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Purified TMEM16A is sufficient to form Ca2+-activated Cl- channels.

ABSTRACT: Ca(2+)-activated Cl(-) channels (CaCCs) are key regulators of numerous physiological functions, ranging from electrolyte secretion in airway epithelia to cellular excitability in sensory neurons and muscle fibers. Recently, TMEM16A (ANO1) and -B were shown to be critical components of CaCCs. It is still unknown whether they are also sufficient to form functional CaCCs, or whether association with other subunits is required. Recent reports suggest that the Ca(2+) sensitivity of TMEM16A is mediated by its association with calmodulin, suggesting that functional CaCCs are heteromultimers. To test whether TMEM16A is necessary and sufficient to form functional CaCCs, we expressed, purified, and reconstituted human TMEM16A. The purified protein mediates Ca(2+)-dependent Cl(-) transport with submicromolar sensitivity to Ca(2+), consistent with what is seen in patch-clamp experiments. The channel is synergistically gated by Ca(2+) and voltage, so that opening is promoted by depolarizing potentials. Mutating two conserved glutamates in the TM6-7 intracellular loop selectively abolishes the Ca(2+) dependence of reconstituted TMEM16A, in a manner similar to what was reported for the heterologously expressed channel. Well-characterized CaCC blockers inhibit Cl(-) transport with Kis comparable to those measured for native and heterologously expressed CaCCs. Finally, direct physical interactions between calmodulin and TMEM16A could not be detected in copurification experiments or in functional assays. Our results demonstrate that purified TMEM16A is necessary and sufficient to recapitulate the biophysical and pharmacological properties of native and heterologously expressed CaCCs. Our results also show that association of TMEM16A with other proteins, such as calmodulin, is not required for function.

SUBMITTER: Terashima H

PROVIDER: S-EPMC3845129 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Project description:BackgroundTransmembrane protein 16A (TMEM16A) Ca2+-activated Cl- channels have diverse physiological functions, such as epithelial secretion of Cl- and fluid and sensation of pain. Recent studies have demonstrated that TMEM16A contributes to the pathogenesis of infectious and non-infectious inflammatory diseases. However, the role of TMEM16A in inflammation has not been clearly elucidated.Aim of reviewIn this review, we aimed to provide comprehensive information regarding the roles of TMEM16A in inflammation by summarizing the mechanisms underlying TMEM16A expression and activation under inflammatory conditions, in addition to exploring the diverse inflammatory signaling pathways activated by TMEM16A. This review attempts to develop the idea that TMEM16A plays a diverse role in inflammatory processes and contributes to inflammatory diseases in a cellular environment-dependent manner.Key scientific concepts of reviewMultiple inflammatory mediators, including cytokines (e.g., interleukin (IL)-4, IL-13, IL-6), histamine, bradykinin, and ATP/UTP, as well as bacterial and viral infections, promote TMEM16A expression and/or activity under inflammatory conditions. In addition, TMEM16A activates diverse inflammatory signaling pathways, including the IP3R-mediated Ca2+ signaling pathway, the NF-κB signaling pathway, and the ERK signaling pathway, and contributes to the pathogenesis of many inflammatory diseases. These diseases include airway inflammatory diseases, lipopolysaccharide-induced intestinal epithelial barrier dysfunction, acute pancreatitis, and steatohepatitis. TMEM16A also plays multiple roles in inflammatory processes by increasing vascular permeability and leukocyte adhesion, promoting inflammatory cytokine release, and sensing inflammation-induced pain. Furthermore, TMEM16A plays its diverse pathological roles in different inflammatory diseases depending on the disease severity, proliferating status of the cells, and its interacting partners. We herein propose cellular environment-dependent mechanisms that explain the diverse roles of TMEM16A in inflammation.

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Purified TMEM16A is sufficient to form Ca2+-activated Cl- channels.

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