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. 2021 Feb 26;4(1):259.
doi: 10.1038/s42003-021-01782-2.

Specific PIP2 binding promotes calcium activation of TMEM16A chloride channels

Zhiguang Jia  1   2 , Jianhan Chen  3   4
Affiliations

Specific PIP2 binding promotes calcium activation of TMEM16A chloride channels

Zhiguang Jia et al. Commun Biol. .

Abstract

TMEM16A is a widely expressed Ca2+-activated Cl- channel that regulates crucial physiological functions including fluid secretion, neuronal excitability, and smooth muscle contraction. There is a critical need to understand the molecular mechanisms of TMEM16A gating and regulation. However, high-resolution TMEM16A structures have failed to reveal an activated state with an unobstructed permeation pathway even with saturating Ca2+. This has been attributed to the requirement of PIP2 for preventing TMEM16A desensitization. Here, atomistic simulations show that specific binding of PIP2 to TMEM16A can lead to spontaneous opening of the permeation pathway in the Ca2+-bound state. The predicted activated state is highly consistent with a wide range of mutagenesis and functional data. It yields a maximal Cl- conductance of ~1 pS, similar to experimental estimates, and recapitulates the selectivity of larger SCN- over Cl-. The resulting molecular mechanism of activation provides a basis for understanding the interplay of multiple signals in controlling TMEM16A channel function.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Structural features of the Ca2+-bound mTMEM16A channel.
A The overall dimer structure with key TM helices highlighted (PDB: 5oyb). B, C Front and top views of the putative conducting pore. The pore profile calculated using HOLE is illustrated using the green dots. The two key hydrophobic residues, L547 and I641, in the “neck” region are shown in yellow sticks. The pore-lining TMs are colored in blue (3 and 4), red (5) and green (6), respectively. The other TMs are colored in yellow. The bound PIP2 molecule is represented as sticks in panels B and C. D Cartoon representation of the functional domain organization of TMs from the top. The pore-forming domain consists of the PIP2-binding regulatory module (TMs 3-5) and Ca2+-binding activation module (TMs 6–8). TMs 1, 2, 9, and 10 form the dimerization and supporting domain.
Fig. 2
Fig. 2. Spontaneous TMEM16A pore opening induced by specific PIP2 binding.
A Distance between the centers of mass of L547 and I641 (upper panel) and number of water molecules (lower panel) in the neck region as a function of simulation time. The black and red traces were derived from simulations with (sim 1, chain B) and without (sim 4, chain B) PIP2, respectively. In the upper panel, snapshots belonging to the open cluster (see Fig. S3) are highlighted in green. In the lower panel: the number of lipid heavy atoms inside the neck region during sim 1 is also shown (blue trace). B Averaged pore radius profiles in the neck region calculated using HOLE. The black trace was derived from snapshots sampled during 2.679–2.683 μs of sim 1 and the red trace from those sampled during 0.0–0.01 μs of sim 4. C, D Representative structures of the closed and open pores, taken from 0 ns and 2681 ns of sim 1, respectively. TMs 3, 5, and 6 are colored in red, TM4 in blue, and the rest of the protein in cyan. Two of the inner gate residues (L547 and I641) and key pore-lining residues above the gate (V543, I640, and P595) are represented as yellow and blue sticks, respectively. The HOLE pore profiles are illustrated as the green tunnels.
Fig. 3
Fig. 3. Spontaneous permeation of Cl− through the TMEM16A pore.
A Number of coordinating waters of permeating Cl− and its distance to the inner gate during the event (780–1030 ns of sim 3). The insert shows the hydrating water number of the same Cl− ion in the whole simulation with the spontaneous permeation event highlighted by a dashed red box. B Representative snapshots during permeation. TMs 3 and 5 are represented as red cartoons and TMs 4 and 6 are colored in blue and green, respectively. The inner gate residues (L547, S592, and I641) are shown in yellow sticks. The permeating Cl− ion is represented as a cyan sphere, with water molecules within 4 Å shown in the ball-and-stick mode.
Fig. 4
Fig. 4. Pathway and free energy profiles of Cl− permeation through TMEM16A CaCC.
A Positions of Cl− (green beads) during a spontaneous permeation event (0.73–1.14 μs, sim 3), overlaid onto the snapshot of the channel sampled at 1.0 μs of the same trajectory. B Positions of permeating Cl− during metadynamics simulations, overlaid onto the initial structure of metadynamics simulations. The Cl− ion is presented as beads and different colors represent different permeation events. TMs 3–4 are represented as blue cartoons and TMs 5–6 as red cartoons. The rest of the protein is presented as transparent cartoons. The phosphate atoms in lipid head groups are represented as transparent spheres. The pathway predicted by HOLE is shown as a transparent mesh for reference in panel B. C Free energy profiles of Cl− (black trace) and SCN− (red traces) permeation through the predicted open state of TMEM16A CaCC calculated from umbrella sampling. The error bars were estimated as the difference between results calculated using the first and second halves of the sampling.
Fig. 5
Fig. 5. Rearrangement of TMs during PIP2-induced TMEM16A pore reactivation.
A TM conformations in the closed (TMs 3, 5, 6: red; TM4: blue) and activated (TMs 3, 5, 6: white; TM4: yellow) states. B Dynamic network analysis reveals that TMs 3 and 4 are clustered into two large independent communities (blue and yellow networks, respectively). In A and B, only the headgroup of PIP2 is shown for clarity. C, D Schematic illustration of the TM movement during TMEM16A pore reactivation (C: side view; D: top view). The binding of PIP2 triggers largely rigid-body movement of TMs 3–5, particularly TM4 (highlighted by the red arrows), leading to the dilation of the pore near the neck region (yellow circles in panel C and dashed circle in panel D).
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