Mechanism of allosteric activation of TMEM16A/ANO1 channels by a commonly used chloride channel blocker

Abstract

Background and PurposeCalcium‐activated chloride channels (CaCCs) play varied physiological roles and constitute potential therapeutic targets for conditions such as asthma and hypertension. TMEM16A encodes a CaCC. CaCC pharmacology is restricted to compounds with relatively low potency and poorly defined selectivity. Anthracene‐9‐carboxylic acid (A9C), an inhibitor of various chloride channel types, exhibits complex effects on native CaCCs and cloned TMEM16A channels providing both activation and inhibition. The mechanisms underlying these effects are not fully defined.Experimental ApproachPatch‐clamp electrophysiology in conjunction with concentration jump experiments was employed to define the mode of interaction of A9C with TMEM16A channels.Key ResultsIn the presence of high intracellular Ca2+, A9C inhibited TMEM16A currents in a voltage‐dependent manner by entering the channel from the outside. A9C activation, revealed in the presence of submaximal intracellular Ca2+ concentrations, was also voltage‐dependent. The electric distance of A9C inhibiting and activating binding site was ~0.6 in each case. Inhibition occurred according to an open‐channel block mechanism. Activation was due to a dramatic leftward shift in the steady‐state activation curve and slowed deactivation kinetics. Extracellular A9C competed with extracellular Cl−, suggesting that A9C binds deep in the channel's pore to exert both inhibiting and activating effects.Conclusions and ImplicationsA9C is an open TMEM16A channel blocker and gating modifier. These effects require A9C to bind to a region within the pore that is accessible from the extracellular side of the membrane. These data will aid the future drug design of compounds that selectively activate or inhibit TMEM16A channels.

Independent activation of ion conduction pores in the double-barreled calcium-activated chloride channel TMEM16A

“…3 B). The fitted values are in general agreement with a previous analysis of TMEM16A (Ta et al, 2016). Our data thus suggest that the increase in current in WT at high Ca 2+ concentrations might result from an increase in P O without strongly affecting the permeation properties of the pore.…”

Extracellular protons enable activation of the calcium‐dependent chloride channel TMEM16A

“…At [H + ] = 10 −7.3 m P o was 0.26 ± 0.02, a value similar to that reported by Ta et al . (). As shown in Fig.…”

“…As assumed by others (Ta et al . ), we presumed that N o remained unchanged because the effects of protons were reversible. At [H + ] = 10 −7.3 m P o was 0.26 ± 0.02, a value similar to that reported by Ta et al .…”

Contrasting effects of phosphatidylinositol 4,5‐bisphosphate on cloned TMEM16A and TMEM16B channels

“…Current versus diC8-PIP 2 concentration ([diC8-PIP 2 ]) relationship. In our recordings conditions, HEK-293T cells presented a small endogenous background current Ta et al, 2016). To assess the sensitivity of TMEM16A and TMEM16B channels to diC8-PIP 2 during inside-out patch-clamp recordings (experiments of Figures 1 and 2), the currents were measured at +70 mV in nominally Ca 2+ -free solution and in solutions containing a given [Ca 2+ ] i .…”

“…For instance, both TMEM16A and TMEM16B are modulated in a complex manner by antracene-9-carboxilic acid (A9C) (Cherian et al, 2015;Ta et al, 2016). These channels are inhibited by A9C via an open channel block mechanism while also being allosterically activated by the compound (Cherian et al, 2015;Ta et al, 2016). TMEM16A and TMEM16B are also blocked by other commonly used Cl À channel blockers such as 4,4 0 -diisothiocyano-2,2 0 -stilbenedisulfonic acid (DIDS) and niflumic acid with comparable potencies (Bradley et al, 2014;Liu et al, 2015;Pifferi et al, 2009).…”