Niflumic acid inhibits chloride conductance of rat skeletal muscle by directly inhibiting the CLC‐1 channel and by increasing intracellular calcium

Background and purpose: Given the crucial role of the skeletal muscle chloride conductance (gCl), supported by the voltage‐gated chloride channel CLC‐1, in controlling muscle excitability, the availability of ligands modulating CLC‐1 are of potential medical as well as toxicological importance. Here, we focused our attention on niflumic acid (NFA), a molecule belonging to the fenamates group of non‐steroidal anti‐inflammatory drugs (NSAID). Experimental approach: Rat muscle Cl− conductance (gCl) and heterologously expressed CLC‐1 currents were evaluated by means of current‐clamp (using two‐microelectrodes) and patch‐clamp techniques, respectively. Fura‐2 fluorescence was used to determine intracellular calcium concentration, [Ca2+]i, in native muscle fibres. Key results: NFA inhibited native gCl with an IC50 of 42 μM and blocked CLC‐1 by interacting with an intracellular binding site. Additionally, NFA increased basal [Ca2+]i in myofibres by promoting a mitochondrial calcium efflux that was not dependent on cyclooxygenase or CLC‐1. A structure‐activity study revealed that the molecular conditions that mediate the two effects are different. Pretreatment with the Ca‐dependent protein kinase C (PKC) inhibitor chelerythrine partially inhibited the NFA effect. Therefore, in addition to direct channel block, NFA also inhibits gCl indirectly by promoting PKC activation. Conclusions and Implications: These cellular effects of NFA on skeletal muscle demonstrate that it is possible to modify CLC‐1 and consequently gCl directly by interacting with channel proteins and indirectly by interfering with the calcium‐dependent regulation of the channel. The effect of NFA on mitochondrial calcium stores suggests that NSAIDs, widely used drugs, could have potentially dangerous side‐effects. British Journal of Pharmacology (2007) 150, 235–247. doi:10.1038/sj.bjp.0706954