  Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop superfamily are
important neuroreceptors that mediate fast synaptic transmission. They are
activated by the binding of a neurotransmitter, but the details of this process
are still not fully understood. As a prototypical pLGIC, here we choose the
insect resistance to dieldrin (RDL) receptor, involved in the resistance to
insecticides, and investigate the binding of the neurotransmitter GABA to its
extracellular domain at the atomistic level. We achieve this by means of
$\mu$-sec funnel-metadynamics simulations, which efficiently enhance the
sampling of bound and unbound states by using a funnel-shaped restraining
potential to limit the exploration in the solvent. We reveal the sequence of
events in the binding process, from the capture of GABA from the solvent to its
pinning between the charged residues Arg111 and Glu204 in the binding pocket.
We characterize the associated free energy landscapes in the wild-type RDL
receptor and in two mutant forms, where the key residues Arg111 and Glu204 are
mutated to Ala. Experimentally these mutations produce non-functional channels,
which is reflected in the reduced ligand binding affinities, due to the loss of
essential interactions. We also analyze the dynamical behaviour of the crucial
loop C, whose opening allows the access of GABA to the binding site, while its
closure locks the ligand into the protein. The RDL receptor shares structural
and functional features with other pLGICs, hence our work outlines a valuable
protocol to study the binding of ligands to pLGICs beyond conventional docking
and molecular dynamics techniques.
