Sara Inga Liin

Synthetic cannabinoid receptor agonists (SCRAs) are a large group of structurally diverse designer drugs (analogues of controlled substances) associated with intense and sometimes fatal intoxication. Cardiac symptoms, including tachycardia and arrhythmia, are common consequences of SCRA consumption. However, little is known about the mechanisms through which SCRAs may perturb cardiac rhythm. Here, we used electrophysiological techniques to screen 36 SCRAs on two ion channels responsible for cardiomyocyte repolarization, hERG (also called K _V 11.1) and K _V 7.1/KCNE1. We report that the majority of tested SCRAs inhibited hERG, primarily by reducing channel conductance, and some also inhibited K _V 7.1/KCNE1. In silico data suggest that SCRAs may use both a known drug binding site in the central cavity of the hERG channel, shared by established hERG blockers like astemizole, and a recently identified side pocket site. Experimental and in silico data suggest SCRA structural features associated with prominent inhibitory effects on hERG, with chemical moieties allowing bond formation and/or the ability to fit into binding sites being important. Structure-activity relationships (SAR) for SCRA effects on hERG, K _V 7.1/KCNE1 and the cannabinoid receptor 1 (CB ₁ ) varied, demonstrating the need to assess SCRA effects on multiple potential targets. In conclusion, we found SCRAs to be inhibitors of cardiac voltage-gated potassium channels important for cardiomyocyte repolarization, highlighting the importance of more extensive investigation of SCRAs on cardiac function.

Although widely prescribed globally, benzodiazepines (BZDs) are commonly misused and involved in a high rate of drug-related deaths worldwide. Novel chemically similar compounds, designer BZDs (DBZDs), have emerged on the recreational market with little information available on their pharmacodynamic effects. An in vitro α ₁ β ₂ γ ₂ GABA _A assay was used to determine the activity and explore structure-activity relationships of 42 DBZDs plus 11 prescription BZDs. Their interaction with the BZD α ⁺ /γ ₂ ^– interface was explored using the BZD antagonist flumazenil. Most of the D/BZDs were positive allosteric modulators, but 4’-chlorodiazepam and 4’-fluorodiazepam were negative allosteric modulators, although the activity was not antagonized by flumazenil. All metabolites tested were active. This data informs the interpretation of intoxications, harm reduction measures, and drugs legislation for DBZDs; demonstrates the importance of testing new drugs as they emerge on the recreational market; and offers insights into how BZD chemical modifications alter GABA _A pharmacodynamics.

KCNQ4 -encoded K _V 7.4 voltage-gated potassium channels are expressed in hair-cells of the inner ear. Loss-of-function variants in KCNQ4 cause non-syndromic progressive hearing loss (DFNA2). K _V 7.4 pore opening requires voltage-dependent conformational changes (activation) of the voltage-sensor domains (VSDs); however, how fast charge displacement during VSD activation is coupled to slow channel opening is currently unclear. Here, we optically tracked K _V 7.4 VSD activation with voltage-clamp fluorometry, leveraging two fluorophores and pulsed excitation, and found that VSD activation comprises several voltage-dependent transitions, some with kinetics and voltage-dependence matching those of channel opening and closing. The DFNA2-causing R216H mutation impairs VSD movement and channel opening by destabilizing the active VSD configuration, a result confirmed by molecular dynamics simulations. We propose that the K _V 7.4 VSD activates in two steps: a fast movement representing a first transition to an intermediate activation state, followed by slower component(s) that fully activate the VSD and drive channel opening.

Synthetic cannabinoid receptor agonists (SCRAs) is a large group of structurally diverse designer drugs (analogues of controlled substances) associated with intense and sometimes fatal intoxication. Cardiac symptoms including tachycardia and arrhythmia are common consequences of SCRA consumption. However, little is known about the mechanisms through which SCRAs may perturb cardiac rhythm. Here we used electrophysiological techniques to screen 36 SCRAs on two ion channels responsible for cardiomyocyte repolarization, hERG (also called KV11.1) and KV7.1/KCNE1. We report that the majority of tested SCRAs inhibited hERG, primarily by reducing channel conductance, and some also inhibited KV7.1/KCNE1. In silico data suggests that SCRAs use a known drug binding site in the pore of the hERG channel, shared by established hERG blockers like astemizole, where a planar SCRA molecule lays perpendicular to the ion conducting pathway. Experimental and in silico data identify SCRA structural features associated with prominent inhibitory effects on hERG, with chemical moieties allowing bond formation and/or the ability to fit into the vestibule being important. Structure-activity-relationships for SCRA effects on hERG, Kv7.1/KCNE1 and the cannabinoid receptor 1 (CB1) varied, demonstrating the importance of assessing SCRA effects on multiple potential targets. In conclusion, we found SCRAs to be inhibitors of cardiac voltage-gated potassium channels important for cardiomyocyte repolarization. These results offer mechanistic insight into potentially detrimental SCRA effects on the heart and highlight the urgency of more extensive investigation of SCRAs on cardiac function.

Objective: Variants in GABRG2 , encoding the γ2 subunit of the γ‐aminobutyric acid type A receptor, are linked to epilepsy phenotypes of varying severity, with gain‐of‐function (GoF) variants associated with the more severe phenotypes than loss‐of‐function variants. Here, we provide a comprehensive analysis of the early clinical features, motor, language, and eating abilities, and structural consequences of the recurrent GABRG2 p.(Ala106Thr) GoF variant, aiming to refine genotype–phenotype correlations and deepen the understanding of GoF‐associated GABRG2 disorders. Methods: Individuals were recruited through international collaborations, literature review, and patient advocacy groups. Clinical data were collected via standardized interviews and physician reports. Functional effects were assessed using electrophysiological recordings. Structural modeling was performed using homology‐based approaches to evaluate conformational changes. Results: We collected 23 unrelated individuals harboring the GABRG2 p.(Ala106Thr) variant. Symptoms began with early dysphagia (median onset age = 1 month), hypotonia, seizures (median onset age = 2 months), and hyperkinetic movement disorders (median onset age = 3 months). Epilepsy was present in 91% of the individuals, 71% featuring a developmental and epileptic encephalopathy; 33% of the individuals with epilepsy achieved seizure freedom during the follow‐up. All individuals exhibited moderate to severe cognitive/neurodevelopmental impairment, with profound deficits in motor, language, and other domains. Functional analysis confirmed a GoF effect of the p.(Ala106Thr) variant. By contrast, another variant affecting the same residue, p.(Ala106Pro), which is observed in population databases, was functionally characterized as neutral. Structural modeling indicated that the p.(Ala106Thr) variant may enhance receptor gating by facilitating a hydrogen bond between the extracellular and transmembrane domains, a mechanism not observed with the p.(Ala106Pro) variant. Significance: Although some individuals exhibited severe developmental impairment without epilepsy, our findings demonstrate that the recurrent GABRG2 p.(Ala106Thr) GoF variant is consistently associated with a severe neurodevelopmental phenotype. Despite its profound clinical impact, in silico tools consistently predict this variant as benign, highlighting a critical gap in current predictive models.

Background and aims: Loss-of-function mutations in KCNQ1 and KCNH2 (α-subunits of the slow delayed IKs and rapid delayed IKr-conducting repolarising K+ channels) lead to long QT syndrome type 1 (LQT1) and 2 (LQT2), respectively. These channelopathies present with longer action potential duration (APD) and prolonged QT interval on ECG, which can ultimately lead to deadly arrhythmias. Here we investigated the therapeutic potential of the polyunsaturated fatty acid docosahexaenoyl glycine (DHA-gly) in normalizing APD and QT interval in LQT2 by increasing IKs. Methods: The effects of DHA-gly on electrical and mechanical parameters were assessed in Xenopus laevis oocytes, wild-type (WT), LQT1 (KCNQ1-Y315S) and LQT2 (KCNH2-G628S) transgenic rabbit models, and human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). Results: DHA-gly increased IKs in oocytes and WT ventricular cardiomyocytes (VCMs) in a dose-dependent manner. Consequently, DHA-gly shortened APD in vitro and QT interval ex vivo in WT and LQT2 rabbits, but not in LQT1. However, DHA-gly was unable to reduce arrhythmia formation in LQT2. Beneficial APD/QT shortening effects were accompanied by a detrimental decrease in both cellular and ventricular contraction across all genotypes, including LQT1, which could be due to a shortening in Ca2+ transient duration observed in VCMs and hiPSC-CMs. Conclusions: DHA-gly-induced IKs enhancement shows promising results in shortening APD/QT in LQT2 rabbits, while having no effect on LQT1 (impaired IKs). However, its adverse effect on cardiac contractility, even in LQT1, makes it unsuitable to treat LQTS patients. Our study highlights the importance of considering both electrical and mechanical effects of potential therapeutic compounds prior to clinical translation.

Neuroinflammation and oxidative dysfunction, and their reciprocal interplay, are critically involved in the pathophysiology of chronic neuropathic pain (NeuP). Numerous studies have investigated the crosstalk between inflammatory biomolecules such as cytokines, chemokines, and neuronal cells. However, the impact of immunomodulatory lipoproteins and oxylipins in NeuP pathophysiology is far less explored. Using a combination of techniques, we uncovered altered lipoprotein composition in high-density lipoproteins (HDLs) and low-density lipoproteins (LDLs) with complementary alterations in the plasma profile of oxylipins and cytokines among patients. Lower level of apolipoproteins in patient HDL and 2 isoforms of acute phase serum amyloid A (SAA) with higher levels in patients was found. The constitutively expressed SAA4 was detected in 6 isoforms in patients, but only 2 isoforms were detected in healthy controls. In LDL, lysozyme C and 2 isoforms of SAA were exclusive to patients. Analysis of protein carbonylation showed oxidation of 6 proteins in HDL, of which 3 were unique to patients. No oxidized proteins were observed in LDL. Oxylipin analysis revealed 13 octadecanoids that were significantly downregulated in patients, of which 7 demonstrated significant activating effects on the Kv7.2/7.3 channel, which is anticipated to dampen neuronal signalling in sensory afferents. Among the significant octadecanoids, 9-HODE showed most prominent facilitating effects on Kv7.2/7.3 channel activation. These results present a previously unexplored network of integrated alterations of lipoproteins, octadecanoids, and cytokines in patients suffering from NeuP, indicative of deviant immuno-protective functioning across several biological systems including lipid metabolic processes, inflammation, and Kv7.2/7.3 signalling.

Oestradiol (17β‐E2) is reported to prolong the cardiac action potential duration and QT interval, in part by affecting cardiac ion channels. Previous studies found inhibiting 17β‐E2 effects on the repolarizating cardiac K _V 7.1/KCNE1 channel, or its native current, in heterologous expression systems or tissue from animal species. However, there is variability in reported 17β‐E2 effects and required concentrations. In this work, we aimed to test whether a contributing factor may be different pharmacological profiles of K _V 7.1/KCNE1 channels from different species. To this end, we used the two‐electrode voltage clamp technique to characterize and quantitatively compare the effects of 17β‐E2 on K _V 7.1/KCNE1 channels from guinea pig, zebrafish, and rabbit expressed in Xenopus oocytes. We found that K _V 7.1/KCNE1 of all tested species is inhibited by 17β‐E2, although with species variability in the response. The guinea pig channel responded similar to previous reports for the human channel with a concentration‐dependent reduction in the overall conductance. In contrast, the rabbit channel was sensitive to lower 17β‐E2 concentrations, whereas the zebrafish channel responded with an additional inhibiting effect seen as a shifted voltage dependence of channel opening toward more positive voltages. By testing the 17β‐E2 response of K _V 7.1 alone, and by combining K _V 7.1 and KCNE1 subunits from different species, we conclude that the species variability is not simply dictated by one of the subunits but rather by the K _V 7.1/KCNE1 complex. The species variability in the 17β‐E2 response of K _V 7.1/KCNE1 could be considered when choosing appropriate animal models or interpreting findings from different experimental models.

Background and Purpose: Congenital long QT syndrome (LQTS) involves genetic mutations affecting ion channels, leading to a prolonged QT interval and increased risk of potentially lethal ventricular arrhythmias. Mutations in the genes encoding K_V7.1/KCNE1 are the most frequent, with channel loss‐of‐function contributing to LQTS. The endocannabinoid N‐arachidonoyl‐L‐serine (ARA‐S) has been shown to facilitate activation of wild type K_V7.1/KCNE1 channels and to counteract a prolonged QT interval in isolated guinea pig hearts. In this study, we examine the ability of ARA‐S to facilitate activation of LQTS‐associated mutations, in various regions of the channel, and hence to counteract loss‐of‐function.Experimental Approach: The two‐electrode voltage clamp technique on Xenopus oocytes expressing human K_V7.1/KCNE1 channels was used to investigate the effects of ARA‐S in 20 LQTS type 1‐associated mutations distributed across the channel. Thereafter, different electrophysiology was used to assess ARA‐S effects in mammalian cells.Key Results: ARA‐S enhanced the function of all mutated channels by shifting V₅₀ and increasing current amplitude. However, the magnitude of effect varied, related to whether mutations were in one of the two putative ARA‐S binding sites on the channel as suggested by molecular dynamics simulations. ARA‐S displayed translational potential by facilitating channel opening in mammalian cells and shortening the action potential duration in cardiomyocytes.Conclusions and Implications: This study demonstrates the rescuing capability of ARA‐S on a diverse set of LQTS mutants. These insights may aid in developing drug compounds using ARA‐S sites and mechanisms and guide interpretation of which LQTS mutants respond well to such compounds.

Voltage-gated, potassium-selective K _V 7.4 channels are expressed in the inner ear and are crucial for hair-cell function and survival. Loss-of-function variants of KCNQ4 , the gene encoding K _V 7.4-channel subunits, cause non-syndromic progressive hearing loss (DFNA2). K _V 7.4 opening requires a voltage-dependent conformational change (activation) of the charged voltage-sensor domains (VSDs), and its transduction to the pore. Previously, fast charge displacement was reported during VSD activation at negative potentials, but it is unclear how this is coupled to slow channel opening occurring at more depolarized potentials. Here, we optically tracked K _V 7.4 VSD activation with voltage-clamp fluorometry, leveraging two different fluorophores and pulsed excitation, to thoroughly characterize VSD movements. We found that VSD activation comprises several voltage-dependent transitions, some of which had kinetics and voltage-dependence matching those of channel opening and closing. The deafness-associated mutation R216H, which substitutes a charged amino-acid in the VSD, impaired both VSD movements and channel opening, shifting them towards more depolarized potentials. This suggested that R216H impaired K _V 7.4 function by destabilizing VSD activation. Using molecular dynamics, we found that H216 reduced intramolecular interactions, thus decreasing the stability of an active VSD conformation. We propose that the K _V 7.4 VSD activates in two steps: a fast movement at negative voltages that represents a first transition to an intermediate state of activation; and this is followed by slower, depolarized component that represents subsequent full VSD activation, which drives channel opening.