Jason Tien

TMEM16F Forms a Ca2+-Activated Cation Channel Required for Lipid Scrambling in Platelets during Blood Coagulation

Collapse of membrane lipid asymmetry is a hallmark of blood coagulation. TMEM16F of the TMEM16 family that includes TMEM16A/B Ca(2+)-activated Cl(-) channels (CaCCs) is linked to Scott syndrome with deficient Ca(2+)-dependent lipid scrambling. We generated TMEM16F knockout mice that exhibit bleeding defects and protection in an arterial thrombosis model associated with platelet deficiency in Ca(2+)-dependent phosphatidylserine exposure and procoagulant activity and lack a Ca(2+)-activated cation current in the platelet precursor megakaryocytes. Heterologous expression of TMEM16F generates a small-conductance Ca(2+)-activated nonselective cation (SCAN) current with subpicosiemens single-channel conductance rather than a CaCC. TMEM16F-SCAN channels permeate both monovalent and divalent cations, including Ca(2+), and exhibit synergistic gating by Ca(2+) and voltage. We further pinpointed a residue in the putative pore region important for the cation versus anion selectivity of TMEM16F-SCAN and TMEM16A-CaCC channels. This study thus identifies a Ca(2+)-activated channel permeable to Ca(2+) and critical for Ca(2+)-dependent scramblase activity during blood coagulation. PAPERFLICK:

A comprehensive search for calcium binding sites critical for TMEM16A calcium-activated chloride channel activity

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Furthermore, TMEM16A mutant channels containing double cysteine substitutions at these residues are sensitive to the redox potential of the internal solution, providing evidence for their physical proximity and solvent accessibility. DOI: http://dx.doi.org/10.7554/eLife.02772.001

Identification of a dimerization domain in the TMEM16A calcium-activated chloride channel (CaCC)

Transmembrane proteins with unknown function 16 (TMEM16A) is a calcium-activated chloride channel (CaCC) important for neuronal, exocrine, and smooth muscle functions. TMEM16A belongs to a family of integral membrane proteins that includes another CaCC, TMEM16B, responsible for controlling action potential waveform and synaptic efficacy, and a small-conductance calcium-activated nonselective cation channel, TMEM16F, linked to Scott syndrome. We find that these channels in the TMEM16 family share a homodimeric architecture facilitated by their cytoplasmic N termini. This dimerization domain is important for channel assembly in eukaryotic cells, and the in vitro association of peptides containing the dimerization domain is consistent with a homotypic protein–protein interaction. Amino acid substitutions in the dimerization domain affect functional TMEM16A-CaCC channel expression, as expected from its critical role in channel subunit assembly.

Four basic residues critical for the ion selectivity and pore blocker sensitivity of TMEM16A calcium-activated chloride channels

Significance TMEM16A is a calcium-activated chloride channel, meaning that it is a protein found at the surface of a variety of cells that permits chloride to enter when internal calcium levels rise. TMEM16A has been implicated in a variety of biological processes, including epithelial fluid secretion, smooth muscle contraction, neuron firing, and cancer cell proliferation. However, it is unclear how chloride ions are guided across the cell membrane by this protein. Here, we have modified and tested positively charged amino acids of TMEM16A and found four that modify chloride flux. We also tested potential blockers from a high-throughput screen and describe two that appear to block chloride’s path, which should contribute to future studies attempting to further analyze TMEM16A’s function. TMEM16A (transmembrane protein 16) (Anoctamin-1) forms a calcium-activated chloride channel (CaCC) that regulates a broad array of physiological properties in response to changes in intracellular calcium concentration. Although known to conduct anions according to the Eisenman type I selectivity sequence, the structural determinants of TMEM16A anion selectivity are not well-understood. Reasoning that the positive charges on basic residues are likely contributors to anion selectivity, we performed whole-cell recordings of mutants with alanine substitution for basic residues within the putative pore region and identified four residues on four different putative transmembrane segments that significantly increased the permeability of the larger halides and thiocyanate relative to that of chloride. Because TMEM16A permeation properties are known to shift with changes in intracellular calcium concentration, we further examined the calcium dependence of anion selectivity. We found that WT TMEM16A but not mutants with alanine substitution at those four basic residues exhibited a clear decline in the preference for larger anions as intracellular calcium was increased. Having implicated these residues as contributing to the TMEM16A pore, we scrutinized candidate small molecules from a high-throughput CaCC inhibitor screen to identify two compounds that act as pore blockers. Mutations of those four putative pore-lining basic residues significantly altered the IC50 of these compounds at positive voltages. These findings contribute to our understanding regarding anion permeation of TMEM16A CaCC and provide valuable pharmacological tools to probe the channel pore.

Cryo-EM structures of the TMEM16A calcium-activated chloride channel.

Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca2+ ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca2+ occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca2+ ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca2+. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.

Chapter 11 – Molecular Properties of Ion Channels

Ion channels are important integral membrane proteins that allow charged ions to pass into and out of a cell. Many ion channels are evolutionarily related, and small mutations may be responsible for the observed diversity in channel gating and ion selectivity. Ion channels can be gated by the movement of a charged voltage sensor through the lipid bilayer, the pull of a calcium-gating ring, the interaction with calmodulin proteins, the binding of G-protein subunits, or the detection of ATP by auxiliary subunits. Any of these events deforms the channel protein, physically distorting the gate to allow ion permeation. Ion selectivity in pore-loop potassium, sodium, and calcium channels is achieved by protein-ion interactions that dehydrate ions as they move through the pore. The energetics involved in the unfavorable removal of water molecules from an ion’s hydration shell ensure that ions of the correct size and charge are selectively conducted through the pore.

Identification of Critical Residues for Anion Conductance in the TMEM16A Channel

TMEM16A (also called DOG-1 and Anoctamin-1) is a transmembrane-localized, homodimeric protein that makes up the pore forming subunit of a calcium-activated chloride channel found in epithelial cells, tumor cells and a subset of DRG neurons. However, the structural and biophysical properties of this channel remain poorly understood. Here, we aimed to identify residues that determine the conductance properties of TMEM16A, by using site-directed mutagenesis and heterologous expression in HEK293 cells followed by patch clamp electrophysiology. Using a recently crystallized fungal orthologue of the TMEM16 family as a model, we identified several residues clustered around the putative pore region that are critical for normal channel conductance of anions. Mutations at these residues also influenced the binding properties of several known channel inhibitors. We conclude that the identified residues are likely to be directly involved in dictating the physical properties of the anion conduction pathway, and may also help to confirm the as yet unknown location of the TMEM16A channels anion pore.

A Comprehensive Search for Calcium Binding Sites Critical for TMEM16A Calcium-Activated Chloride Channel Activity

TMEM16A forms calcium-activated chloride channels (CaCCs) that regulate physiological processes such as the secretions of airway epithelia and exocrine glands, the contraction of smooth muscles, and the excitability of neurons. Notwithstanding intense interest in the mechanism behind TMEM16A-CaCC calcium-dependent gating, comprehensive surveys to identify and characterize potential calcium sensors of this channel are still lacking. By aligning distantly related calcium-activated ion channels in the TMEM16 family and conducting systematic mutagenesis of all conserved acidic residues thought to be exposed to the cytoplasm, we identify four acidic amino acids as putative calcium-binding residues. Alterations of the charge, polarity, and size of amino acid side chains at these sites alter the ability of different divalent cations to activate the channel. Our results thus demonstrate that direct binding of calcium to TMEM16A triggers channel activation independently of calmodulin, identify novel interaction sites between calcium ions and TMEM16A, and lay the groundwork for future studies examining the mechanism of calcium-dependent TMEM16 channel activation.