Two groups (Lim et al. 2016 & Jeng et al. 2016) with companion papers in the Journal of General Physiology have tried to answer the question, does TMEM16A have 1 or 2 Cl– conducting pores. They have done this with functional studies using covalently linked mouse TMEM16A (mTMEM16A) subunits over expressed in HEK293T cells with one of the two subunits carrying mutations that change the functional properties of TMEM16A ion channels.
Functional members of the TMEM16 family were known to consist of 2 identical subunits (Fallah et al., 2011; Sheridan et al., 2011; Tien et al., 2013) however, with Brunner et al. (2014) solving the crystal structure of the phospholipid scramblase TMEM16 family member from the fungus Nectria haematococca (nhTMEM16) confirmed that TMEM16 molecules adopt a homodimeric architecture. With each subunit harbouring a hydrophilic groove, the “subunit cavity”, located at the periphery of the dimer that is exposed to the lipid bilayer (Figure 1A). The location of the Ca2+ binding site in the hydrophobic part of the phospholipid bilayer offers a plausible explanation for the observed voltage dependence of calcium activation in TMEM16A ion channels, as Ca2+ has to cross part of the transmembrane electric field to reach their binding site.
However, because of the unique architecture of the subunit cavity, forming a half-channel that is exposed to lipids on one side, a potential alternative arrangement of subunits in ion channels of TMEM16A and B was envisioned. In this alternative arrangement, the 2 exposed half-channels can theoretically form a single enclosed aqueous pore that would be completely surrounded by protein residues, akin to other know channel architecture (Figure 1B). In such an arrangement, the Ca2+ binding site and the residues lining the ion conduction path would be in close proximity, and it could thus be expected that any changes in the pore or the Ca2+ binding site in one of the subunits may affect the activation and conduction properties of the entire protein. In contrast, in the case of the separated 2 pore ion conduction pathways, the same mutation may only affect activation and conduction in one of the 2 pores (Figure 1A).
Figure 1. A) Schematic representation of TMEM16A containing two pores that are independently regulated by Ca2+. B) Hypothetical alternative arrangement of subunits resulting in a single pore. Lim et al. 2016 J. Gen. Physiol. 148:5, 375-392.
In CLC proteins, this question was addressed by kinetic analysis of single channel recordings which are known to consist of 2 independent ion transport pathways (Miller 1982). But, because the low single-channel conductance of TMEM16A precludes such a strategy, Lim et al. (2016) studied macroscopic currents of the over expressed, covalently linked subunits of mTMEM16A in HEK293T cells with one of the 2 subunits carrying mutations that change the functional properties of the channel and therefore attempting to answer whether mTMEM16A ion channels consist of 1 or 2 pores, and if 2, whether the 2 pores function independently.
Recordings were performed in the inside-out patch configuration and not whole cell, which under some of their test conditions was potentially non-physiological. They showed that these linked proteins were stable and dimeric, and that the covalent link between the 2 subunits does not significantly alter the functional properties of the TMEM16A protein. The covalent linked dimers showed the established Ca2+ and voltage dependent gating, Ca2+ binding cooperativity and chloride selectivity of TMEM16A ion channels. However, they have also shown a biphasic Ca2+ activation is evident upon careful correction of the irreversible rundown that becomes more severe at higher Ca2+ concentrations with a predominant Ca2+ activation at low and a second shallow step at high Ca2+ concentration, for the linked WT-WT dimer the EC50 for Ca2+ activation was 0.209 µM and 724µM respectively. The second activation lacks any voltage dependence and might thus reflect the interaction of Ca2+ with an unknown low-affinity site located at the cytoplasmic part of the channel. Their results suggest that exposure to 1mM Ca2+ does not change the high anion over cation selectivity of the channel, nor its conductance, but that it results from an increase in the open probability.
More importantly they have also demonstrated that both subunits act independently with respect to Cl– permeation and gating characteristics. They have shown WT linked dimers have the same properties as wild type TMEM16A channels and when they link WT-WT and WT-mutated channels, the signature of the linked dimer retains the functional signature of each subunit of the dimer, inferring 2 independent conducting pores in the dimer. Also, besides the unaltered anion selectivity and conductance of the construct containing only a single activatable subunit, provides additional evidence for the spatial separation of both pores. Functional independence is also corroborated by experiments on constructs where 2 subunits show different potency of Ca2+ activation and where each activation step retains the signature of the non-concatenated counterparts (Figure 2).
Figure 2. Rundown-corrected concentration-reponse relation of the WT-E702Q concatemer at 80mV. The solid line is the best fit to the triphasic Hill equation. Dashed lines indicate the first activation of WT (green) and E702Q (orange) at 80mV. Lim et al. 2016 J. Gen. Physiol. 148:5, 375-392.
Their experiments with mutant containing dimers thus provide strong functional evidence for independent activation of 2 separate ion conduction pores in the covalently linked dimeric mTMEM16A channel. Although the results presented in this study suggest that activation of different subunits of TMEM16A opens distinct pores, the exact mechanism of TMEM16A activation is still a subject of much speculation. Although, their evidence implies that the TMEM16A ion channel may contain two pores, and Ca2+ activation of individual subunits opens the pore associated with that activated subunit (Figure 3).
Figure 3. Cartoon summarizing the functional properties of TMEM16A. Ion conduction pores in the dimeric protein are indicated in light blue. Ca2+ is displayed as dark blue, and Cl- is displayed as red spheres. Lim et al. 2016 J. Gen. Physiol. 148:5, 375-392.
As yet no structural information on either mouse or human TMEM16A protein has been published, neither do we have, at the moment, the resolution to see TMEM16A crystal structure in either the open or closed state. However, Lim et al. (2016) have provided functional evidence that mTMEM16A ion channel has 2 independently functional pores, at high Ca2+ concentrations increases their open probability, potentially giving us a condition for high resolution crystallography to confirm whether TMEM16A has 2 pores, or not 2 pores.
Blog written by Roy Fox
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