Abstract
Pentameric ligand gated ion channels (pLGICs) are ionotropic receptors that mediate fast intercellular communications at synaptic level and include either cation selective (e.g., nAChR and 5-HT3) or anion selective (e.g., GlyR, GABAA and GluCl) membrane channels. Among others, 5-HT3 is one of the most studied members, since its first cloning back in 1991, and a large number of studies have successfully pinpointed protein residues critical for its activation and channel gating. In addition, 5-HT3 is also the target of a few pharmacological treatments due to the demonstrated benefits of its modulation in clinical trials. Nonetheless, a detailed molecular analysis of important protein features, such as the origin of its ion selectivity and the rather low conductance as compared to other channel homologues, has been unfeasible until the recent crystallization of the mouse 5-HT3A receptor. Here, we present extended molecular dynamics simulations and free energy calculations of the whole 5-HT3A protein with the aim of better understanding its ion transport properties, such as the pathways for ion permeation into the receptor body and the complex nature of the selectivity filter. Our investigation unravels previously unpredicted structural features of the 5-HT3A receptor, such as the existence of alternative intersubunit pathways for ion translocation at the interface between the extracellular and the transmembrane domains, in addition to the one along the channel main axis. Moreover, our study offers a molecular interpretation of the role played by an arginine triplet located in the intracellular domain on determining the characteristic low conductance of the 5-HT3A receptor, as evidenced in previous experiments. In view of these results, possible implications on other members of the superfamily are suggested.
Highlights
X-ray crystallographic structures of the pentameric ligand-gated ion channels have boosted the research in computational biology, owing to their importance in vital neuronal activities and their suitability as targets for pharmacological treatments. Pentameric ligand gated ion channels (pLGICs) form the so-called Cys-Loop receptor superfamily and are involved in a number of different physiological and pathological roles[1]: in signal transduction processes at synaptic level,[2] pLGICs can change membrane potential by allowing ions to transiently translocate through them via a complex gating mechanism triggered by neurotransmitter
Structural Features of the 5-HT3A Receptor In Fig 1A, the structure of the 5-HT3A receptor is depicted, where the three domains forming the integral homopentameric membrane protein are evidenced: 1) the ECD is mostly made up by ten β-sheet strands along with the corresponding loops interconnecting them; 2) the TMD is formed by an α-helix bundle of four α-helices per monomer, namely M1, M2, M3 and M4, spanning the lipid bilayer, where the assembly of the internal M2 helices frames the channel core (Fig 1B)
It is believed that the gating mechanism, which is triggered by agonist binding, does involve a rather complex series of movements leading to the pore expansion/ compression at the TMD level; 3) the ICD is formed by an α-helix (MA-helix) and a long, partially unresolved, loop connected to the M3 helix in the TMD
Summary
X-ray crystallographic structures of the pentameric ligand-gated ion channels (pLGICs) have boosted the research in computational biology, owing to their importance in vital neuronal activities and their suitability as targets for pharmacological treatments. pLGICs form the so-called Cys-Loop receptor superfamily and are involved in a number of different physiological and pathological roles[1]: in signal transduction processes at synaptic level,[2] pLGICs can change membrane potential by allowing ions to transiently translocate through them via a complex (not fully uncovered) gating mechanism triggered by neurotransmitter. Through the use of atomistic molecular dynamics simulations and free energy calculations, we have studied three aspects of the receptor: 1) the structural and dynamical features of the whole channel, including ECD, TMD and part of the ICD (MA helices); 2) the potential of mean force of the single-ion translocation through TMD and ICD (the latter formed by post-M3 loops and MA helices) in order to identify and evaluate the energetic barriers experienced by the ions crossing regions pivotal for ionic conductance and selectivity; 3) the molecular determinants of the low single-channel conductance In the latter case, we have simulated three systems, i.e. a wild-type protein and two high-conductive mutants Peculiarities, similarities and differences in structural and biophysical properties, as compared to other well-known homologue channels, are thoroughly presented and discussed
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