Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA352

Poster Communications

Investigation into the bimodal effects of Niflumic acid and Anthracene-9-Carboxylic acid on TMEM16A currents expressed in Human Embryo Kidney cells

S. J. Fedigan1, T. Webb1, M. Hollywood1, N. G. McHale1, K. D. Thornbury1, G. P. Sergeant1

1. Applied Sciences, Dundalk Institute of Technology, Dundalk, Louth, Ireland.

Calcium-activated chloride channels (CaCCs) are involved in a range of physiological functions including fluid secretion and smooth muscle contraction. CaCCs are now thought to be encoded by TMEM16A (ANO1) [1]. Both niflumic acid and anthracene-9-carboxylic acid (A9C) have been used to characterise CaCC/TMEM16A activity in a range of cell types. However, the effects of these agents are complex, as they both produce a paradoxical potentiation of native calcium-activated chloride currents and TMEM16A currents (ITMEM16A), in HEK cells [2,3]. The mechanisms underlying this effect are still unclear. The purpose of this study was to investigate if the potentiating effects of niflumic acid and A9C on ITMEM16A were related to intracellular Ca2+ concentration ([Ca2+]i). Currents were recorded from Human Embryo Kidney (HEK) 293 cells stably expressing human TMEM16A (transcript 1), at room temperature using the whole cell patch clamp technique. The effects of niflumic acid and A9C were compared on ITMEM16A recorded with pipette solutions containing either 58 or 335 nM [Ca2+]i. In pipette solution containing 58 nM [Ca2+]i, niflumic acid and A9C inhibited ITMEM16A, evoked by a step to +80 mV, with IC50 values of 7.2 and 293 µM respectively (n=8). When repeated in solution containing 335 nM [Ca2+]I, the IC50 values decreased to 2.6 and 174 µM, for niflumic acid and A9C respectively (n=6-10). In the presence of 58 nM [Ca2+]i tail currents, evoked by a step back to -140 mV, were enhanced in amplitude. For example, 10 μM niflumic acid increased the amplitude of tail currents from -1.2 ± 0.2 nA to -2.5 ± 0.3 nA (p≤0.01, n=5). When repeated in 335 nM [Ca2+]i this potentiation effect was abolished and a slight inhibition was observed (-4.5 ± 0.7 nA under control conditions versus -4.2 ± 0.7 nA in 10 μM niflumic acid, n=6 p>0.05) . Similar potentiating effects were observed with A9C in 58 nM [Ca2+]i. A9C (1 mM) increased the amplitude of TMEM16A tail currents, at -140 mV, by approximately 3-fold from -1.9 ± 0.7 nA to -5.5 ± 1.1 nA (p≤0.01, n=5). When repeated in 335 nM [Ca2+]i this enhancement effect was reduced, whereby at -140 mV an approximate 2-fold increase from -2.5 ± 0.4 nA in control, compared to -5.4 ± 0.8 nA in 1 mM A9C was observed (p≤0.05, n=8). In summary, in 58 nM [Ca2+]i both niflumic acid and A9C produced a bimodal effect on TMEM16A currents, causing inhibition at positive potentials and potentiation at negative potentials. However, in 335 nM [Ca2+]i the paradoxical stimulatory effects niflumic acid were abolished and the stimulatory effects of A9C were reduced. These data suggest that the paradoxical stimulatory effects of niflumic acid and, to a lesser extent, A9C on TMEM16A currents, observed at negative potentials, is reduced when the intracellular Ca2+ concentration is increased.

Where applicable, experiments conform with Society ethical requirements