Voltage-Dependent Protonation of the Calcium Pocket Enable Activation of the Calcium-Activated Chloride Channel Anoctamin-1 (TMEM16A)

Abstract

Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca 2+-activated cl − channel responsible for essential physiological processes. each monomer harbours a pore and a ca 2+-binding pocket; the voltage-dependent binding of two intracellular ca 2+ ions to the pocket gates the pore. However, in the absence of intracellular ca 2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with ca 2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/ Asp residues in the ca 2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca 2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca 2+. Anoctamin-1 (ANO1 or TMEM16A) and Anoctamin-2 (ANO2 or TMEM16B) are the pore-forming subunits of Ca 2+-activated Cl − channels (CaCCs) 1-3. Several tissues express CaCCs that participate in vital physiological functions 4,5. Thus, a role for TMEM16A and TMEM16B in smooth muscle contraction, control of blood pressure, control of gastrointestinal movements, regulation of cardiac and neuronal excitability, fluid secretion in exocrine glands, secretion of melatonin, mucin and insulin, sperm capacitation and motility, inhibition of polyspermy, and sensory transduction was established using tissue-specific knockout mice 6-16. In addition, TMEM16A modulates the partitioning of membrane phosphoinositides and endocytic transport by controlling the [Cl − ] i 17. Overexpression of TMEM16A is associated with hypertension, increased cell proliferation and cancer progression 18-21. Activation of CaCCs is triggered by voltage-dependent binding of two Ca 2+ ions to the channel when the intracellular Ca 2+ concentration ([Ca 2+ ] i) increases 22-26. Structural and mutagenesis analysis show that Ca 2+ ions bind to an acidic Ca 2+ pocket formed by four Glu, one Asp and one Asn 25-27. The pocket is located near the cytosolic side facing the permeation pathway. However, other divalent cations and maybe trivalent cations too can support TMEM16A activation. Based on the cation concentrations to obtain the half-maximum response, the cation selectivity of TMEM16A gating machinery is Ca 2+ »Sr 2+ »Ba 2+ »Cd 2+ , 23,27-29. Gd 3+ may also activate TMEM16A since its application removed the inward rectification of the Gly644Pro TMEM16A mutant channel 30 .

Molecular underpinning of intracellular pH regulation on TMEM16F

“…Phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) was recently shown to play a critical role in regulating TMEM16A and TMEM16F ion channel rundown or desensitization ( Ta et al, 2017 ; De Jesús-Pérez et al, 2018 ; Ye et al, 2018 ; Le et al, 2019a ; Tembo et al, 2019 ; Yu et al, 2019 ). In addition, both intracellular and extracellular pH have also been reported to regulate endogenous CaCCs ( Arreola et al, 1995 ; Park and Brown, 1995 ; Qu and Hartzell, 2000 ) and heterologous expressed TMEM16A CaCCs ( Chun et al, 2015 ; Cruz-Rangel et al, 2017 ; Segura-Covarrubias et al, 2020 ). The Oh laboratory recently reported that intracellular protons can inhibit TMEM16A CaCC by competing with Ca 2+ on binding to the Ca 2+ binding sites instead of affecting intracellular histidine residues ( Chun et al, 2015 ).…”

Molecular underpinning of intracellular pH regulation on TMEM16F

“…C. Le et al ., 2019; Tembo et al ., 2019; Yu et al ., 2019). In addition, both intracellular and extracellular pH have also been reported to regulate endogenous CaCCs (Arreola, Melvin and Begenisich, 1995; Park and Brown, 1995; Qu and Hartzell, 2000) and heterologous expressed TMEM16A CaCCs (Chun et al ., 2015; Cruz-Rangel et al ., 2017; Segura-Covarrubias et al ., 2020). The Oh laboratory recently reported that intracellular proton can inhibit TMEM16A CaCC by competing with Ca 2+ on binding to the Ca 2+ binding sites instead of affecting intracellular histidine residues (Chun et al ., 2015).…”

TMEM16A and TMEM16B Modulate Pheromone-Evoked Action Potential Firing in Mouse Vomeronasal Sensory Neurons

“…These results indicate that the Ca 2+ -activated chloride channels TMEM16A and TMEM16B do not contribute to passive membrane properties of mouse VSNs and do not alter major voltage-gated inward and outward currents in the range from −100 up to +40 mV. As TMEM16A can also be activated by high positive voltages in absence of intracellular Ca 2+ ( Xiao et al, 2011 ; Segura-Covarrubias et al, 2020 ), these results show that TMEM16A does not alter passive properties at membrane potentials up to +40 mV.…”