Pharmacological characterization of TMEM16A currents

Abstract

Recent studies have shown that transmembrane protein 16 A (TMEM16A) is a subunit of calcium-activated chloride channels (CACCs). Pharmacological agents have been used to probe the functional role of CACCs, however their effect on TMEM16A currents has not been systematically investigated. In the present study, we characterized the voltage and concentration-dependent effects of 2 traditional CACC inhibitors (niflumic acid and anthracene-9-carboxcylic acid) and 2 novel CACC / TMEM16A inhibitors (CACC(inh)A01 and T16A(inh)A01) on TMEM16A currents. The whole cell patch clamp technique was used to record TMEM16A currents from HE K 293 cells that stably expressed human TMEM16A. Niflumic acid, A-9-C, CACC(inh)A01 and T16A(inh)A01 inhibited TMEM16A currents with IC50 values of 12, 58, 1.7 and 1.5 μM, respectively, however, A-9-C and niflumic acid were less efficacious at negative membrane potentials. A-9-C and niflumic acid reduced the rate of TMEM16A tail current deactivation at negative membrane potentials and A-9-C (1 mM) enhanced peak TMEM16A tail current amplitude. In contrast, the inhibitory effects of CACC(inh)A01 and T16A(inh)A01 were independent of voltage and they did not prolong the rate of TMEM16A tail current deactivation. The effects of niflumic acid and A-9-C on TMEM16A currents were similar to previous observations on CACCs in vascular smooth muscle, strengthening the hypothesis that they are encoded by TMEM16A. However, CACC(inh)A01 and T16A(inh)A01 were more potent inhibitors of TMEM16A channels and their effects were not diminished at negative membrane potentials making them attractive candidates to interrogate the functional role of TMEM16A channels in future studies.

9–Anthracene carboxylic acid is more suitable than DIDS for characterization of calcium-activated chloride current during canine ventricular action potential

“…This phenomenon is quite opposite compared with that observed with expressed TMEM16A channels, where a maintained component also can be seen (Bradley et al 2014). This can be a result of the physiological [Ca 2+ ] i changes and the remarkable compartmentalization, factors which are much more pronounced in native cardiac myocytes (Trafford et al 1998;Zhang et al 2014).…”

“…Therefore, this must be kept in mind when comparing these half-effective doses with data available in the literature. The half-inhibitory dose of 9-AC on TMEM16A currents in HEK 293 cells that stably expressed human TMEM16A was reported to be 58 μM (Bradley et al 2014). Calcium-sensitive chloride current was reduced by 9-AC with a half-inhibitory dose of 80 μM on brown fat cells (Pappone and Lee 1995).…”

A novel exon in the human Ca2+-activated Cl− channel Ano1 imparts greater sensitivity to intracellular Ca2+

“…Mean current from 0.5-1.0 s after the start of each test pulse was expressed as a fraction of cell capacitance [I/C (in pA/pF)], which was estimated by the amount of whole cell capacitance compensation dialed in during the seal test. As previously published by our group and others, we used voltage steps of 1 s in duration (1,7,10,15,20,36,42). Our reason for this was twofold.…”

“…Outside of the GI tract, Ano1 is present in the vascular smooth muscle (3,36), salivary glands, thyroid, pancreas, prostate, and urinary and gall bladders (29). 11q13 chromosome amplification and subsequent Ano1 overexpression are indicators of poor prognosis for a variety of tumors (1,19,27,40).…”

Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

“…It is also important to identify and characterize pharmacological modulators of the TMEM16A channel pore. Of the channel blockers that have been identified, some are of low potency [DIDS and tannic acid (30)] and/or nonspecific [niflumic acid, anthracene-9-carboxylic acid, NPPB, CaCC-inh001, and benzbromarone (6,(31)(32)(33)], whereas others with potency in the low micromolar range (30,34) seem to be only partially effective (T16Ainh-001) in blocking the current (31), and none have been conclusively shown to interact with the pore.…”