Effects of unsaturated fatty acids on the kinetics of voltage-gated proton channels heterologously expressed in cultured cells

Abstract

Key points: Arachidonic acid (AA) greatly enhances the activity of the voltage-gated proton (Hv) channel, although its mechanism of action and physiological function remain unclear. In the present study, we analysed the effects of AA on proton currents through Hv channels heterologously expressed in HEK293T cells. The dramatic increase in proton current amplitude elicited by AA was accompanied by accelerated activation kinetics and a leftward shift in the voltage-dependence of activation. Mutagenesis studies suggest the two aforementioned effects of AA reflect two distinct structural mechanisms. Application of phospholipase A2, which liberates AA from phospholipids in the membrane, also enhances Hv channel activity, supporting the idea that AA modulates Hv channel activity within physiological contexts. Unsaturated fatty acids are key components of the biological membranes of all cells, and precursors of mediators for cell signalling. Arachidonic acid (AA) is an unsaturated fatty acid known to modulate the activities of various ion channels, including the voltage-gated proton (Hv) channel, which supports the rapid production of reactive oxygen species (ROS) in phagocytes through regulation of pH and membrane potential. However, the molecular mechanisms and physiological functions of the effects of AA on Hv channels remain unclear. In the present study, we report an electrophysiological analysis of the effects of AA on the mouse Hv channel (mHv1) heterologously expressed in HEK293T cells. Application of AA to excised inside-out patch membranes rapidly induced a robust increase in the amplitude of the proton current through mHv1. The current increase was accompanied by accelerated activation kinetics and a small leftward shift of the current-voltage relationship. In monomeric channels lacking the coiled-coil region of the channel protein, the shift in the current-voltage relationship was diminished but activation and deactivation remained accelerated. Studies with several AA derivatives showed that double bonds and hydrophilic head groups are essential for the effect of AA, although charge was not important. The application of phospholipase A2 (PLA2), which generates AA from cell membrane phospholipids, stimulated mHv1 activity to a similar extent as direct application of ∼20 μm AA, suggesting that endogenous AA may regulate Hv channel activity.