Functional mechanisms of neurotransmitter transporters regulated by lipid-protein interactions of their terminal loops

Abstract

The physiological functions of neurotransmitter:sodium symporters (NSS) in reuptake of neurotransmitters from the synapse into the presynaptic nerve have been shown to be complemented by their involvement, together with non-plasma membrane neurotransmitter transporters, in the reverse transport of substrate (efflux) in response to psychostimulants. Recent experimental evidence implicates highly anionic phosphatidylinositol 4,5-biphosphate (PIP(2)) lipids in such functions of the serotonin (SERT) and dopamine (DAT) transporters. Thus, for both SERT and DAT, neurotransmitter efflux has been shown to be strongly regulated by the presence of PIP(2) lipids in the plasma membrane, and the electrostatic interaction of the N-terminal region of DAT with the negatively charged PIP(2) lipids. We examine the experimentally established phenotypes in a structural context obtained from computational modeling based on recent crystallographic data. The results are shown to set the stage for a mechanistic understanding of physiological actions of neurotransmitter transporters in the NSS family of membrane proteins. This article is part of a Special Issue entitled: Lipid-protein interactions.

Keywords: Amphetamine-induced efflux; Cell signaling and phosphorylation; Continuum mean-field theory; Electrostatic interactions; Lipid segregation in the membrane; Membrane composition and PIP(2) lipids; Molecular dynamics; Molecular dynamics simulations; Psychostimulant drugs of abuse.

Figure 1

Sequences of the N-terminal regions of the human dopamine transporter (hDAT), serotonin transporter (hSERT), norepinephrine transporter (hNET), and vesicular monoamine transporter 2 (hVMAT2); positively charged Arg/Lys residues are indicated in red, Ser/Thr residues that are potentially targeted for phosphorylation, are in green.

Figure 2

Electrostatic potential isosurfaces (EPIs) (+1kt/e values shown as blue wireframes and −1kt/e as red wireframes) derived from the predicted structures for: (A) human VMAT2 (hVMAT2, residues 1-20); (B) hDAT N-terminus (residues 1-59). In both panels, the locations of basic residues and Serine residues are indicated by labels and black arrows, and highlighted in yellow and white space-fill representations, respectively. Note the extended EPI in the hDAT N-term, generated by a belt-like arrangement of K/R residues. The electrostatic potential was calculated from the Non-linear Poisson-Boltzmann equation using APBS software [105].

Figure 3

Illustration of modes of interaction of the hDAT N-term with PIP₂ lipids in the membrane in selected snapshots from long atomistic MD simulation (A-B), and from the simulations of the chimera construct composed of the hDAT N-term and the dDAT TM bundle (C) (see Ref. [99] for more details). (D-E) Snapshots from various time-points in the atomistic MD simulation with a PIP₂-depleted membrane. The hDAT N-terminus peptide (in pink) is next to the lipid membrane (orange spheres trace the positions of the lipid head group phosphate atoms). In all panels, lipid head group phosphate atoms are shown as orange spheres, the hDAT N-term (residues 1-65) is depicted in pink cartoon, and residues K3, K5, K27, K19, K35, and R51 are shown in space fill and are labeled. Panels (A-C) also illustrate PIP₂ lipids within 3Å of the hDAT N-term (rendered in licorice representation). In panel C the dDAT TM bundle is shown in white cartoon.

Figure 4

View from the intracellular side, of hDAT N-terminus (cartoon) adsorbing on the lipid membrane. Panels show wild type (WT) and various S/D mutants, as indicated. In each panel, the level of local PIP₂ enrichment near the N-terminus is illustrated in color code representing the ratio of local lipid fraction values to the value in the distal bulk regions. Regions of PIP₂ aggregation are shaded white (enrichment values > 1), whereas membrane areas depleted of PIP₂ are shown in dark blue colors (enrichment values < 1). The positively charged residues are highlighted in space fill and labeled on each panel. Steady state distribution of PIP₂ around a given peptide construct was calculated using the SCMFM approach [101, 102], by placing the N-term 2Å away from the lipid surface with average surface charge density of −0.0031e, which corresponds to a lipid mixture with ~5% PIP₂. The Non-linear Poisson-Boltzmann equation was then solved numerically using APBS software [105] to obtain reduced electrostatic potential in space, as described previously [101], in a 0.1 M ionic solution of monovalent counterions (corresponding to λ=9.65Å Debye length), and using a dielectric constant of 2 for membrane interior and protein, and 80 for the solution.

Figure 5

Electrostatic potential isosurfaces (EPIs) (+1kt/e shown as blue wireframes and −1kt/e as red wireframes) derived from the predicted structures for various S/D mutants of the hDAT N-term, as indicated. Locations of basic residues are highlighted in yellow space-fill representations, and are designated by corresponding labels and black arrows. The electrostatic potential was calculated with the Non-linear Poisson-Boltzmann equation using APBS software [105].

Figure 6

Steady-state adsorption free energies of hDAT N-term wild type, and phospho-mimic constructs. Units are k_BT.