Author: Damon Frampton

Publisher: Linköping University Electronic Press

Published: 2024-10-02

Total Pages: 148

ISBN-13: 9180757529

The superfamily of voltage-gated potassium (KV) channels is crucial for the normal function of several tissues and represents an attractive pharmacological target for treating disorders such as epilepsy and cardiac arrhythmias. However, any drug designed to target a KV channel must be capable of discriminating between different members within the superfamily, lest they plague the user with deleterious side effects. Such rational design requires structural and functional insight into how the selectivity of a molecule can be tailored to suit the intended target. This thesis combines the use of electrophysiological and computational techniques to investigate the molecular basis for how the function of hKV7 and hERG channels can be modulated by different lipophilic compounds with known or suspected effects on ion channels. These include polyunsaturated fatty acids (PUFAs), cannabidiol (CBD), and synthetic cannabinoid receptor agonists (SCRAs). Using the two-electrode voltage clamp technique on Xenopus oocytes, we find that both PUFAs and CBD modulate the function of hKV7 channels in subtype-specific manners. PUFAs facilitated the activation of hKV7 channels, except for hKV7.4 channels which were instead inhibited. Molecular dynamics simulations revealed that structural differences in the voltage-sensing domain of hKV7.4 conferred a unique, inhibitory PUFA interaction site absent in the other hKV7 subtypes. Once this site was neutralised by mutagenesis, PUFAs facilitated hKV7.4 activation. In the case of CBD, we observed three different responses: inhibition of channels with hKV7.1 subunits, potentiated voltage-sensitivity of channels with hKV7.2 or hKV7.3 subunits and enhanced maximum conductance of channels with hKV7.4 or hKV7.5 subunits. However, these responses were evoked from the same interaction site in the pore domain, indicating a more complex subtype-specific mechanism of action. Finally, using an automated patch-clamp system we screened 36 different SCRAs on the cardiac channels responsible for repolarisation: hERG and hKV7.1/KCNE1. We find 28 of the SCRAs to be inhibitors of hERG and 22 to be inhibitors of hKV7.1/KCNE1. Molecular dynamics simulations suggest the increased susceptibility of hERG to SCRA-mediated inhibition may be due to a unique central cavity site that is absent from the pore domain of hKV7.1/KCNE1. In conclusion, structurally diverse lipophilic molecules of endogenous and exogenous origins can interact with KV channels and influence their function by enhancing or interfering with functional domains. In some instances, structural differences in the channel protein can explain the discrepancies in pharmacology. These findings have implications for both pharmacology (informing rational drug design) and toxicology (identifying targets through which adverse effects may occur).