Fenamates Alter Bilayer Properties

Abstract

Fenamates are a family of non-steroidal anti-inflammatory drugs (NSAIDs). They are widely prescribed to manage pain and inflammation and, like other NSAIDs, inhibit the cyclooxygenases; they have also been proposed to have anti-epileptic and neuroprotective effects. The fenamates modulate a variety of ion channels, with mechanism(s) of action that range from direct fenamate-protein interactions (binding) to non-specific membrane-mediated effects. We therefore examined whether fenamates alter bilayer properties at concentrations where they modify membrane protein function. To this end, we used a gramicidin-based fluorescence assay to investigate whether the fenamates could alter bilayer properties (as sensed by a bilayer-spanning channel), and to establish dose response curves for the fenamates bilayer-modifying effects. The fenamates we examined were flufenamic acid, meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid. All of them increased the rate of fluorescence quenching, meaning that they shifted the gramicidin monomer-dimer equilibrium in favor of the monovalent cation permeable dimers. These results thus show that fenamates alter bilayer properties, most likely by softening the bilayer. Niflumic acid was the most potent modifier of bilayer properties and tolfenamic acid the least potent. All the fenamates have limited solubility, which limited the concentration range that could be studied to 300 μM (or less). To examine the alteration of bilayer properties in more detail, mefenamic acid was tested using single-channel electrophysiology in planar bilayers. The electrophysiological results support the data from the fluorescence assay. The fenamates alter bilayer properties at the concentrations where they have been reported to alter membrane protein function. This suggests that there may indeed be a membrane contribution to the fenamates’ mechanism of action.