Revealing the activation pathway for TMEM16A chloride channels from macroscopic currents and kinetic models

Abstract

TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth, and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca2+]i), membrane depolarization, extracellular Cl− or permeant anions, and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. a) TMEM16A is activated by voltage in the absence of intracellular Ca2+. b) The Cl− conductance is decreased after reducing extracellular Cl− concentration ([Cl−]o). c) ICl is regulated by physiological concentrations of [Cl−]o. d) In cells dialyzed with 0.2 µM [Ca2+]i, Cl− has a bimodal effect: at [Cl−]o < 30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM [Cl−]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca2+ and Cl− to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca2+ ions coupled to a Vm-dependent binding of an external Cl− ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl− does not alter the apparent Ca2+ affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl− acts by stabilizing the open configuration induced by Ca2+ and by contributing to the Vm dependence of activation.

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

“…; Contreras‐Vite et al . ). Cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 2 m m l ‐glutamine and 1 mg ml −1 Geneticin at 37°C in a 95% O 2 –5% CO 2 atmosphere.…”

“…The gating of TMEM16A is a complex process (Contreras‐Vite et al . ). It results from the interaction between the binding of intracellular Ca 2+ , membrane depolarization (Arreola et al .…”

Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1)

“…In addition to its regulation by Ca 2+ , TMEM16A is also regulated by permeant anions that facilitate TMEM16A channel gating (Arreola et al, 1996; Kuruma and Hartzell, 2000; Arreola et al, 2010; Xiao et al, 2011; Terashima et al, 2013; Betto et al, 2014; Contreras-Vite et al, 2016) because ion permeation and gating are coupled (Perez-Cornejo et al, 2004; Betto et al, 2014). Also, protons exert a strong regulatory effect on channel activity.…”

Recent advances in TMEM16A: Structure, function, and disease

“…Xiao et al () found that replacement of extracellular Cl − with NO 3 − significantly reduced the voltage‐dependent gating of TMEM16A, suggesting the voltage gating of TMEM16A is affected by permeant anions. On the contrary, Contreras‐Vite et al () demonstrated that extracellular Cl − does not alter the apparent Ca 2+ affinity of TMEM16A. Rather, extracellular Cl − can stabilize the open conformation of TMEM16A and contribute to the voltage dependence of activation.…”

“…TMEM16A is widely expressed in many cells including epithelia, airway and vascular smooth muscle cells, vascular endothelium, cardiac muscles, olfactory sensory neurons, somatosensory neurons interstitial cells of Cajal (ICC), and nociceptive neurons (K. Ma, Wang, Yu, Wei, & Xiao, ; U. Oh & Jung, ; H. Wang et al, ). The electrophysiological properties of TMEM16A involve calcium and voltage dependence (Contreras‐Vite et al, ; Cruz‐Rangel et al, ; Terashima, Picollo, & Accardi, ; Xiao et al, ), rectifying properties (K. Ma et al, ), ion selectivity (Peters et al, ; Xiao et al, ), activation kinetics (Contreras‐Vite et al, ; Espinosa et al, ), and rundown (Yu, Zhu, Qu, Cui, & Hartzell, ). In addition, the dysfunction of TMEM16A can lead to many diseases, including various cancers (Cao et al, ), gastrointestinal (GI) disorders (Mazzone et al, ), hypertension (Wang, Li, Huai, & Qu, ), and cystic fibrosis (Sondo, Caci, & Galietta, ).…”