only is the contribution of IKr to ventricular repolarization reduced but its ability to protect against unwanted premature stimuli also becomes impaired., (© 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.)

The antianginal drug ranolazine, which combines inhibitory actions on rapid and sustained sodium currents with inhibition of the hERG/IKr potassium channel, shows promise as an antiarrhythmic agent. This study investigated the structural basis of hERG block by ranolazine, with lidocaine used as a low potency, structurally similar comparator. Recordings of hERG current (IhERG) were made from cell lines expressing wild-type (WT) or mutant hERG channels. Docking simulations were performed using homology models built on MthK and KvAP templates. In conventional voltage clamp, ranolazine inhibited IhERG with an IC50 of 8.03μM; peak IhERG during ventricular action potential clamp was inhibited ~62% at 10μM. The IC50 values for ranolazine inhibition of the S620T inactivation deficient and N588K attenuated inactivation mutants were respectively ~73-fold and ~15-fold that for WT IhERG. Mutations near the bottom of the selectivity filter (V625A, S624A, T623A) exhibited IC50s between ~8 and 19-fold that for WT IhERG, whilst the Y652A and F656A S6 mutations had IC50s ~22-fold and 53-fold WT controls. Low potency lidocaine was comparatively insensitive to both pore helix and S6 mutations, but was sensitive to direction of K(+) flux and particularly to loss of inactivation, with an IC50 for S620T-hERG ~49-fold that for WT IhERG. Docking simulations indicated that the larger size of ranolazine gives it potential for a greater range of interactions with hERG pore side chains compared to lidocaine, in particular enabling interaction of its two aromatic groups with side chains of both Y652 and F656. The N588K mutation is responsible for the SQT1 variant of short QT syndrome and our data suggest that ranolazine is unlikely to be effective against IKr/hERG in SQT1 patients., (Copyright © 2014. Published by Elsevier Ltd.)

human Ether-à-go-go-Related Gene (hERG) encodes the pore-forming subunit of cardiac rapid delayed rectifier K(+) current (I Kr) channels, which play important roles in ventricular repolarization, in protecting the myocardium from unwanted premature stimuli, and in drug-induced Long QT Syndrome (LQTS). KCNE1, a small transmembrane protein, can coassemble with hERG. However, it is not known how KCNE1 variants influence the channel's response to premature stimuli or if they influence the sensitivity of hERG to pharmacological inhibition. Accordingly, whole-cell patch-clamp measurements of hERG current (I hERG) were made at 37°C from hERG channels coexpressed with either wild-type (WT) KCNE1 or with one of three KCNE1 variants (A8V, D76N, and D85N). Under both conventional voltage clamp and ventricular action potential (AP) clamp, the amplitude of I hERG was smaller for A8V, D76N, and D85N KCNE1 + hERG than for WT KCNE1 + hERG. Using paired AP commands, with the second AP waveform applied at varying time intervals following the first to mimic premature ventricular excitation, the response of I hERG carried by each KCNE1 variant was reduced compared to that with WT KCNE1 + hERG. The I hERG blocking potency of the antiarrhythmic drug quinidine was similar between WT KCNE1 and the three KCNE1 variants. However, the I hERG inhibitory potency of the antibiotic clarithromycin and of the prokinetic drug cisapride was altered by KCNE1 variants. These results demonstrate that naturally occurring KCNE1 variants can reduce the response of hERG channels to premature excitation and also alter the sensitivity of hERG channels to inhibition by some drugs linked to acquired LQTS.

Purpose: Exudative AMD (wet AMD) is treated by monthly injection into the eye of anti-VEGF proteins. VEGF is alternatively spliced to produce numerous isoforms that differ in angiogenic activity. Serine-rich protein kinase-1 (SRPK1) has been identified as a regulator of pro-angiogenic VEGF splicing by phosphorylating serine-rich splicing factor-1 (SRSF1), which binds to VEGF pre-mRNA. We tested the hypothesis that topical (eye drop) SRPK1-selective inhibitors could be generated that reduce pro-angiogenic isoforms, and prevent choroidal neovascularization in vivo., Methods: Novel inhibitors were tested for SRPK inhibition in vitro, pro-angiogenic VEGF production in RPE cells by PCR and ELISA, and for inhibition of choroidal neovascularisation in mice and rats., Results: A novel disubstituted furan inhibitor was selective for the SRPK family of kinases and reduced expression of pro-angiogenic but not antiangiogenic VEGF isoforms. This inhibitor and previously identified SRPK inhibitors significantly reduced choroidal neovascularisation in vivo. Topical administration of SRPK inhibitors dose-dependently blocked CNV with an EC50 of 9 μM., Conclusions: These results indicate that novel SRPK1 selective inhibitors could be a potentially novel topical (eye drop) therapeutic for wet AMD.

Background and Purpose: KB-R7943 is an isothiourea derivative that is used widely as a pharmacological inhibitor of sodium-calcium exchange (NCX) in experiments on cardiac and other tissue types. This study investigated KB-R7943 inhibition of hERG (human ether-à-go-go-related gene) K(+) channels that underpin the cardiac rapid delayed rectifier potassium current, I(Kr) ., Experimental Approach: Whole-cell patch-clamp measurements were made of hERG current (I(hERG) ) carried by wild-type or mutant hERG channels and of native rabbit ventricular I(Kr) . Docking simulations utilized a hERG homology model built on a MthK-based template., Key Results: KB-R7943 inhibited both I(hERG) and native I(Kr) rapidly on membrane depolarization with IC(50) values of ∼89 and ∼120 nM, respectively, for current tails at -40 mV following depolarizing voltage commands to +20 mV. Marked I(hERG) inhibition also occurred under ventricular action potential voltage clamp. I(hERG) inhibition by KB-R7943 exhibited both time- and voltage-dependence but showed no preference for inactivated over activated channels. Results of alanine mutagenesis and docking simulations indicate that KB-R7943 can bind to a pocket formed of the side chains of aromatic residues Y652 and F656, with the compound's nitrobenzyl group orientated towards the cytoplasmic side of the channel pore. The structurally related NCX inhibitor SN-6 also inhibited I(hERG) , but with a markedly reduced potency., Conclusions and Implications: KB-R7943 inhibits I(hERG) /I(Kr) with a potency that exceeds that reported previously for acute cardiac NCX inhibition. Our results also support the feasibility of benzyloxyphenyl-containing NCX inhibitors with reduced potential, in comparison with KB-R7943, to inhibit hERG., (© 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.)