Synaptic neurotransmission has recently been proposed to function via either a membrane-independent or a membrane-dependent mechanism, depending on the neurotransmitter type. In the membrane-dependent mechanism, amphipathic neurotransmitters first partition to the lipid headgroup region and then diffuse along the membrane plane to their membrane-buried receptors. However, to date, this mechanism has not been demonstrated for any neurotransmitter-receptor complex. Here, we combined isothermal calorimetry measurements with a diverse set of molecular dynamics simulation methods to investigate the partitioning of an amphipathic neurotransmitter (dopamine) and the mechanism of its entry into the ligand-binding site. Our results show that the binding of dopamine to its receptor is consistent with the membrane-dependent binding and entry mechanism. Both experimental and simulation results showed that dopamine favors binding to lipid membranes especially in the headgroup region. Moreover, our simulations revealed a ligand-entry pathway from the membrane to the binding site. This pathway passes through a lateral gate between transmembrane alpha-helices 5 and 6 on the membrane-facing side of the protein. All in all, our results demonstrate that dopamine binds to its receptor by a membrane-dependent mechanism, and this is complemented by the more traditional binding mechanism directly through the aqueous phase. The results suggest that the membrane-dependent mechanism is common in other synaptic receptors, too.
Keywords: ligand entry pathway prediction; lipid membrane; molecular dynamics; random acceleration molecular dynamics; synaptic neurotransmission; umbrella sampling.