TMEM16A: Closing the circle


Raimund Dutzler and his lab have been the first group to solve the crystal structure of the mammalian mouse TMEM16A ion channel using cryo-electron microscopy (Paulino et al., 2017). This study is the first essential step to resolving the controversies which has raged in the ion channel community since the discovery of this protein responsible for the calcium activated chloride channel back in 2008 by 3 independent groups (Caputo et al; Schroeder et al; Yang et al., 2008). More recently Brunner et al. (2014) was the first to solve the crystal structure for the related fungal protein Nectria haematococca TMEM16 (nhTMEM16), a lipid scramblase.  Their hypothesis being that the TMEM16 protein family, consisting of ten genes (TMEM16A-k, missing out I) split from a common ancestral lipid scramblase and evolved in mammals to produce the only 2 known chloride ion channels, TMEM16A and TMEM16B, in the TMEM16 family. Up until this point functional studies had shown Ca2+ activation, voltage dependency and through mutagenesis which amino acids had an impact on chloride conductance in TMEM16A. Brunner et al’s X-ray structure, to a 2.6 Å resolution, gave rise to a number of profound questions about the chloride ion channels TMEM16A & B.

Their structure of the TMEM16 homodimer did not conform to ion channel dogma. Based on the homology model of the lipid scramblase X-ray structure for nhTMEM16A, Whitlock and Hartzel (2016) proposed that the calcium activated chloride ion conductance protein TMEM16A shared structural similarities to the lipid scramblases i.e. it was also an homodimer and each dimer had a subunit cavity or pore, but the chloride permeation channel was made up of half lipid, half protein and the Ca2+ binding site was located within the transmembrane domain of the subunit cavity of the protein. In June 2017 (TMEM16A: 2pores, or not 2 pores) I discussed 2 studies (Lim et al. 2016 & Jeng et al. 2016) using covalently linked mouse TMEM16A subunits with one of the two subunits carrying mutations, which confirmed that the TMEM16A homodimer had 2 independent chloride pores.

Raimund Dutzler’s lab are the first to report the structure of mammalian TMEM16A ion channel with cryo-electron microscopy of mouse TMEM16A at a resolution of 6.6Å (Paulino et al., 2017). They are proposing that the putative pore region of mTMEM16A is now an enclosed aqueous proteinaceous pore with a large intracellular vestibule that narrows to a chloride conducting pathway surrounded by alpha –helices (Figure 1). They show this is brought about by a realignment of helices, when compared to the nhTMEM16A X-ray structure, helices 4 and 6 move from the edges of the subunit cavity, in to enclose the aqueous proteinaceous pore and closing it off to membrane lipids (Figure 1). However, a drawback of this study is the resolution of the cyro-electron microscopy technique, as crucially it does not give the locations of amino acids side chains or the pitch of helices within the protein. We will await the increased resolution of an X-ray protein structure to confirm these important initial findings. Also, the protein was purified in the presence of high levels of Ca2+ and therefore could represent a non-conducting form of mTMEM16A.

Roy 1

Figure 1. Mechanistic relationships within TMEM16 family.

(A) Depiction of the mTMEM16A pore. The molecular surface of the pore region is shown as grey mesh. The boundaries of hydrophobic (black) and polar regions (grey) of the membrane are indicated by rectangular planes. The positions of positively charged residues affecting ion conduction are depicted as blue and bound Ca2+ ions as green spheres. Hypothetical Cl ions (radius 1.8 Å) placed along the pore are displayed as red spheres. (B) Schematic depiction of features distinguishing lipid scramblases (left) from ion channels (right) in the TMEM16 family. The view is from within the membrane (top panels) and from the outside (bottom panels). The helices constituting the membrane accessible polar cavity in scramblases have changed their location in channels to form a protein-enclosed conduit. A and B, Permeating ions and lipid headgroups are indicated in red. (Paulino et al. (2017) eLife;6: e26232).

They tested their hypothesis with functional studies, mutating basic amino acids for neutral Alanines in both the vestibule and within the pore region of the mTMEM16A to see what impact this had on chloride conductance. They found, as predicted, that altering the charge in the vestibule had little impact on chloride conductance whereas, altering the charge in the pore had a more pronounced effect.

The Brunner et al. (2014) X-ray structure and the homology models that arose from it, gave rise to the controversy in the literature, trying to reconcile ion channel dogma with the available TMEM16A functional data and the nhTMEM16 X-ray structure. Paulino et al. (2017) has with this essential first mTMEM16A structure, albeit with the cryo-electron microscopy technique at 6.6Å resolution which needs to be confirmed with the increased resolution of an X-ray structure, resolved our understanding of how chloride ions pass through TMEM16A. And in mTMEM16A anyway, we still await elucidation of the human structure, this has come with the realignment of helices 4 and 6 closing the circle of the subunit furrow to form an aqueous chloride conducting pore.

Blog written by Roy Fox

References:

  1. Paulino et al. (2017) eLife; 6:e26232
  2. Caputo et al. (2008) Science 322:590–594.
  3. Schroeder (2008) Cell 134:1019–1029.
  4. Yang et al. (2008) Nature 455:1210–1215.
  5. Brunner et al. (2014) Nature 516:207–212.
  6. Whitlock JM and Hartzell HC (2016) Eur. J. Physiol. 468: 455-473.
  7.  Lim et al. (2016) J. Gen. Physiol. 148:5, 375-392.
  8.  Jeng et al. (2016) J. Gen. Physiol. 148:5, 393-404.

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