Probing the Molecular Basis of Opposing PUFA Effects on KV7 Channels

Abstract

The human KV7 ion channel family consists of five members, KV7.1-7.5, distribution of which differs between tissues. As different KV7 subtypes play different roles in different physiological processes, developing pharmacological drugs that differentiate between KV7 subtypes is important in order to limit off-target effects. In pilot experiments, we have observed that free fatty acids, such as the omega-3 fatty acid docosahexaenoic acid, activated all KV7 channels save for KV7.4. In contrast, docosahexaenoic acid induced a slight inhibition of the KV7.4 channel. The aim of this study was to understand the molecular mechanism underlying the difference in observed effects of free fatty acid and their analogues on KV7.4. Using the two-electrode voltage clamp technique to measure potassium currents in Xenopus oocytes expressing hKV7.4 and hKV7.5, we compared the effect of free fatty acids and their analogues on these two KV7 channel subtypes. Our experiments found that neutralisation of the negative charge in the carboxylic head group of docosahexaenoic acid quenched both the inhibition and activation of KV7.4 and KV7.5, respectively, indicating an electrostatic element is involved in the mechanism of action. However, the importance of lipid tail properties differs between the two effects. To determine the contributions of ion channel structure to the observed difference in response, we created KV7.4 mutants in which native amino acids had been substituted for their KV7.5 counterparts. Our experiments found that mutation of residues in a putative fatty acid site either impaired or completely abolished fatty acid-mediated inhibition, but did not result in channel activation when docosahexaenoic acid was applied. Our preliminary studies have identified regions in KV7.4 that contribute to fatty acid-mediated inhibition. Further studies will be performed to fully understand the mechanistic basis for the opposing PUFA effects on KV7.4 and KV7.5.