Spontaneous Inward Opening of the Dopamine Transporter Is Triggered by PIP₂-Regulated Dynamics of the N-Terminus

Abstract

We present the dynamic mechanism of concerted motions in a full-length molecular model of the human dopamine transporter (hDAT), a member of the neurotransmitter/sodium symporter (NSS) family, involved in state-to-state transitions underlying function. The findings result from an analysis of unbiased atomistic molecular dynamics simulation trajectories (totaling >14 μs) of the hDAT molecule immersed in lipid membrane environments with or without phosphatidylinositol 4,5-biphosphate (PIP2) lipids. The N-terminal region of hDAT (N-term) is shown to have an essential mechanistic role in correlated rearrangements of specific structural motifs relevant to state-to-state transitions in the hDAT. The mechanism involves PIP2-mediated electrostatic interactions between the N-term and the intracellular loops of the transporter molecule. Quantitative analyses of collective motions in the trajectories reveal that these interactions correlate with the inward-opening dynamics of hDAT and are allosterically coupled to the known functional sites of the transporter. The observed large-scale motions are enabled by specific reconfiguration of the network of ionic interactions at the intracellular end of the protein. The isomerization to the inward-facing state in hDAT is accompanied by concomitant movements in the extracellular vestibule and results in the release of an Na+ ion from the Na2 site and destabilization of the substrate dopamine in the primary substrate binding S1 site. The dynamic mechanism emerging from the findings highlights the involvement of the PIP2-regulated interactions between the N-term and the intracellular loop 4 in the functionally relevant conformational transitions that are also similar to those found to underlie state-to-state transitions in the leucine transporter (LeuT), a prototypical bacterial homologue of the NSS.

A Markov State-based Quantitative Kinetic Model of Sodium Release from the Dopamine Transporter

“…Indeed, studies of LeuT and other related bacterial transporters162233 have proposed that an occupied Na2 site is essential for maintaining the transporter in the inward-closed state and that inward-opening and the subsequent substrate release process are energetically more favorable once the Na + ion has left the Na2 site. Consistent with this model, MD simulations of DAT28 and SERT31 have displayed spontaneous outward-open to inward-open transition upon destabilization and inward release of the sodium from the Na2 site. From analysis of microsecond-long MD trajectories28 we discovered that the release of the sodium from the Na2 site (referred to hereafter as Na + /Na2) in the human DAT (hDAT) can be allosterically triggered by interactions of the N-terminus region of the transporter (residues 1–59) with various other regions of the transporter, and that some of these interactions are electrostatically driven and supported by the highly charged (−4e at neutral pH) PI(4,5)P 2 (phosphatidylinositol 4,5-biphosphate) lipids in the membrane283435.…”

“…They have offered an essential molecular context for the investigation of the mechanism of uphill neurotransmitter reuptake transport enabled by the coupling with the transmembrane Na + gradient110. Our current understanding of functional mechanisms in NSS has been further shaped by the large body of structure-function studies of LeuT and other related bacterial transporters, carried out both experimentally11121314151617 and computationally1112131416181920212223, and more recently by findings from molecular dynamics (MD) simulations of DAT242526272829 and SERT3031 constructs. Together, these studies have suggested for the transport cycle in NSS proteins an allosteric process that is consistent with the alternating access mechanism32 in which concerted dynamic rearrangements on the extracellular (EC) and intracellular (IC) sides of the transporter result from ion- and ligand-specific conformational rearrangements.…”

“…Here we describe results quantifying the rates of Na + ion release from the Na2 site of hDAT from the analysis of the trajectories of extensive equilibrium atomistic MD simulations (~50 μs) using a Markov State Model (MSM)37. We find that starting from the transporter in the occluded state, the average time for Na + ion to diffuse from the Na2 site to the IC solution is ~800 ns after the initial destabilization of the functionally important IC gates262838 that maintain hDAT in the inward-closed state. From this analysis we also identify the existence of a meta-stable intermediate state in the Na + /Na2 release process, in which the Na + ion that has left the Na2 site is temporarily retained within the IC vestibule of hDAT by strong interactions with residue E428.…”

Mapping Cholesterol Interaction Sites on Serotonin Transporter through Coarse-Grained Molecular Dynamics

“…This mechanism would enable the kinase-dependent phosphorylation required for the amphetamine-induced reverse transport of the substrate, which is, in turn, responsible for the psychostimulant effect. Moreover, subsequent simulations on DAT have shown that PIP2 is able to trigger the inward-open state and hence ions/substrate release, by contacting, among the others, residue R443 on IL4, which is conserved in SERT (K460), and K352 (IL3) [32]. However, experimental evidence has also demonstrated that depletion of this lipid does not affect physiological substrate reuptake, neither in SERT nor in DAT [30, 31], suggesting that direct and reverse transport may be independently managed by membrane machinery [32].…”

“…In this respect, recent studies have been carried out on both LeuT- and d DAT-based homology models to investigate the effects of h DAT missense mutations on the onset of brain diseases [29]. A similar conclusion can be drawn the investigation of NSSs-lipid interactions, where the above mentioned homology models have found broad application [30–32]. …”