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Author summaryThe neurovascular coupling (NVC) is the basis for functional magnetic resonance imaging (fMRI), since the NVC connects neural activity with the observed hemodynamic changes. This connection is highly complex, which warrants a model-based analysis. However, even though NVC-data from several species and many relevant variables are available, a mathematical model for all these data is still missing. Herein, we combine experimental data from mice, monkeys, and humans, to develop a comprehensive model for NVC. Importantly, our new approach to modelling propagates the qualitative insights from each species to the subsequent analysis of data from other species. In mice, we unravel the role of different neuronal sub-populations when producing a biphasic response to prolonged sensory stimulations. The qualitative role of these sub-populations is preserved when analysing primate data. These primate data add knowledge on the interplay between local field potential (LFP) and vascular changes. Similarly, these pre-clinical qualitative insights are propagated to analysis of human data, which contain additional insights regarding blood flow and volume in arterioles and venules, during both positive and negative responses. This work illustrates how data with complementary information from different species can be combined, so that qualitative insights from animals are preserved in the quantitative analysis of human data. Neurons regulate the activity of blood vessels through the neurovascular coupling (NVC). A detailed understanding of the NVC is critical for understanding data from functional imaging techniques of the brain. Many aspects of the NVC have been studied both experimentally and using mathematical models; various combinations of blood volume and flow, local field potential (LFP), hemoglobin level, blood oxygenation level-dependent response (BOLD), and optogenetics have been measured and modeled in rodents, primates, or humans. However, these data have not, Funding Agencies|Swedish Research Council [IDs: 2018-05418, 2018-03319, 2018-03391]; CENIIT, Center for Industrial Information Technology; Swedish Foundation for Strategic Research [F2019-0010]; SciLifeLab National COVID-19 Research Program - Knut and Alice Wallenberg Foundation; Swedish Fund for Research without Animal Experiments [2020.0182]; ELLIIT, Excellence Center at Linkoeping - Lund in Information Technology [777107]; VINNOVA (VisualSweden); VINNOVA; H2020 project PRECISE4Q, Personalised Medicine by Predictive Modelling in Stroke for better Quality of Life; MedTech4Health; SweLife; Swedish Brain Foundation; [ITM17-0245]; [2020-A12]; [15.09]; [2020-04711]

Voltage-gated potassium (K-V) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker KV channel increased resin-acid-induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker KV channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid-binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1-S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner., QC 20210409

Objective About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hK(V)) channel hK(V)7.2/7.3 is a validated antiseizure target for compounds that activate this channel. In a previous study we have shown that resin acid derivatives can activate the hK(V)7.2/7.3 channel. In this study we investigated if these channel activators have the potential to be developed into a new type of antiseizure drug. Thus we examined their structure-activity relationships and the site of action on the hK(V)7.2/7.3 channel, if they have unwanted cardiac and cardiovascular effects, and their potential antiseizure effect.Methods Ion channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model.Results Fourteen resin acid derivatives were tested on hK(V)7.2/7.3. The most efficient channel activators were halogenated and had a permanently negatively charged sulfonyl group. The compounds did not bind to the sites of other hK(V)7.2/7.3 channel activators, retigabine, or ICA-069673. Instead, they interacted with the most extracellular gating charge of the S4 voltage-sensing helix, and the effects are consistent with an electrostatic mechanism. The compounds altered the voltage dependence of hK(V)7.4, but in contrast to retigabine, there were no effects on the maximum conductance. Consistent with these data, the compounds had less smooth muscle-relaxing effect than retigabine. The compounds had almost no effect on the voltage dependence of hK(V)11.1, hNa(V)1.5, or hCa(V)1.2, or on the amplitude of hK(V)11.1. Finally, several resin acid derivatives had clear antiseizure effects in a zebrafish-larvae model.

Objective About one third of all patients with epilepsy have pharmacoresistant seizures. Thus there is a need for better pharmacological treatments. The human voltage-gated potassium (hK(V)) channel hK(V)7.2/7.3 is a validated antiseizure target for compounds that activate this channel. In a previous study we have shown that resin acid derivatives can activate the hK(V)7.2/7.3 channel. In this study we investigated if these channel activators have the potential to be developed into a new type of antiseizure drug. Thus we examined their structure-activity relationships and the site of action on the hK(V)7.2/7.3 channel, if they have unwanted cardiac and cardiovascular effects, and their potential antiseizure effect. Methods Ion channels were expressed in Xenopus oocytes or mammalian cell lines and explored with two-electrode voltage-clamp or automated patch-clamp techniques. Unwanted vascular side effects were investigated with isometric tension recordings. Antiseizure activity was studied in an electrophysiological zebrafish-larvae model. Results Fourteen resin acid derivatives were tested on hK(V)7.2/7.3. The most efficient channel activators were halogenated and had a permanently negatively charged sulfonyl group. The compounds did not bind to the sites of other hK(V)7.2/7.3 channel activators, retigabine, or ICA-069673. Instead, they interacted with the most extracellular gating charge of the S4 voltage-sensing helix, and the effects are consistent with an electrostatic mechanism. The compounds altered the voltage dependence of hK(V)7.4, but in contrast to retigabine, there were no effects on the maximum conductance. Consistent with these data, the compounds had less smooth muscle-relaxing effect than retigabine. The compounds had almost no effect on the voltage dependence of hK(V)11.1, hNa(V)1.5, or hCa(V)1.2, or on the amplitude of hK(V)11.1. Finally, several resin acid derivatives had clear antiseizure effects in a zebrafish-larvae model. Significance The desc, Funding Agencies|Det Frie ForskningsradDet Frie Forskningsrad (DFF) [9039-00409B]; Hjarnfonden [FO2019-0247]; VetenskapsradetSwedish Research Council [2016-02615]; LundbeckfondenLundbeckfonden [R323-2018-3674]; Novo Nordisk FondenNovo Nordisk Foundation [NNF18OC0033216]; Swedish Heart-Lung FoundationSwedish Heart-Lung Foundation [20180404]

Voltage-gated potassium (K-V) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker KV channel increased resin-acid-induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker KV channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid-binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1-S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner., Funding Agencies|Swedish Research CouncilSwedish Research CouncilEuropean Commission [20180404, 2018-04905]; Gustafsson Foundation, and Science for Life Laboratory; Swedish Brain Foundation [FO2019-0247]; Swedish Heart-Lung FoundationSwedish Heart-Lung Foundation [20180404]

The opening and closing of voltage-gated ion channels are regulated by voltage sensors coupled to a gate that controls the ion flux across the cellular membrane. Modulation of any part of gating constitutes an entry point for pharmacologically regulating channel function. Here, we report on the discovery of a large family of warfarin-like compounds that open the two voltage-gated type 1 potassium (KV1) channels KV1.5 and Shaker, but not the related KV2-, KV4-, or KV7-type channels. These negatively charged compounds bind in the open state to positively charged arginines and lysines between the intracellular ends of the voltage-sensor domains and the pore domain. This mechanism of action resembles that of endogenous channel-opening lipids and opens up an avenue for the development of ion-channel modulators., Funding agencies: Swedish Research CouncilSwedish Research Council [2016-02615]; Swedish Heart-Lung FoundationSwedish Heart-Lung Foundation [20150672]; Swedish Brain Foundation [2016-0326]

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Voltage-gated ion channels are key molecules for the generation of cellular electrical excitability. Many pharmaceutical drugs target these channels by blocking their ion-conducting pore, but in many cases, channel-opening compounds would be more beneficial. Here, to search for new channel-opening compounds, we screen 18,000 compounds with high-throughput patch-clamp technology and find several potassium-channel openers that share a distinct biaryl-sulfonamide motif. Our data suggest that the negatively charged variants of these compounds bind to the top of the voltage-sensor domain, between transmembrane segments 3 and 4, to open the channel. Although we show here that biaryl-sulfonamide compounds open a potassium channel, they have also been reported to block sodium and calcium channels. However, because they inactivate voltage-gated sodium channels by promoting activation of one voltage sensor, we suggest that, despite different effects on the channel gates, the biaryl-sulfonamide motif is a general ion-channel activator motif. Because these compounds block action potential-generating sodium and calcium channels and open an action potential-dampening potassium channel, they should have a high propensity to reduce excitability. This opens up the possibility to build new excitability-reducing pharmaceutical drugs from the biaryl-sulfonamide scaffold., Funding Agencies|Swedish Research Council [2016-02615]; Swedish Heart-Lung Foundation [20150672]; Swedish Brain Foundation [2016-0326]; Swedish Society for Medical Research

Dehydroabietic acid (DHAA) is a naturally occurring component of pine resin that was recently shown to open voltage-gated potassium (KV) channels. The hydrophobic part of DHAA anchors the compound near the channel’s positively charged voltage sensor in a pocket between the channel and the lipid membrane. The negatively charged carboxyl group exerts an electrostatic effect on the channel’s voltage sensor, leading to the channel opening. In this study, we show that the channel-opening effect increases as the length of the carboxyl-group stalk is extended until a critical length of three atoms is reached. Longer stalks render the compounds noneffective. This critical distance is consistent with a simple electrostatic model in which the charge location depends on the stalk length. By combining an effective anchor with the optimal stalk length, we create a compound that opens the human KV7.2/7.3 (M type) potassium channel at a concentration of 1 µM. These results suggest that a stalk between the anchor and the effector group is a powerful way of increasing the potency of a channel-opening drug., Funding agencies: Swedish Research Council [2016-02615]; Swedish Heart-Lung Foundation [20150672]; Swedish Brain Foundation [2016-0326]

AimAtrial fibrillation is the most common persistent cardiac arrhythmia, and it is not well controlled by present drugs. Because some resin acids open voltage-gated potassium channels and reduce neuronal excitability, we explored the effects of the resin acid isopimaric acid (IPA) on action potentials and ion currents in cardiomyocytes. MethodsSpontaneously beating mouse atrial HL-1 cells were investigated with the whole-cell patch-clamp technique. Results1-25 mol L-1 IPA reduced the action potential frequency by up to 50%. The effect of IPA on six different voltage-gated ion channels was investigated; most voltage-dependent parameters of ion channel gating were shifted in the negative direction along the voltage axis, consistent with a hypothesis that a lipophilic and negatively charged compound binds to the lipid membrane close to the positively charged voltage sensor of the ion channels. The major finding was that IPA inactivated sodium channels and L- and T-type calcium channels and activated the rapidly activating potassium channel and the transient outward potassium channel. Computer simulations of IPA effects on all of the ion currents were consistent with a reduced excitability, and they also showed that effects on the Na channel played the largest role to reduce the action potential frequency. Finally, induced arrhythmia in the HL-1 cells was reversed by IPA. ConclusionLow concentrations of IPA reduced the action potential frequency and restored regular firing by altering the voltage dependencies of several voltage-gated ion channels. These findings can form the basis for a new pharmacological strategy to treat atrial fibrillation., Funding Agencies|Swedish Research Council; Swedish Heart-Lung Foundation; Swedish Brain Foundation; ALF