Project description:Anoctamin (ANO)/TMEM16 proteins exhibit diverse functions in cells throughout the body and are implicated in several human diseases. Although the founding members ANO1 (TMEM16A) and ANO2 (TMEM16B) are Ca2+-activated Cl- channels, most ANO paralogs are Ca2+-dependent phospholipid scramblases that serve as channels facilitating the movement (scrambling) of phospholipids between leaflets of the membrane bilayer. Phospholipid scrambling significantly alters the physical properties of the membrane and its landscape and has vast downstream signaling consequences. In particular, phosphatidylserine exposed on the external leaflet of the plasma membrane functions as a ligand for receptors vital for cell-cell communication. A major consequence of Ca2+-dependent scrambling is the release of extracellular vesicles that function as intercellular messengers by delivering signaling proteins and noncoding RNAs to alter target cell function. We discuss the physiological implications of Ca2+-dependent phospholipid scrambling, the extracellular vesicles associated with this activity, and the roles of ANOs in these processes.
Project description:The Tmem16 gene family was first identified by bioinformatic analysis in 2004. In 2008, it was shown independently by 3 laboratories that the first two members (Tmem16A and Tmem16B) of this 10-gene family are Ca(2+)-activated Cl(-) channels. Because these proteins are thought to have 8 transmembrane domains and be anion-selective channels, the alternative name, Anoctamin (anion and octa=8), has been proposed. However, it remains unclear whether all members of this family are, in fact, anion channels or have the same 8-transmembrane domain topology. Since 2008, there have been nearly 100 papers published on this gene family. The excitement about Tmem16 proteins has been enhanced by the finding that Ano1 has been linked to cancer, mutations in Ano5 are linked to several forms of muscular dystrophy (LGMDL2 and MMD-3), mutations in Ano10 are linked to autosomal recessive spinocerebellar ataxia, and mutations in Ano6 are linked to Scott syndrome, a rare bleeding disorder. Here we review some of the recent developments in understanding the physiology and structure-function of the Tmem16 gene family.
Project description:Airway smooth muscle hyperresponsiveness is a key component in the pathophysiology of asthma. Although calcium-activated chloride channel (CaCC) flux has been described in many cell types, including human airway smooth muscle (HASM), the true molecular identity of the channels responsible for this chloride conductance remains controversial. Recently, a new family of proteins thought to represent the true CaCCs was identified as the TMEM16 family. This led us to question whether members of this family are functionally expressed in native and cultured HASM. We further questioned whether expression of these channels contributes to the contractile function of HASM. We identified the mRNA expression of eight members of the TMEM16 family in HASM cells and show immunohistochemical evidence of TMEM16A in both cultured and native HASM. Functionally, we demonstrate that the classic chloride channel inhibitor, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB), inhibited halide flux in cultured HASM cells. Moreover, HASM cells displayed classical electrophysiological properties of CaCCs during whole cell electrophysiological recordings, which were blocked by using an antibody selective for TMEM16A. Furthermore, two distinct TMEM16A antagonists (tannic acid and benzbromarone) impaired a substance P-induced contraction in isolated guinea pig tracheal rings. These findings demonstrate that multiple members of this recently described family of CaCCs are expressed in HASM cells, they display classic electrophysiological properties of CaCCs, and they modulate contractile tone in airway smooth muscle. The TMEM16 family may provide a novel therapeutic target for limiting airway constriction in asthma.
Project description:The TMEM16 family of membrane proteins is composed of both Ca2+-gated Cl- channels and Ca2+-dependent phospholipid scramblases. The functional diversity of TMEM16s underlies their involvement in numerous signal transduction pathways that connect changes in cytosolic Ca2+ levels to cellular signaling networks. Indeed, defects in the function of several TMEM16s cause a variety of genetic disorders, highlighting their fundamental pathophysiological importance. Here, we review how our mechanistic understanding of TMEM16 function has been shaped by recent functional and structural work. Remarkably, the recent determination of near-atomic-resolution structures of TMEM16 proteins of both functional persuasions has revealed how relatively minimal rearrangements in the substrate translocation pathway are sufficient to precipitate the dramatic functional differences that characterize the family. These structures, when interpreted in the light of extensive functional analysis, point to an unusual mechanism for Ca2+-dependent activation of TMEM16 proteins in which substrate permeation is regulated by a combination of conformational rearrangements and electrostatics. These breakthroughs pave the way to elucidate the mechanistic bases of ion and lipid transport by the TMEM16 proteins and unravel the molecular links between these transport activities and their function in human pathophysiology.
Project description:The transmembrane protein 16 (TMEM16) family consists of Ca2+-activated ion channels and Ca2+-activated phospholipid scramblases (CaPLSases) that passively flip-flop phospholipids between the two leaflets of the membrane bilayer. Owing to their diverse functions, TMEM16 proteins have been implicated in various human diseases, including asthma, cancer, bleeding disorders, muscular dystrophy, arthritis, epilepsy, dystonia, ataxia, and viral infection. To understand TMEM16 proteins in health and disease, it is critical to decipher their molecular mechanisms of activation gating and regulation. Structural, biophysical, and computational characterizations over the past decade have greatly advanced the molecular understanding of TMEM16 proteins. In this review, we summarize major structural features of the TMEM16 proteins with a focus on regulatory mechanisms and gating.
Project description:Two TMEM16 family members, TMEM16A and TMEM16F, have different ion transport properties. Upon activation by intracellular Ca2+, TMEM16A-a Ca2+-activated Cl- channel-is more selective for anions than cations, whereas TMEM16F-a phospholipid scramblase-appears to transport both cations and anions. Under saturating Ca2+ conditions, the current-voltage (I-V) relationships of these two proteins also differ; the I-V curve of TMEM16A is linear, while that of TMEM16F is outwardly rectifying. We previously found that mutating a positively charged lysine residue (K584) in the ion transport pathway to glutamine converted the linear I-V curve of TMEM16A to an outwardly rectifying curve. Interestingly, the corresponding residue in the outwardly rectifying TMEM16F is also a glutamine (Q559). Here, we examine the ion transport functions of TMEM16 molecules and compare the roles of K584 of TMEM16A and Q559 of TMEM16F in controlling the rectification of their respective I-V curves. We find that rectification of TMEM16A is regulated electrostatically by the side-chain charge on the residue at position 584, whereas the charge on residue 559 in TMEM16F has little effect. Unexpectedly, mutation of Q559 to aromatic amino acid residues significantly alters outward rectification in TMEM16F. These same mutants show reduced Ca2+-induced current rundown (or desensitization) compared with wild-type TMEM16F. A mutant that removes the rundown of TMEM16F could facilitate the study of ion transport mechanisms in this phospholipid scramblase in the same way that a CLC-0 mutant in which inactivation (or closure of the slow gate) is suppressed was used in our previous studies.
Project description:All vertebrate cells regulate their cell volume by activating chloride channels of unknown molecular identity, thereby activating regulatory volume decrease. We show that the Ca(2+)-activated Cl(-) channel TMEM16A together with other TMEM16 proteins are activated by cell swelling through an autocrine mechanism that involves ATP release and binding to purinergic P2Y(2) receptors. TMEM16A channels are activated by ATP through an increase in intracellular Ca(2+) and a Ca(2+)-independent mechanism engaging extracellular-regulated protein kinases (ERK1/2). The ability of epithelial cells to activate a Cl(-) conductance upon cell swelling, and to decrease their cell volume (regulatory volume decrease) was dependent on TMEM16 proteins. Activation of I(Cl,swell) was reduced in the colonic epithelium and in salivary acinar cells from mice lacking expression of TMEM16A. Thus TMEM16 proteins appear to be a crucial component of epithelial volume-regulated Cl(-) channels and may also have a function during proliferation and apoptotic cell death.
Project description:K and Cl channels play critical role for sweat secretion, however, individual K or Cl channels and their functions are largely unknown. By comparing expreession profilings between wild-type and Eda mutant Tabby footpads we identified 4 K and 2 Cl channels highly expressed in mouse eccrine sweat glands.
Project description:K and Cl channels play critical role for sweat secretion, however, individual K or Cl channels and their functions are largely unknown. By comparing expreession profilings between wild-type and Eda mutant Tabby footpads we identified 4 K and 2 Cl channels highly expressed in mouse eccrine sweat glands. Total RNAs isolated from microdissected wild-type and Tabby footpads were profiled with Agilent 44K NIA mouse microarrays.