Morten Grunnet

Background and Purpose: Small conductance calcium‐activated potassium (K_Ca2) channels represent a promising atrial‐selective target for treatment of atrial fibrillation. Here, we establish the mechanism of K_Ca2 channel inhibition by the new compound AP14145.Experimental Approach: Using site‐directed mutagenesis, binding determinants for AP14145 inhibition were explored. AP14145 selectivity and mechanism of action were investigated by patch‐clamp recordings of heterologously expressed K_Ca2 channels. The biological efficacy of AP14145 was assessed by measuring atrial effective refractory period (AERP) prolongation in anaesthetized rats, and a beam walk test was performed in mice to determine acute CNS‐related effects of the drug.Key Results: AP14145 was found to be an equipotent negative allosteric modulator of K_Ca2.2 and K_Ca2.3 channels (IC₅₀ = 1.1 ± 0.3 μM). The presence of AP14145 (10 μM) increased the EC₅₀ of Ca²⁺ on K_Ca2.3 channels from 0.36 ± 0.02 to 1.2 ± 0.1 μM. The inhibitory effect strongly depended on two amino acids, S508 and A533 in the channel. AP14145 concentration‐dependently prolonged AERP in rats. Moreover, AP14145 (10 mg·kg⁻¹) did not trigger any apparent CNS effects in mice.Conclusions and Implications: AP14145 is a negative allosteric modulator of K_Ca2.2 and K_Ca2.3 channels that shifted the calcium dependence of channel activation, an effect strongly dependent on two identified amino acids. AP14145 prolonged AERP in rats and did not trigger any acute CNS effects in mice. The understanding of how K_Ca2 channels are inhibited, at the molecular level, will help further development of drugs targeting K_Ca2 channels.

Na_v1.1 (SCN1A) channels primarily located in gamma‐aminobutyric acid (GABA)ergic fast‐spiking interneurons are pivotal for action potential generation and propagation in these neurons. Inappropriate function of fast‐spiking interneurons, leading to disinhibition of pyramidal cells and network desynchronization, correlates with decreased cognitive capability. Further, reduced functionality of Na_v1.1 channels is linked to various diseases in the central nervous system. There is, at present, however no subtype selective pharmacological activators of Na_v1.1 channels available for studying pharmacological modulation of interneuron function. In the current study, we identified a small molecule Na_v1.1 activator, 3‐amino‐5‐(4‐methoxyphenyl)thiophene‐2‐carboxamide, named AA43279, and provided an in vitro to in vivo characterization of the compound. In HEK‐293 cells expressing human Na_v1.1 channels, AA43279 increased the Na_v1.1‐mediated current in a concentration‐dependent manner mainly by impairing the fast inactivation kinetics of the channels. In rat hippocampal brain slices, AA43279 increased the firing activity of parvalbumin‐expressing, fast‐spiking GABAergic interneurons and increased the spontaneous inhibitory post‐synaptic currents (sIPSCs) recorded from pyramidal neurons. When tested in vivo, AA43279 had anti‐convulsive properties in the maximal electroshock seizure threshold test. AA43279 was tested for off‐target effects on 72 different proteins, including Na_v1.2, Na_v1.4, Na_v1.5, Na_v1.6 and Na_v1.7 and exhibited reasonable selectivity. Taken together, AA43279 might constitute a valuable tool compound for revealing biological functions of Na_v1.1 channels.

Background and Purpose: The PDE enzymes (PDE1–11) hydrolyse and thus inactivate cyclic nucleotides and are important in the regulation of the cardiovascular system. Here,we have investigated the effects on the cardiovascular system, of two novel selective PDE1 inhibitors, Lu AF41228 and Lu AF58027.Experimental Approach: We used rat mesenteric small arteries (internal diameters of 200–300 μm), RT‐PCR and measured isometric wall tension. Effects of Lu AF41228 and Lu AF58027 on heart rate and BP were assessed in both anaesthetized and conscious male rats.Key Results: Nanomolar concentrations of Lu AF41228 and Lu AF58027 inhibited PDE1A, PDE1B and PDE1C enzyme activity, while micromolar concentrations were required to observe inhibitory effects at other PDEs. RT‐PCR revealed expression of PDE1A, PDE1B and PDE1C in rat brain, heart and aorta, but only PDE1A and PDE1B in mesenteric arteries. In rat isolated mesenteric arteries contracted with phenylephrine or U46619, Lu AF41228 and Lu AF58027 induced concentration‐dependent relaxations which were markedly reduced by inhibitors of guanylate cyclase, ODQ, and adenylate cyclase, SQ22536, and in preparations without endothelium. In anaesthetized rats, Lu AF41228 and Lu AF58027 dose‐dependently lowered mean BP and increased heart rate. In conscious rats with telemetric pressure transducers, repeated dosing with Lu AF41228 lowered mean arterial BP 10–15 mmHg and increased heart rate.Conclusions and Implications: These novel PDE1 inhibitors induce vasodilation and lower BP, suggesting a potential use of these vasodilators in the treatment of hypertension and vasospasm.

Introduction: SK channels have functional importance in the cardiac atrium of many species, including humans. Pharmacological blockage of SK channels has been reported to be antiarrhythmic in animal models of atrial fibrillation; however, the exact antiarrhythmic mechanism of SK channel inhibition remains unclear. Objectives: We speculated that together with a direct inhibition of repolarizing SK current, the previously observed depolarization of the atrial resting membrane potential (RMP) after SK channel inhibition reduces sodium channel availability, thereby prolonging the effective refractory period and slowing the conduction velocity (CV). We therefore aimed at elucidating these properties of SK channel inhibition and the underlying antiarrhythmic mechanisms using microelectrode action potential (AP) recordings and CV measurements in isolated rat atrium. Automated patch clamping and two-electrode voltage clamp were used to access I_Na and I_K,ACh, respectively. Results: The SK channel inhibitor N-(pyridin-2-yl)-4-(pyridin-2-yl)thiazol-2-amine (ICA) exhibited antiarrhythmic effects. ICA prevented electrically induced runs of atrial fibrillation in the isolated right atrium and induced atrial postrepolarization refractoriness and depolarized RMP. Moreover, ICA (1–10 μM) was found to slow CV; however, because of a marked prolongation of effective refractory period, the calculated wavelength was increased. Furthermore, at increased pacing frequencies, SK channel inhibition by ICA (10–30 μM) demonstrated prominent depression of other sodium channel–dependent parameters. ICA did not inhibit I_K,ACh, but at concentrations above 10 μM, ICA use dependently inhibited I_Na. Conclusions: SK channel inhibition modulates multiple parameters of AP. It prolongs the AP duration and shifts the RMP towards more depolarized potentials through direct I_SK block. This indirectly leads to sodium channel inhibition through accumulation of state dependently inactivated channels, which ultimately slows conduction and decreases excitability. However, a contribution from a direct sodium channel inhibition cannot be ruled. We here propose that the primary antiarrhythmic mechanism of SK channel inhibition is through direct potassium channel block and through indirect sodium channel inhibition.

Background: Mutations in SCN5A can result in both long QT type 3 (LQT3) and Brugada syndrome (BrS), and a few mutations have been found to have an overlapping phenotype. Long QT syndrome is characterized by prolonged QT interval, and a prerequisite for a BrS diagnosis is ST elevation in the right precordial leads of the electrocardiogram. Methods and Results: In a Danish family suffering from long QT syndrome, a novel missense mutation in SCN5A, changing a leucine residue into a glutamine residue at position 1786 (L1786Q), was found to be present in heterozygous form co-segregating with prolonged QT interval. The proband presented with an aborted cardiac arrest, and his mother died suddenly and unexpectedly at the age of 65. Flecainide treatment revealed coved ST elevation in all mutation carriers. Electrophysiological investigations of the mutant in HEK293 cells indicated a reduced peak current, a negative shift in inactivation properties and a positive shift in activation properties, compatible with BrS. Furthermore, the sustained (INa,late) tetrodotoxin-sensitive sodium current was found to be drastically increased, explaining the association between the mutation and LQT syndrome. Conclusions: The L1786Q mutation is associated with a combined LQT3 and concealed BrS phenotype explained by gating characteristics of the mutated ion channel protein. Hence, sodium channel blockade should be considered in clinical evaluation of apparent LQT3 patients. (Circ J 2014; 78: 1136–1143)

BACKGROUND AND PURPOSE: Positive allosteric modulation of α4β2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively.EXPERIMENTAL APPROACH: Effects of NS9283 were evaluated in vitro using fluorescence‐based Ca²⁺ imaging and electrophysiological voltage clamp experiments in Xenopus oocytes, mammalian cells and thalamocortical neurons. In vivo the compound was tested in models covering a range of cognitive domains in mice and rats.KEY RESULTS: NS9283 was shown to increase agonist‐evoked response amplitude of (α4)₃(β2)₂ nACh receptors in electrophysiology paradigms. (α2)₃(β2)₂, (α2)₃(β4)₂ and (α4)₃(β4)₂ were modulated to comparable extents, but no effects were detected at α3‐containing or any 2α : 3β stoichiometry nACh receptors. Native nACh receptors in thalamocortical neurons similarly displayed DHβE‐sensitive currents that were receptive to modulation. NS9283 had favourable effects on sensory information processing, as shown by reversal of PCP‐disrupted pre‐pulse inhibition. NS9283 further improved performance in a rat model of episodic memory (social recognition), a rat model of sustained attention (five‐choice serial reaction time task) and a rat model of reference memory (Morris water maze). Importantly, the effects in the Morris water maze could be fully reversed with mecamylamine, a blocker of nACh receptors.CONCLUSIONS AND IMPLICATIONS: These results provide compelling evidence that positive allosteric modulators acting at the (α4)₃(β2)₂ nACh receptors can augment activity across a broad range of cognitive domains, and that α4β2 nACh receptor allosteric modulation therefore constitutes a promising therapeutic approach to symptomatic treatment of cognitive impairment.

The hERG (Kv11.1) potassium channel underlies cardiac I(Kr) and is important for cardiac repolarization. Recently, hERG agonists have emerged as potential antiarrhythmic drugs. As modulation of outward potassium currents has been suggested to modulate cardiac conduction, we tested the hypothesis that pharmacological activation of I(Kr) results in impaired cardiac conduction. Cardiac conduction was assessed in Langendorff-perfused guinea pig hearts. Application of the hERG agonist NS3623 (10 microM) prolonged the QRS rate dependently. A significant prolongation (16 +/- 6%) was observed at short basic cycle length (BCL 90 ms) but not at longer cycle lengths (BCL 250 ms). The effect could be reversed by the I(Kr) blocker E4031 (1 microM). While partial I(Na) inhibition with flecainide (1 microM) alone prolonged the QRS (34 +/- 3%, BCL 250 ms), the QRS was further prolonged by 19 +/- 2% when NS3623 was added in the presence of flecainide. These data suggest that the effect of NS3623 was dependent on sodium channel availability. Surprisingly, in the presence of the voltage sensitive dye di-4-ANEPPS a similar potentiation of the effect of NS3623 was observed. With di-4-ANEPPS, NS3623 prolonged the QRS significantly (26 +/- 4%, BCL 250 ms) compared to control with a corresponding decrease in conduction velocity. Pharmacological activation of I(Kr) by the hERG agonist NS3623 impairs cardiac conduction. The effect is dependent on sodium channel availability. These findings suggest a role for I(Kr) in modulating cardiac conduction and may have implications for the use of hERG agonists as antiarrhythmic drugs.