In 2008, TMEM16A was identified as the molecular basis of calcium-activated chloride channels (CaCCs), which mediate Cl− permeation in response to an increase in intracellular Ca2+ concentration [1–3], it is widely expressed and have important functions such as transepithelial ion transport, smooth muscle contraction, olfaction, phototransduction, nociception, bone resorption and control of neuronal excitability [4–7]. Its dysfunction is closely related to a variety of diseases, such as hypertension [8], gastrointestinal dysfunction [9], neuropathic pain [10], prostate cancer [11, 12], lung cancer [13] and gastric cancer [14]. TMEM16A is a ligand-dependent anion selective channel that is dually regulated by calcium ions and membrane potential, and can be gated by Ca2+ or by voltage in the absence of Ca2+, but Ca2+- and voltage-dependent gating are very closely coupled [15, 16], it can also be inhibited or activated by some specific molecules [17–19]. Therefore, it is necessary to understand the relationship between the structure and function of TMEM16A, which will help us to regulate the channel.
In 2017, the structure of mouse TMEM16A was published for the first time [20]. Subsequently, a number of high-resolution mouse TMEM16A structures in different state were published, including the double Ca2+-bound state [21–23], the single Ca2+-bound state [21], the Ca2+-free state [22], as well as the mutants both in Ca2+-bound and Ca2+-free state [23]. Similar to all the other members of TMEM16 family [24], the TMEM16A protein is a homodimer. Each subunit of the protein contains cytosolic N- and C-terminal domains, a transmembrane unit consisting of ten membrane-spanning α-helices (TM1-10) and an extracellular component. TM3-8 of each subunit forms an ion conduction pore (groove), which is shape resembles an hourglass, with a small extracellular and a large intracellular vestibule bridged by a narrow neck region that is about 20Å long [21, 22]. Both pores function independently and are activated by the binding of two Ca2+ ions to the site formed by residues in TM6-8 (E654, E702, E705, E734, and D738) [25, 26]. The binding of Ca2+ triggers a conformational change in TM6 [22], and the movement of TM6 is in turn related to the release of the space door in the narrow neck of the hourglass-shaped pore. Recently, Lam et al. have determined by electrophysiological experiments that three hydrophobic residues (I550, I551, and I641) located at the intracellular end of the narrow neck are involved in channel activation [23, 27]. Additionally, the bound Ca2+ ions change the charge distribution at the wide intracellular vestibule, thereby removing a second electrostatic barrier and shaping the electrostatic potential for conduction [20, 28], which will help anions inflow. However, in these understandings of channel structure, calcium activation and pore gating, there is still no results about the fully activated pore for chloride ion penetration and mechanism explaining of the dual regulation of the channel by membrane potential and calcium ion. Therefore, molecular simulations are the best way for us to simulate the dynamic process of channel permeation and its allosteric process under different physical and chemical condition.
In this study, the cryo-EM structures of mouse Ca2+-free (PDB ID: 5OYG) and Ca2+-bound (PDB ID: 5OYB) TMEM16A are used as templates to construct the corresponding TMEM16A models. For these two models, molecular dynamics simulations with and without electric field are carried out respectively to achieve the dynamic process of chloride ion passing through the entire pore, and to investigate the gating mechanism of TMEM16A at membrane potential. The results show that the depolarized membrane potential is sufficient to induce the opening of the pore channels to increase the minimum radius of the neck region from ~ 1 Å to ~ 2 Å, and this transition mainly involves the movement of TM3 and TM4. Movement of TM4 in turn affects the opening of the hydrophobic gate between TM4 and TM6. The data indicate that the membrane potential affects the side chain of K645, which plays a key role in the permeability of chloride ions. In this work, the resulting gating mechanism provides a basis for understanding the role of membrane potential in controlling TMEM16A channel function.