MOLECULAR MECHANISMS FOR DUAL ALCOHOL MODULATION AND GATING OF CYS-LOOP RECEPTORS
KTH Applied Physics seminars
Thursday 22 November 2012
to 10:00 at
Erik Lindahl (Inst f Teoretisk Fysik, KTH)
Despite high sequence identity, pentameric ligand-gated ion channels exhibit remarkable diversity in function, with anionic and cationic channels that are either potentiated or inhibited by allosteric ligands. The recently available structures of bacterial homologs of these receptors provide an excellent framework for understanding this allosteric modulation and function, but the modeling can be complex; our first simulations of the prokaryotic anionic glycine receptor (GlyR) suggested inter-subunit binding for ethanol (Murail, Biophys J 100, 1642, 2011), which then appeared to be incompatible with the experimental Gloeobacter violaceus ligand-gated ion channel (GLIC) structure showing other modulators binding intra-subunit.
Here, we present new simulations of GLIC that confirm the occurrence of multiple binding sites by showing intra-subunit binding for ethanol. By experimentally introducing the single-site F238A mutation in GLIC we can turn it into a highly ethanol-sensitive channel (Howard, PNAS 108, 12149, 2011), similar to GlyR, and simulations of the mutated species confirm the occurrence of multiple binding sites. To critically test the results, we performed extensive docking and free energy calculations to identify modulator-binding sites and determine their affinity. In the wild-type GLIC, most modulators preferentially bind intra-subunit, with a very weak binding site inter-subunit. However, with the F238A mutation the inter-subunit site achieves a significantly lower free energy, and even becomes the highest-affinity site in the channel for some alcohols and anesthetics.
The F238A mutant appears to stabilize the closed form of GLIC in experiments, which led us to perform several long simulations of this form at neutral pH. Over a four microsecond timescale, the channel pore is observed to dehydrate after which then M2 helices in all five subunits tilt inwards and appear to close the channel. The final conformation of the transmembrane domain obtained after 4 ms is remarkably close to the new locally closed GLIC structure determined by Corringer & Delarue (in press), and provides detailed information about the relative motions of the transmembrane and extracellular domains.