The M1 helix connects to and anchors the ECD, which is outside of the cell membrane, while the M2 lines the channel. M3 and M4 connect to the ICD, with the M4 at the interface with the membrane. Figure illustrates the entire protein structure, including the three domains, the subunits, the membrane, and the bounded agonists. It is believed that two or three agonists binding to the pockets of the ECD are sufficient to activate the receptor, triggering the ion channels to open through a series of conformational changes, including hard-to-capture "flip" states. The ligand-binding site in the ECD is made of two twisted β-sheets. Three β-strands make up the outside β-sheet, while seven β-strands make up the inner (vestibule-facing) β-sheet. Seven loops (named A-C and loop 2 in the principal subunit and D-F in the complementary subunit) border the ligand binding site, as shown in Figure (i). The agonists are categorised as full agonists and partial agonists with respect to their efficacy in triggering the ion channel opening. For example, in the zebrafish α1 homomeric GlyR, glycine is the full agonist with EC 50 = 190 ± 20µM, but there are several partial agonists: β-alanine is a structural analogue of glycine with EC 50 = 0.97 mM; taurine is less efficient with EC 50 = 1.05 ± 0.08 mM; 17 γ-gamma aminobutyric acid (GABA) is a very weak agonist with EC 50 = 28.4 ± 0.9 mM. Little information is known about the cooperation and interaction between subunits upon (un)binding. Experimental studies, based on single-channel recording on the rat recombinant α1 GlyR, showed that gating efficacy increased with the level of ligation, reaching a maximum with three bounded ligands, although any number of glycines from one to five could open the ion channel. The dissociation rate constants from the channel closed state increased when the number of bound molecules increased from one to three, indicating negatively cooperative interactions between binding sites. However, the association rate constants appeared to show positively cooperative interactions, being slow for the first binding but increasing for subsequent bindings. Recent mutagenesis experiments on the human α 1 β (4:1) GlyR showed that two binding sites at α1 -α1 interfaces were sufficient for activation and necessary for high efficacy. Very recent coarse-grained free energy simulations, where different binding patterns were tested in the homomeric α1 GlyR, concluded that the activation barrier decreased with an increasing number of glycines, reaching a threshold value for efficient activation with three non-consecutive or more occupied binding sites. Moreover, they showed that the desensitization barrier initially decreased but increased upon more glycine bounding. MD simulations on the the bacterial homomeric pLGIC GLIC, allosterically bounded to the anaesthetic propofol in the TMD, showed that symmetry breaking by different propofol occupancies promoted changes in channel conformation. The effects of different numbers of trans-cis proline switches, proposed as mediators for channel gating, at the interface between the ECD and TMD were also computationally explored in a model of the 5-HT 3A receptor. Elucidating the interactions between bound and unbound pockets will be key to understanding the cooperative mechanisms underlying channel opening. With this goal, we built four GlyR ECD models with different ligand occupancy, as sketched in Figure . We employed MD, which has proved very useful in pLGIC investigations, to perform all-atom simulations of these four models. By evaluating the differences between the pockets dynamics, we probed how five subunits interact at the ECD level and how neighbouring pockets may cooperate. Atomic-level insights into the dynamics and interplay of the binding pockets are important to better understand how these receptors function and malfunction, and may ultimately contribute to opening new avenues for therapeutic drug development.