TMEM16A: Function follows form


In their recent review for Current Opinion in Structural Biology, Brunner et al. (2016) have followed on from the groups break through TMEM16 protein structure paper, putting forward an hypothesis of potential mechanisms of activation of TMEM16 scramblase activity.  But is this applicable to the 2 confirmed ion channels, within the TMEM16 protein family, TMEM16A and TMEM16B?

Obviously, their hypothesis is based on their nhTMEM16 crystal structure with TMEM16 proteins forming homodimers with 10 transmembrane helices in each protein (Figure 4 of review below).  Mutational studies have identified Cl binding sites which align to the entrance of the proposed subunit cavity, along with Ca2+ binding residues that activate TMEM16 protein added to the functional data (Yu et al. 2012) which helps to link the ion channel to the subunit cavity of TMEM16A and B.  This is given further strength by Yu et al. (2015) who produced a chimeric TMEM16A protein, with a substituted 15 amino acid domain from TMEM16F, which gained scrambling activity and that this substitution was located within the subunit cavity.  This model, along with homology models based on it, seems now to be the accepted structure but does not explain the gating mechanism of the close cousins the chloride channels.  Further family member’s X-ray structure will need to be elucidated to confirm Brunner et al.’s archetypal structure.

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Figure 4.   Potential mechanisms for Ca2+ activation. Inactive proteins are shown on the left, active proteins on the right. Ca2+ is indicated as blue spheres. Top, ‘clogging’, activation proceeds by a conformational change in the subunit cavity. Center, ‘plug’, the protein is activated by a movement of the cytoplasmic domains that block the path in the inactive conformation. Bottom, ‘electrostatic gate’, the Ca2+ neutralizes the excess negative net charge in the binding site (red) and this removes an electrostatic barrier for lipids traversing the cavity. This effect could be general for phospholipids (the negative charge of the phosphate at the lipid headgroup is indicated).

Brunner et al. Current Opinion in Structural Biology 2016, 39: 61-70

In figure 4 of their review (above) Brunner et al. speculate about potential activation mechanisms of TMEM16; first, a structural conformational change in the hydrophilic groove of the subunit cavity, which might take place to block the passage of lipids (‘clogging’).  Second, the N-terminus of TMEM16 might move through conformational changes within the protein to occlude the cytosolic entry of the subunit cavity, therefore ‘plugging’ the cavity/pore.  And third, no large conformational changes take place, but instead the negatively charged amino acids in the Ca2+ binding site act as an electrostatic gate which is nullified on binding Ca2+.  However, scrambling of ceramides (that do not contain a negative charge) still requires activation of the scramblase by Ca2+ (Suzuki et al. 2013).  All are conceivable along with other combinations of the outlined mechanisms of activation of TMEM16 proteins.

The Brunner et al. structure is easily perceived for scramblase activity with the scramblase ‘pore’ having access to membrane lipid via the proposed subunit cavity.  But the question is, can these proposed mechanisms be transferred to the TMEM16A and TMEM16B chloride channels, raising the fundamental question do they have the same structure, is the subunit cavity the ion channel pore, and what is the gating mode?  As alluded to mutational evidence suggests that the location of the ion channel might be within the subunit cavity proposed by Brunner et al. (2016) but how any of the TMEM16 family proteins activate scrambling/ion permeation is currently unknown.

An alternative to Brunner et al.’s hypothesis is put forward by Ma et al. (2016) who clearly and precisely review the evidence for TMEM16A as a chloride channel; the Ca2+ dependent properties, voltage-dependent properties and regulation by other molecules.  In figure 6 of their review (below) they propose a different gating model of TMEM16A regulated by Ca2+, the cartoon of their model indicates the ion channel is proteinaceous in nature and that at high intracellular Ca2+ concentrations the Ca2+ binding site moves outside the membrane, which they suggest is consistent with the evidence that high intracellular Ca2+ concentrations causes TMEM16A inactivation.  But this would not explain the sensitivity of TMEM16A and TMEM16B to the presence of chloride or their voltage-dependency each of which might confer subtle conformational changes which regulate ion channel activity.

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Figure 6.  Proposed gating models of TMEM16A regulated by Ca2+. TMEM16A currents activated by zero Ca2+, low Ca2+ (<1 µM) and high Ca2+ (>1 µM) exhibit different electrophysiological properties in terms of outward rectification, activation kinetics, and deactivation kinetics, as well as rundown. The crystal structure of nhTMEM16 reveals two Ca2+ ions in each monomer binding site located within the membrane (Brunner et al., ). We propose that only one Ca2+ binds to the monomer binding site at low Ca2+ concentrations, whereas two Ca2+ ions bind to the binding site at high Ca2+ concentrations. Therefore, the channel is proposed to have at least four states: “0 Ca” state, “low Ca” state, “high Ca” state, and “rundown” state. In the “0 Ca” state, the channel can be activated by strong depolarization in the absence of Ca2+. In the “low Ca” state, only one Ca2+ ion binds to the monomer binding site, and depolarization facilitates Ca2+ binding to its binding site, thus increasing channel activity. In the “high Ca” state, two Ca2+ ions binds to the monomer binding site, and induce a great conformational change of the channel. The Ca2+ binding site moves out of the membrane, and thus the channel does not respond to membrane depolarization. In the “rundown” state, the channel is closed even in the presence of high Ca2+ concentrations.

Neither model suggests where and what regulatory protein interactions do to confirmation of the ion channel in TMEM16A and TMEM16B, although potentially pull down experiments for associated proteins would give us the answer.  One such protein, calmodulin has been proposed although this is still controversial.

How do we go about reconciling these two models of TMEM16 protein, ultimately we need more structural information principally on vertebrate/human TMEM16 proteins, with the required resolution to see TMEM16 crystal structure in either the ‘open’ or ‘closed’ state or potentially even the more subtle conformational changes in crystal structure generated in chloride free conditions.  This would confirm one or the other proposed structures that makes up the lipid permeation/ion channel pore and gain insight into the gating mode of the TMEM16 protein family and therefore gain an understanding of the function following form.

References

Brunner JD, Schenck S, and Dutzler R. 2016. Structural basis for phospholipid scrambling in the TMEM16 family. Curr. Op. Structural Biol. 39: 61-70.

Ma K, Wang H, Yu J, Wei M and Xiao Q. 2016. New insights on the regulation of Ca2+-activated chloride channel TMEM16A. J. Cell. Physiol. 9999: 1-10.

Suzuki J, Fujii T, Imao T, Ishihara K, Kuba H and Nagata S. 2013. Calcium-dependent phospholipid scramblase activity of TMEM protein family members. J. Biol. Chem. 288: 13305-13316.

Yu K, Duran C, Qu Z and Hartzell HC. 2012. Explaining calcium-dependent gating of anoctamin-1 chloride channels requires a revised topology. Cir. Res. 110: 990-999.

Yu K, Whitlock JM, Lee K, Ortlund EA, Yuan Cui Y and Hartzell HC 2015. Identification of a lipid scrambling domain in AN06/TMEM16F. eLife. 4.

Blog written by Roy Fox

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