Activation and Inhibition of TMEM16A Calcium-Activated Chloride Channels
Figure 8
NFA block of ANO1 is affected by the occupancy of permeant ions in the pore but not by the degree of channel activation.
(A) Blocking ANO1 current with 50 µM NFA in different intracellular anions. All intracellular anion concentrations were 140 mM. Vm = ±20 mV. The 5-V digital signal at the bottom panel indicates the time of the switch of perfusion pipes. (B) Concentration-dependent NFA blockage of ANO1 in different intracellular anions. Inset shows the fitted K1/2 of the block in different intracellular anions. Data obtained in the Cl− solution were averaged from 9–11 patches, while the rest were obtained from 3–5 patches. (C) Comparison of the NFA dose-response inhibition curves obtained in symmetrical [Cl−] of 140 mM (black) and 4 mM (red) at −40 mV. For the latter solution, Na-Glutamate (136 mM) was added to replace the reduced [NaCl]. The two regression curves in 140 mM NaCl (K1/2 = 18.4 µM) and 4 mM NaCl (K1/2 = 7.9 µM) are statistically different from each other. Data were from 6 different patches. (D) Comparing NFA sensitivities of the ANO1 current activated by 20 µM [Ca2+] (K1/2 = 17.9 µM) and 404 µM [Ba2+] (K1/2 = 22.6 µM). Vm = −20 mV. N = 5–11. No statistical difference between the two curves. (E) Sensitivity of ANO1 to Ca2+ activation is not reduced by NFA. Percentage of the ANO1 current induced by 666 nM Ca2+ at ±40 mV was compared in the absence (solid bars) and in the presence (open bars) of 20 µM NFA (N = 4). Experiments were performed in the same way as those shown in Fig. 1. No statistically significant difference between the experiments with and without NFA (p>0.05, t test).