Research

Current Projects

TMEM16 A major focus recently has been on the TMEM16 (Anoctamin) family of proteins. In mammals, there are 10 TMEM16 genes named TMEM16A – TMEM16F (ANO1 – ANO10). The family is functionally split. TMEM16A and TMEM16B are Ca2+-activated Cl- channels that play diverse, but crucial, physiological roles including epithelial fluid secretion (e.g.; saliva, tears, and hydrating fluid of the airways and reproductive tract), regulation of smooth muscle contraction, and control of cellular excitability. Surprisingly, other members of the family including TMEM16E and TMEM16F are phospholipid scramblases, despite their sequence similarity to TMEM16A. Phospholipid scramblases are essentially ion channels that allow phospholipids to flip between one leaflet of the bilayer. But unlike ion channels that form a hydrophilic pore completely surrounded by protein, scramblases form a sluice with one side open to the core of the membrane.  Lipids are conducted across the membrane with their head groups in the sluice and their acyl chains dangling in the hydrophobic core of the membrane. Our lab has been interested in answering 2 main questions regarding the TMEM16 proteins: how do they work as molecular machines and what physiological functions do they play?

Figure Legend: Comparison of the structure of a K+ ion channel (KcsA, top) and a TMEM16 phospholipid scramblase (nhTMEM16, bottom) embedded in phospholipid bilayers. Phospholipids are represented as sticks. KcsA is composed of 4 subunits that surround a central pore that conducts K+ ions (purple) across the membrane. The ion-conducting pore is completely surrounded by protein. nhTMEM16 is composed of 2 subunits. Each subunit functions independently to transport lipids across the membrane. The lipid conduction pathway is located on the surface of each subunit facing the lipid bilayer. A conducting lipid is shown in each subunit with atoms represented as spheres. nhTMEM16 also conducts ions, probably through this same pathway. ANO1 conducts Cl- ions through a homologous structure. nhTMEM16 data is from Jiang et al.  and KcsA data is from Mark Sansom’s CGD.

Structure-function studies of the TMEM16 proteins

Figure 2We would like to understand how proteins with similar amino acid sequences are specialized to transport either Cl- or phospholipids. In other words, how does Cl- interact with the TMEM16A protein and how does phospholipid interact with TMEM16F and how do these proteins effect the transport of their substrates? Both the TMEM16 Cl- channels and TMEM16 phospholipid scramblases are turned on by increases in cytosolic Ca2+ concentration. We are exploring how binding of Ca2+ to the TMEM16 proteins moves the protein into an open, conducting state and how TMEM16 gating by Ca2+ is modulated or dependent on membrane phospholipids, in particular phosphatidylinositol 4,5-bisphosphate (PIP2).

Figure Legend: This image shows a frame from an atomistic molecular dynamics simulation of lipid translocation by nhTMEM16 (from Jiang et al. 2017). For clarity, only one nhTMEM16 subunit and only one phospholipid are shown. Scramblase activity is turned on by binding of Ca2+ (green spheres) to several glutamic acid residues in the sixth and seventh transmembrane domains. The phospholipid has its hydrophilic head (red and grey spheres) in the cleft of the protein and its hydrophobic tails (blue sticks) extend into the lipid bilayer (removed for clarity). 

Role of TMEM16E (ANO5) in muscle physiology

ANO5 has been genetically linked to a type of limb-girdle muscular dystrophy, LGMD2L. LGMD2L appears to be one of the most common muscular dystrophies in northern Europe. LGMD2L generally appears later in life, suggesting that muscle forms normally during embryogenesis but somehow fails with aging. We are testing the hypothesis that ANO5-mediated phospholipid scrambling plays an important role in muscle repair and regeneration. During activity muscle is continually damaged, but it is repaired by several mechanisms. An indispensable mechanism of muscle repair involves the fusion of muscle progenitor cells (satellite cells) with each other and with damaged muscle fibers. We have found that fusion of myoblasts from ANO5 knockout mice is defective and have evidence that phospholipid scrambling by ANO5 plays an important role in the fusion defect.

Figure 3Figure Legend:  Live-cell imaging of myoblasts expressing a cytoplasmic red fluorescent protein or a membrane green fluorescent protein. The green cell fuses with the two nearby red cells over a period of about 2 minutes.

Figure 4

Figure Legend: Loss of Ano5 expression impairs myoblast fusion. (A, B) Confocal images of primary myoblast cultures isolated from adult WT (A) and Ano5-/- (B) mice differentiated for 3 days, fixed and stained for MHC with antibody (red) and nucleic acid (green). (C) There was a significant reduction in fusion index between WT (73%) and Ano5-/- (48%) myoblasts (P<0.03) (D, E). The average number of nuclei per myotube was measured from the same three experiments for WT (D) and Ano5-/- (E). In c-e, individual data points indicate mean values for each of 3 independent experiments with a total of > 12,000 nuclei counted.

Biogenesis of the primary cilium

Several years ago, while we were studying the trafficking of TMEM16A (ANO1) in epithelial cells, we discovered that ANO1 became concentrated in a toroidal apical structure (the “nimbus”) prior to the development of the primary cilium. The primary cilium is a sensory structure that exists in most cells and plays a key role in directing morphogenesis. Disruption of the primary cilium results in a spectrum of human diseases called ciliopathies that are characterized by developmental defects in tissue organization and structure. The spatio-temporal association of the nimbus with the primary cilium led us to propose that the nimbus is some type of platform for ciliogenesis. Knockout of ANO1 in mice produces a developmental defect that resembles ciliopathies and inhibition or knockdown of ANO1 in cultured cells results in a defect in primary ciliogenesis. We are interested in understanding the functional role of ANO1 in primary ciliogenesis and the relationship of the nimbus to the primary cilium.

Figure 5

Figure Legend: The nimbus is a hub of the microtubule cytoskeleton. The ANO1 (green) nimbus contains both acetylated (blue) and non-acetylated (red) α-tubulin. Top row of panels: Representative confocal XY planes of a z-stack of a nimbus that was subjected to deconvolution. Bottom panels: The z-stack was deconvolved and isosurfaces were then constructed from the deconvolved image. Top view: viewed from the apical surface of the cell. Side view: viewed from a plane near the apical surface. Scale bar = 2.5µm.

Figure 6

Figure Legend:  shRNA knockdown of ANO1 causes shorter cilia. mpkCCD14 cells were treated with lentiviral particles encoding shRNA against ANO1 or GAPDH or a non-silencing control sequence as indicated. Cilium length in cells treated with ANO1 shRNA were significantly shorter than control cells. Also, fewer cells were ciliated (not shown). Data are from Ruppersburg et al. (2014).

Other projects

We also are collaborating on several studies on voltage-gated Ca2+ channels (CaV) that aim explore the pathogenic nature of CaV mutations linked to neurological diseases. Also, we continue our studies on bestrophins, another type of Ca2+-activated Cl- channel.

Publications

Jiang T, Yu K, Hartzell HC, Tajkhorshid E. Lipids and ions traverse the membrane by the same physical pathway in the nhTMEM16 scramblase. Elife. 2017 Sep 16;6. pii: e28671. doi: 10.7554/eLife.28671. PMID: 28917060.

De Jesús-Pérez JJ, Cruz-Rangel S, Espino-Saldaña ÁE, Martínez-Torres A, Qu Z, Hartzell HC, Corral-Fernandez NE, Pérez-Cornejo P, Arreola J. Phosphatidylinositol 4,5-bisphosphate, cholesterol, and fatty acids modulate the calcium-activated chloride channel TMEM16A (ANO1). Biochim Biophys Acta. 2018 Mar;1863(3):299-312. PMID: 29277655.

Whitlock JM, Hartzell HC. Anoctamins/TMEM16 Proteins: Chloride Channels Flirting with Lipids and Extracellular Vesicles. Annu Rev Physiol. 2017 Feb10;79:119-143. doi: 10.1146/annurev-physiol-022516-034031. PMID: 27860832.

Griffin DA, Johnson RW, Whitlock JM, Pozsgai ER, Heller KN, Grose WE, Arnold WD, Sahenk Z, Hartzell HC, Rodino-Klapac LR. Defective membrane fusion and repair in Anoctamin5-deficient muscular dystrophy. Hum Mol Genet. 2016 May 15;25(10):1900-1911.

Yu K, Whitlock JM, Lee K, Ortlund EA, Yuan Cui Y, Hartzell, HC. Identification of a lipid scrambling domain in ANO6/TMEM16F. eLife. 2015; 4. PMC4477620

Ruppersburg CC and Hartzell HC. The Ca2+-activated Cl- channel ANO1 (TMEM16A) regulates primary ciliogenesis. Mol. Biol. Cell. 2014 Jun 1;25(11):1793-807. PMC4038505.

Perez-Cornejo P, Gokhale A, Duran C, Cui Y, Xiao Q, Hartzell HC, Faundez V. Anoctamin 1 (Tmem16A) Ca2+-activated chloride channel stoichiometrically interacts with an ezrin-radixin-moesin network. Proc Natl Acad Sci U S A. 2012 Jun 26;109(26):10376-81. PMC3387097

Yu K, Duran C, Qu Z, Cui YY, Hartzell HC. Explaining Calcium-Dependent Gating of Anoctamin-1 Chloride Channels Requires a Revised Topology. Circ Res. 2012 Mar 30;110(7):990-9. PMC3558997

Yu K, R Lujan, A Marmorstein, S Gabriel, and HC Hartzell. Bestrophin-2 mediates bicarbonate transport by goblet cells in mammalian colon. Journal of Clinical Investigation 120, 1722-35 (2010). PMC2860923

Hartzell, H.C., Z. Qu, K. Yu, Q. Xiao, L.T. Chien. Molecular Physiology of Bestrophins: A Family of Multi-functional Chloride Channels Linked to Macular Degeneration. Physiological Reviews 88: 639-672 (2008).

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