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

Abstract

The dopamine transporter (DAT) belongs to the neurotransmitter:sodium symporter (NSS) family of membrane proteins that are responsible for reuptake of neurotransmitters from the synaptic cleft to terminate a neuronal signal and enable subsequent neurotransmitter release from the presynaptic neuron. The release of one sodium ion from the crystallographically determined sodium binding site Na2 had been identified as an initial step in the transport cycle which prepares the transporter for substrate translocation by stabilizing an inward-open conformation. We have constructed Markov State Models (MSMs) from extensive molecular dynamics simulations of human DAT (hDAT) to explore the mechanism of this sodium release. Our results quantify the release process triggered by hydration of the Na2 site that occurs concomitantly with a conformational transition from an outward-facing to an inward-facing state of the transporter. The kinetics of the release process are computed from the MSM, and transition path theory is used to identify the most probable sodium release pathways. An intermediate state is discovered on the sodium release pathway, and the results reveal the importance of various modes of interaction of the N-terminus of hDAT in controlling the pathways of release.The dopamine transporter (DAT) is a transmembrane (TM) secondary transporter protein belonging to the Neurotransmitter:Sodium Symporter (NSS) family, which also includes the closely related serotonin (SERT) and norepinephrine (NET) transporters 1 . The NSS are responsible for clearance of released neurotransmitters (e.g. dopamine (DA), by DAT) from the synaptic cleft through their translocation back into the presynaptic nerve termini. The function of NSS transporters in neuronal signaling implicate them in the mechanisms of action of abused psychostimulants, such as cocaine and amphetamine 2,3 , and in various psychiatric and neurological disorders including drug addiction, schizophrenia, and Parkinson's disease 3 . These essential neurophysiological roles have made NSS transporters primary targets for antidepressant medications.Breakthrough crystallographic determinations of members of the NSS family includes the bacterial homolog LeuT 4,5 , and most recently the structures of Drosophila DAT (dDAT) [6][7][8] . Structures of these transporters have been determined in complex with various substrates and ligands in the primary (S1) and secondary (S2) binding sites and with sodium ions bound in sites Na1 and Na2. 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 + gradient 1,10 . 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 experimentally 11-17 and computationally [11][12][13][14]16,[18][19][20][21][22][23] , and more recently by findings from molecular dynamics (MD) simulat...

Deep Mutagenesis of a Transporter for Uptake of a Non-Native Substrate Identifies Conformationally Dynamic Regions

“…The stability of the OC state promotes the solvation of the intracellular vestibule, thereby weakening the hydrogen bonding network involving various intracellular gating residues (Arg79-Glu452, Glu80-Lys275, Asp87-Trp282, Tyr350-Glu444). Opening of the intracellular permeation pathway is associated with displacement of Na + from the Na2 site into the cytoplasm (consistent with extensive experimental evidence (36,40,67)), followed by cytosolic exit of APP+. Simulations show contacts made by the neurotransmitter to specific subsites (32) within the orthosteric binding pocket change as import progresses.…”

“…In addition, structural, biochemical and pharmacological studies have shown that substrates entering the outward-facing extracellular vestibule engage an allosteric (or S2) site immediately adjacent to critical gating residues, before moving past the open gate to the orthosteric site (26,(33)(34)(35). Cryo-EM analysis of detergent-solubilized SERT in the presence of ibogaine, a psychoactive plant product that stabilizes IF conformations, allowed for the characterization of OF, OC and IF-like states at moderate resolution (19), showing that regions already implicated in bacterial and mammalian NSS homologues through multiple methods (18,(36)(37)(38)(39)(40)(41) move to open a solvent-accessible intracellular vestibule. In particular, transmembrane helix (TM) 1a moves away from the helical bundle.…”

The Substrate Import Mechanism of the Human Serotonin Transporter

“…[45][46][47] Structural investigations into human NSS transporters have further benefited from the more recent resolution of outward-facing conformations of eukaryotic monoamine transporters Drosophila DAT (d DAT) and human SERT (hSERT). [48][49][50] The screening and dock-ing studies using these crystal structures provide the structural basis of antidepressant recognition and inhibition. [51][52][53][54][55][56] Most recently, cryogenic electron microscopy (cryo-EM) structures of hSERT complexed with the psychedelic non-competitive inhibitor ibogaine reveal the occluded and inward-facing states with similar structural arrangements as seen in LeuT.…”

Thermodynamic Coupling Function Analysis of Allosteric Mechanisms in the Human Dopamine Transporter

“…We have previously described long timescale atomistic MD simulations of hDAT in which we observed spontaneous inward-opening and release of Na þ from Na2 into the intracellular environment (10). Subsequently, we constructed a kinetic model for the release process of Na þ /Na2 and inward-opening of hDAT by using the Markov State Model (MSM) approach on an ensemble of MD simulations totaling >50 ms, and exhibiting multiple spontaneous Na þ /Na2 release events (13). The MSM analysis revealed a complex molecular mechanism in which Na þ /Na2 release can occur via several alternative pathways, each displaying different degrees of net flux that appear to be enabled by different modes of Submitted July 26, 2017, and accepted for publication October 18, 2017.…”

“…The TCF analysis of 50 ms of MD trajectories of hDAT described in ( 13) serves first to quantify the thermodynamic coupling between Na þ /Na2 release and inward-opening, and second to identify the coordinating residues in the Na2 site that mediate the coupling. CVs were constructed from time structurebased independent component analysis (tICA) (16) performed previously (13). The first CV, denoted as ''Na þ /Na2 release,'' combines distances between Na þ /Na2 and coordinating residues in Na2, as well as Na þ /Na2 and Na þ /Na1; the second CV, denoted as inward-opening, combines distances between the intracellular gate residues (see Fig.…”