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- Larsson, Johan, 1990-
(författare)
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Molecular mechanisms of modulation of KV7 channels by polyunsaturated fatty acids and their analogues
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ion channels are membrane proteins that regulate the permeability of ions across the cell membrane. The sequential opening of different types of ion channels produces action potentials in excitable cells. Action potentials are a way for the body to, for example, transmit signals quickly over a long distance.The KV7 family is an important group of voltage-gated potassium channels. Mutations that cause dysfunction in members of the KV7 family are associated with several forms of disease. Compounds that can activate KV7 channels have previously been shown to work as medical treatments. However, the previously available antiepileptic drug retigabine, has been withdrawn due to adverse effects. Thus, there is a need for further development of compounds that target these channels. PUFA and PUFA analogs have previously been demonstrated to activate KV7.1 through an electrostatic mechanism. This thesis investigates new aspects of the interaction between KV7 channels and PUFA-related compounds.The data in this thesis are from human KV7 channels expressed in Xenopus laevis oocytes. The currents produced by the channels expressed in the oocytes have been studied using twoelectrode voltage clamp. Our aim was to study the mechanism for the activation of KV7 channels by PUFA and PUFA analogs. More specifically, we intended to study why the beta subunit KCNE1 abolishes the activating effect of PUFA on KV7.1 and how PUFAs activate KV7.2 and KV7.3. Additionally, we wanted to study aspects that may affect whether these compounds are viable as medical treatments. For instance, whether these compounds can activate channels containing disease-causing mutations and whether we can improve compound selectivity towards certain KV7 channels.In Paper I, we introduce disease-causing mutations found in patients into KV7.1 and KCNE1. The characterization showed that these channels had altered biophysical properties compared to wild type channels. A PUFA analog was found to activate and, to a large degree, restore wild type-like biophysical properties in the mutated channels regardless of the localization of the mutation in the channel.In Paper II, we demonstrate why PUFA is unable to activate KV7.1 co-expressed with beta subunit KCNE1. KCNE1 induces a conformational change of KV7.1 that moves the S5-Phelix loop closer to the PUFA binding site. This causes negative charges of the loop to attract protons that reduce local pH at the PUFA binding site. The decreased local pH leads to protonation of PUFA and the PUFAs therefore lose their negative charge. Thus, PUFA cannot activate KV7.1 when it is co-expressed with KCNE1.In Paper III, we study a group of PUFA-related substances, endocannabinoids, on KV7 channels. One endocannabinoid, Arachidonoyl-L-Serine (ARA-S), was identified as a potent activator of the neuronal M-channel, comprising KV7.2 and KV7.3 heteromers. We study the activating mechanism of ARA-S in KV7.2 and KV7.3, demonstrating how the activating effect is linked to two parts of the channel protein, one in the voltage sensor domain and the other in the pore domain. ARA-S was also found to activate KV7.1 and KV7.5 but not KV7.4, which instead was inhibited. Retigabine, a compound that activates the M-channel but has a different KV7 subtype selectivity compared to ARA-S, was used in combination with ARA-S to maintain a potent effect on the M-channel while limiting the activation of other KV7 channels.In conclusion, the activating effect of PUFA analogs on KV7 channels may be helpful in the development of future drug candidates for diseases such as arrhythmia and epilepsy.
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| 2. |
- Sten, Sebastian, 1993-
(författare)
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Mathematical modeling of neurovascular coupling
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The brain is critically dependent on the continuous supply of oxygen and glucose, which is carried and delivered by blood. When a brain region is activated, metabolism of these substrates increases rapidly, but is quickly offset by a substantially higher increase in blood flow to that region, resulting in a brief oversupply of these substrates. This phenomenon is referred to as functional hyperemia, and forms the foundation of functional neuroimaging techniques such as functional Magnetic Resonance Imaging (fMRI), which captures a Blood Oxygen Level-Dependent (BOLD) signal. fMRI exploits these BOLD signals to infer brain activity, an approach that has revolutionized the research of brain function over the last 30 years. Due to the indirect nature of this measure, a deeper understanding of the connection between brain activity and hemodynamic changes — a neurovascular coupling (NVC) — is essential in order to fully interpret such functional imaging data. NVC connects the synaptic activity of neurons with local changes in cerebral blood flow, cerebral blood volume, and cerebral metabolism of oxygen, through a complex signaling network, consisting of multiple different brain cells which release a myriad of distinct vasoactive messengers with specific vascular targets. To aid with this complexity, mathematical modeling can provide vital help using methods and tools from the field of Systems Biology. Previous models of the NVC exist, conventionally describing quasi-phenomenological steps translating neuronal activity into hemodynamic changes. However, no mechanistic mathematical model that describe the known intracellular mechanisms or hypotheses underlying the NVC, and which can account for a wide variety of NVC related measurements, currently exists. Therefore, in this thesis, we apply a Systems Biology approach to develop such intracellular mechanisms based models using in vivo experimental data consisting of different NVC related measures in rodents, primates, and humans.Paper I investigates two widely discussed hypotheses describing the NVC: the metabolic feedback hypothesis, and the vasoactive feed-forward hypothesis. We illustrate through multiple model rejections that only a model describing a combination of the two hypotheses can capture the qualitative features of the BOLD signal, as measured in humans. This combined model can describe data used for training, as well as predict independent validation data not previously seen by the model before.Paper II extends this model to describe the negative BOLD response, where the blood oxygenation drops below basal levels, which is commonly observed in clinical and cognitive studies. The model explains the negative BOLD response as the result of neuronal inhibition, describing and adequately predicting experimental data from two different experiments.In Paper III, we develop a first model including the cell-specific contributions of GABAergic interneurons and pyramidal neurons to functional hyperemia, using data of optogenetic and sensory stimuli in rodents for both awake and anesthesia conditions. The model captures the effect of the anesthetic as purely acting on the neuronal level if a Michaelis-Menten expression is included, and it also correctly predicts data from experiments with different pharmacological inhibitors.Finally, in Paper IV, we extend the model in Paper III to describe and predict a majority of the relevant hemodynamic NVC measures using data from rodents, primates, and humans. The model suggests an explanation for observed bi-modal behaviors, and can be used to generate new insights regarding the underpinnings of other complicated observed behaviors. This model constitutes the most complete mechanistic model of the NVC to date.This new model-based understanding opens the door for a more integrative approach to the analysis of neuroimaging data, with potential applications in both basic science and in the clinic.
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