It is becoming increasingly apparent that membrane lipids

It is becoming increasingly apparent that membrane lipids play a critical role in modulating and regulating protein function. For example, embedding the homologous SLC1 transporter GltPh in a POPE bilayer allowed a third Na+ binding site to be identified from MD simulations [42], and the binding energy of Na+ at each of the specific Na+ Clonidine HCl to be calculated [43]. Notably, Lev and Noskov showed that when GltPh is embedded in a DPPC membrane, it is too flexible for the Na1 and Na2 sites to discriminate between monovalent ion species [44]. The regulatory effect of bilayer composition is well documented for many membrane proteins, both experimentally and in MD simulations (for example, see references [[45], [46], [47], [48], [49]]), highlighting the essential role lipids play in regulating protein function. Mass spectrometry analyses have also demonstrated that specific lipid interactions with membrane proteins stabilize structure and promote protein oligomerisation [37,50]. MD simulation techniques are powerful tools that allow the user to combine crystallographic data with model lipid bilayers to elucidate lipid-protein interactions in atomic detail. While the majority of MD simulations of SLC6 transporters have used POPE or POPC lipid bilayers, there has been little discussion of the effect of the appropriate bilayer environment on protein dynamics. For example, simulations of the bacterial LeuT transporter have been conducted in the bacterial model membrane, POPE [23,28,29], as well as the eukaryotic membrane models, POPC [51] and DMPC [52]. These differences in bilayer composition, particularly when experimentally comparing the transporter dynamics in bacterial bilayers to simulations in eukaryotic phosphatidylcholine membranes, can create inconsistencies when attempting to draw conclusive comparative results. With the exception of the study by LeVine and colleagues [12], all of the aforementioned MD simulations of SLC6 transporters were performed with the protein embedded in a bilayer composed of a single lipid species. However, neuronal membranes are comprised of a variety of lipids [53], and with the aid of tools like the CHARMM-GUI membrane builder [54] and insane [55] it is becoming easier to computationally model bilayers that are more reflective of the native membrane, or of the experimental conditions. To date, only a small amount of research has addressed this. One study explored the effect of embedding LeuT in either a pure POPC bilayer or a 3:1 mixture of POPE and POPG, which better represents the bacterial membrane [56]. The membrane became thinner at the protein interface, and water penetrated into the membrane in the vicinity of a membrane-facing polar residue, K288, from TM7 in both membrane types [56]. This water defect exposed hydrophobic residues from both TM7 and TM1a to the aqueous solution, creating a hydrophobic mismatch, which may influence the energetics and rate of transitions between conformational states [56,57]. In other MD simulations, which contained arseno-choline-bound BetP embedded in a 50:50 diastereoisomeric mixture of POPG lipids, a progression involving sequential Na+ and substrate binding, dehydration, and protein coordination was observed as the transport cycle was initiated [58]. Furthermore, microsecond-timescale MD simulations of a human DAT homology model embedded in a sophisticated asymmetric bilayer—modelling a near physiological environment—have been performed. This asymmetric bilayer contained a mixture of POPC, DPPC and cholesterol in the extracellular leaflet (103:11:16 ratio) and POPE, POPC, PIP2, POPS and cholesterol (54:24:15:15:15 ratio) in the intracellular leaflet [59]. In this lipid environment, the ab initio model of the N-terminus of DAT bound spontaneously, and specifically, to the PIP2 phospholipids. This interaction was integral to transporter function: binding to PIP2 allowed the N-terminus to anchor to the bilayer and define the transporter’s location and orientation in the bilayer. This anchoring behaviour was abolished if PIP2 was removed from the bilayer [59]. Kinetic insight into sodium ion transport has been gained from MD simulations based on this hDAT homology model embedded in an asymmetric bilayer containing POPC, sphingomyelin and cholesterol (125:12:29 ratio) extracellularly and POPE, POPC, PIP2, POPS and cholesterol (92:26:18:20:24) intracellularly [60]. Notably, PIP2 has also recently been implicated in stabilising SERT oligomer complexes experimentally [61]. Clearly, lipids can have important and relatively underappreciated impacts on the structure and function of SLC6 transporters.