Key pointsr Calcium-activated chloride channels TMEM16A and TMEM16B support important physiological processes such as fast block of polyspermy, fluid secretion, control of blood pressure and sensory transduction.r Given the physiological importance of TMEM16 channels, it is important to study how incoming stimuli activate these channels. Here we study how channels open and close and how the process of gating is regulated.r We show that TMEM16A and TMEM16B display fast and slow gating. These gating modes are regulated by voltage and external chloride.r Dual gating explains the complex time course of the anion current. r Residues within the first intracellular loop of the channel influence the slow gating mode. r Dual gating is an intrinsic property observed in endogenous calcium-activated chloride channels and could be relevant to physiological processes that require sustained chloride ion movement.Abstract TMEM16A and TMEM16B are molecular components of the physiologically relevant calcium-activated chloride channels (CaCCs) present in many tissues. Their gating is dictated by membrane voltage (V m ), intracellular calcium concentrations ([Ca 2+ ] i ) and external permeant anions. As a consequence, the chloride current (I Cl ) kinetics is complex. For example, TMEM16A I Cl activates slowly with a non-mono-exponential time course while TMEM16B I Cl activates rapidly following a mono-exponential behaviour. To understand the underlying mechanism responsible for the complex activation kinetics, we recorded I Cl from HEK-293 cells transiently transfected with either TMEM16A or TMEM16B as well as from mouse parotid acinar cells. Two distinct V m -dependent gating modes were uncovered: a fast-mode on the millisecond time scale followed by a slow mode on the second time scale. Using long (20 s) depolarizing pulses both gating modes were activated, and a slowly rising I Cl was recorded in whole-cell and inside-out patches. The amplitude of I Cl at the end of the long pulse nearly doubled and was blocked by 100 μM tannic acid. The slow gating mode was strongly reduced by decreasing the [Cl − ] o from 140 to 30 mM and by altering the sequence of the first intracellular loop. Mutating 480 RSQ 482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 448 AVK 451 to RSQ in TMEM16A has little effect. Deleting 448 EAVK 451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic V m -and Cl − -sensitive dual gating that elicits complex I Cl kinetics.

Key points• Plasma membrane ClC-2 chloride channels are widely distributed in our body and are important for vision and fertility.• ClC-2 channels are gated by changes in transmembrane voltage despite of lacking a voltage sensor device. It has been hypothesized that the interaction of an external proton with the gating machinery is responsible for voltage-dependent gating.

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.

The Vickers micro-hardness of this alloy was unusually dependent on the heat treatment from 300 to 634K, inferring important micro-structural changes and the presence of amorphous grains before its phase transition. Once the alloy is crystallized, the microhardness is characteristic of a brittle alloy, the main problem of these alloys. Within the amorphous state, other properties like free-volume, magnetic states and Fe-Fe distances were followed by PALS and MS, respectively, to analyze those micro-structural changes, thermally induced, which are of paramount interest to understand their brittleness problem.  

Los CaCCs son proteínas formadoras de poros que se ubican en la membrana celular. Los CaCCs permiten el paso de iones a través de la membrana, lo cual es clave para una adecuada realización de funciones celulares y para el desarrollo de algunas enfermedades. En este contexto, se brinda una reseña del papel fisiopatológico de los CaCCs. La metodología empleada fue hacer una extensa consulta en U.S. National Library of Medicine-PubMed.gov. Los resultados encontrados indican que, a pesar del papel fundamental que tienen los CaCCs en el desarrollo de enfermedades crónicas, en nuestro país se realiza poca investigación y difusión en este campo.