SLC6 transporter oligomerization

Abstract

Transporters of the solute carrier 6 (SLC6) family mediate the reuptake of neurotransmitters such as dopamine, norepinephrine, serotonin, GABA, and glycine. SLC6 family members are 12 transmembrane helix-spanning proteins that operate using the transmembrane sodium gradient for transport. These transporters assume various quaternary arrangements ranging from monomers to complex stoichiometries with multiple subunits. Dopamine and serotonin transporter oligomerization has been implicated in trafficking of newly formed proteins from the endoplasmic reticulum to the plasma membrane with a pre-fixed assembly. Once at the plasma membrane, oligomers are kept fixed in their quaternary assembly by interaction with phosphoinositides. While it remains unclear how oligomer formation precisely affects physiological transporter function, it has been shown that oligomerization supports the activity of release-type psychostimulants. Most recently, single molecule microscopy experiments unveiled that the stoichiometry differs between individual members of the SLC6 family. The present overview summarizes our understanding of the influence of plasma membrane constituents on transporter oligomerization, describes the known interfaces between protomers and discusses open questions.

Keywords: PIP2; Quaternary structure; neurotransmitter transporter; oligomerization; psychostimulant; transporter-mediated efflux.

FIGURE 1

The sodium‐bound outward‐open conformation represents the resting state of SLC6 transporters. Upon substrate binding to the substrate‐binding site halfway through the membrane, the transporter occludes around the substrate, reaching first the outward‐occluded, then the inward‐occluded state. Substrate and co‐transported ions are then released to the cytosol from the inward‐open conformation. The transport cycle typically completes by returning empty to the outward‐facing state, with the exception of human SERT that carries a bound potassium ion

FIGURE 2

Experimentally and computationally determined oligomerization interfaces. (a) SLC6 transporter fold highlighting the experimentally identified oligomerization interfaces. (b) Oligomerization interfaces predicted by computational approaches. According to (Jayaraman et at, 2019) except the bundle domain, all transmembrane helices can contribute to oligomer interfaces (indicated as orange arc)

FIGURE 3

Electrostatic fields generated by monoamine transporters: (a) The positive electrostatic field in close proximity to intracellular loop 4 of hSERT is shown in blue rendering. A PIP2 molecule is modeled into the membrane for visualization purposes. The fourfold negatively charged headgroup of PIP2 can interact with the positive electrostatic field of hSERT while remaining membrane embedded. hSERT is shown in white surface rendering, the membrane as sticks. Standard coloring (negative potential in red, positive potential in blue) is used for the electrostatic fields. Image reproduced from Anderluh et al., 2017. (b) The electrostatic fields (shown as red and blue surfaces) of hDAT extends far into the plasma membrane, if two positively charged regions on the hDAT surface are juxtaposed, representing attractive fields for PIP2 binding. Image reproduced from Jayaraman et al., 2018

FIGURE 4

Transporter oligomerization: (a) The distribution of oligomeric size of hSERT follows an exponential decay as measured by single molecule imaging. (B) Model of transporter oligomerization and kinetic trapping by PIP2: SLC6 transporters are in a dynamic equilibrium of monomers, dimers, and higher oligomers in the case of hSERT. The occurrence of higher order oligomers decreases with higher oligomeric size. Oligomers are kinetically trapped by PIP2 interactions, which is a consequence of the orders of magnitude faster diffusion of PIP2, therefore effectively stabilizing existing oligomers by fast re‐binding

FIGURE 5

Motions of the bundle domain are important for effective transport. The bundle domain rotates during the transport cycle by switching between the inward and the outward facing state; this leads to lateral bundle movements of retraction and extension (cyan area). (a) The bundle domain can freely move during the transport cycle unless it interacts with other protomers. (b) Interactions that include the bundle domain need to be overcome. Additional energy is required for displacement of the adjacent protomer or for breaking of existing interactions. This effectively slows down the rate of substrate transport