The membrane protein GlyT2 is a key neurotransmitter transporter in the inhibitory pain pathways. GlyT2 is expressed in presynaptic inhibitory neurons where it controls the synaptic concentration of glycine, which binds to the postsynaptic glycine receptor to inhibit pain signalling. Lipid based inhibitors of GlyT2 show enormous promise as therapeutic leads in the treatment of pain. Here we use multiscale simulations of GlyT2 in a complex neuronal membrane to examine the impact of a of lipid inhibitors on GlyT2 function, and the recruitment of membrane cholesterol in specific GlyT2 conformations. This work highlights the role of computational techniques in structure-based drug design and the importance of considering physiological membrane lipid distributions when characterising protein function.