Molecular mechanisms of modulation of KV7 channels by polyunsaturated fatty acids and their analogues
Sammanfattning: 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 However, the previously available antiepileptic drug retigabine, has been withdrawn due to adverse effects.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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