Voltage- and calcium-dependent gating of TMEM16A/Ano1 chloride channels are physically coupled by the first intracellular loop

Abstract

Ca 2+-activated Cl − channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca 2+ has recently become possible with the discovery that Ano1 (TMEM16a) − channels (CaCCs) play manifold roles in cell physiology (1, 2), including epithelial secretion (3, 4), sensory transduction and adaptation (5-8), regulation of smooth muscle contraction (9), control of neuronal and cardiac excitability (10), and nociception (11). This myriad of functions has attracted attention for more than 25 years (12, 13), but a lack of consensus regarding their molecular composition has stymied a mechanistic understanding of their gating. Recently, two members of the TMEM16/anoctamin family (Ano1 and Ano2) were identified as CaCC channels (14-16) and shown to be essential for salivary exocrine secretion (14,17,18), gut slow-wave activity (18, 19), tracheal secretion (18,20,21), and olfactory transduction (5-7).Ano1 and Ano2 are well suited for their diverse roles because they are dually gated by voltage (V m ) and intracellular Ca 2+ concentration ([Ca 2+ ] i ), so that their activity is tuned by the interplay between metabotropic and ionotropic inputs (14,16,(22)(23)(24) and depolarization. In the absence of Ca 2+ , no current was evident at V m between −100 mV and +100 mV, but as [Ca 2+ ] i was increased, an outward current was activated by depolarization and deactivated by hyperpolarization (Fig. 1 A-D and F). As [Ca 2+ ] i was increased, outward rectification (Fig. 1F) and the fraction of total current exhibiting time-dependence (Fig. 1E) were reduced. V m -dependent activation of Ano1 was evaluated by plotting normalized conductance versus V m (G/G max vs. V m curves; Fig. 1G). The data were well fit by the Boltzmann equation,where G/G max is normalized conductance; z is the equivalent gating charge associated with voltage-dependent channel opening; V 0.5 is the membrane potential (V m ) where G/G max is halfmaximal and is related to the conformational energy associated with voltage-independent channel opening; and F/RT = 0.039 mV −1 . At 1 μM Ca 2+ , V 0.5 was 64 ± 0.9 mV (Fig. 1G, black squares); doubling [Ca 2+ ] to 2 μM shifted the G/G max vs. V m curve to the left by −145 mV (Fig. 1G, red circles) with no significant effect on z. Because Ca 2+ shifts the G/G max vs. V m curves so dramatically, a complete G/G max vs. V m curve could be recorded for only a narrow range of [Ca 2+ ] i . For these [Ca 2+ ], z was not obviously Ca 2+ -dependent (z = 0.40-0.46). This indicates that Ca 2+ does not change the V m sensitivity (z) of the Ano1 channel, but rather shifts V 0.5 , the energy associated with V m -independent gating.Although these data may imply that Ano1 is a simple ligandgated channel, Ano1 is actually more complicated, because Ca 2+ gating is st...

Activation of Ca2+‐activated Cl− channel ANO1 by localized Ca2+ signals

“…At sub‐micromolar [Ca 2+ ] i ANO1 exhibits outward rectification and slow, time‐dependent activation at depolarizing voltages while at [Ca 2+ ] i above several micromolar both these features largely disappear (Xiao et al . 2011). A recent study added an unexpected twist to this ‘tale of two currents’ suggesting that SOCE activates ANO1 in oocytes not directly but also in an ER‐dependent way (Courjaret & Machaca, 2014).…”

“…2008; Xiao et al . 2011), would make them insensitive to ‘distal’ Ca 2+ elevations. Differential sensitivity to ‘local’ and ‘global’ Ca 2+ signals has been successfully probed for Ca 2+ ‐activated K + (and some other) channels (Berkefeld et al .…”

“…In turn, at negative membrane potentials the channel requires much higher [Ca 2+ ] i for activation than at positive potentials (Xiao et al . 2011; Yu et al . 2012).…”

“…2008; Xiao et al . 2011). This means that in cells with negative membrane potentials (such as neurons or muscle cells) ANO1 channels are unlikely to be activated by global Ca 2+ signals which in most cells do not normally reach micromolar levels.…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…13) 120 Concomitantly, the time-dependent component of ANO1 activation disappears. 1,3,5,119 ANO1 -Pharmacological profile Verkmanns group identified 2 ANO1 inhibitors (T16 inh -A01; CaCC inh -A01), 50,51 which have no effect on VRAC (Fig. 3) although some side effects of CaCC inh -A01 on Ca 2C signaling was reported.…”