Towards Identifying Biologically Relevant Intermediate Conformational States in Dopamine Transporter

Abstract

Psychostimulant abuse leads to debilitating disorders, the treatment of which remains an alarming challenge. Cocaine and amphetamine act on the dopamine transporter (DAT), which belongs to the neurotransmitter:sodium symporter family that terminates neurotransmission by reuptake of neurotransmitters from the synaptic cleft. The reuptake process can be described by the Na+-coupled alternating-access mechanism in which the transporter adopts outward-open, occluded and inward-open conformations. Cocaine is known to inhibit DAT function by trapping the protein in an outward-open conformation. Interestingly, several other DAT inhibitors, such as benztropine, modafinil, and some of their derivatives, appear to have low abuse liability. Compared to cocaine, these atypical DAT inhibitors show a preference for the conformation of DAT that can be stabilized by a mutation at the intracellular gate, Y335A, and were deduced to prefer less outward-open conformations than cocaine. Nevertheless their differential inhibiting mechanisms at the atomistic level is not well understood.We carried out comparatively molecular dynamics simulations of both DAT-WT and DAT-Y335A constructs, stabilized by a variety of DAT inhibitors, and used Markov State Models to analyze the resulting trajectories. This analysis identifies an ensemble of conformational states exhibited spontaneously by the molecule at local equilibrium, and has the advantage in its ability to represent both thermodynamic and kinetic characteristics of protein conformational changes. Thus we capture the conformational states and kinetics of transitions between outward-occluded and outward-open states of DAT. Identification and characterization of the intermediate states that are stabilized by the atypical inhibitors and/or Y335A mutation is crucial in understanding the mechanism of action of these inhibitors. This understanding of how these states eventually trigger different downstream cellular effects from cocaine holds new promises to develop targeted treatment of cocaine dependence.